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Jie Gao, Feng Wu, Sifang Wu, Xing Yang Department of Pulmonary and Critical Care Medicine, Huizhou the Third People’s Hospital, Guangzhou Medical College, Huizhou 516002, People’s Republic of ChinaCorrespondence: Sifang Wu; Xing YangHuizhou the Third People’s Hospital, Guangzhou Medical College, 1# Xuebei Ave., Huizhou, Guangdong, 516002, ChinaTel +86-13502436132; +86-13528085770Fax +86 752-2359648Email wsf197906@126.com; 87575589@qq.comBackground: Measurement of sputum is used to define airway inflammatory phenotypes. Cough variant asthma (CVA) is considered to be the initial stage of classic asthma (CA). The aim of this study was to describe the association between the different subtypes of CVA and CA.Methods: A total of 459 patients with CVA and CA were screened for the study. All included patients performed spirometry, underwent a bronchial challenge with methacholine and induced sputum according to the guidelines.Results: A higher frequency of female patients were found with CVA and the eosinophilic airway inflammation of CVA than in CA and the noneosinophilic airway inflammation of CA (p=0.004 and p=0.024, respectively). Bronchial hyper-responsiveness (BHR) was lower in eosinophilic CVA and CA (p=0.006), while no difference was found in noneosinophilic CVA and CA. Association between the percentage of sputum eosinophils and the FEV1 level fell below 20% of the baseline value (PD20) in CVA and CA (r= − 0.1245, p=0.0357 and r= − 0.2148, p=0.0014, respectively).Conclusion: Eosinophilia may be associated with more severe disease, yet there was no difference in spirometry between the eosinophilic and noneosinophilic groups, and the BHR difference was not dramatic.Keywords: cough variant asthma, airway inflammatory types, sputum induction, bronchial hyper-responsiveness

Jie Gao,1 Rehan M Hussain,2 Christina Y Weng1 1Department of Ophthalmology, Baylor College of Medicine, Houston, TX, USA; 2Retina Associates, Elmhurst, IL, USACorrespondence: Christina Y WengBaylor College of Medicine, Alkek Eye Center, 1977 Butler Blvd, Houston, TX 77030, USAEmail christina.weng@bcm.eduAbstract: Subretinal gene therapy trials began with the discovery of RPE65 variants and their association with Leber congenital amaurosis. The RPE65 protein is critical for the normal functioning of the visual phototransduction cascade. RPE65 gene knockout animal models were developed and showed similar diseased phenotypes to their human counterparts. Proof of concept studies were carried out in these animal models using subretinal RPE65 gene replacement therapy, resulting in improvements in various visual function markers including electroretinograms, pupillary light responses, and object avoidance behaviors. Positive results in animal models led to Phase 1 human studies using adeno-associated viral vectors. Results in these initial human studies also showed positive impact on visual function and acceptable safety. A landmark Phase 3 study was then conducted by Spark Therapeutics using a dose of 1.5 x1011 vector genomes after dose-escalation studies confirmed its efficacy and safety. Multi-luminance mobility testing was used to measure the primary efficacy endpoint due to its excellent reliability in detecting the progression of inherited retinal diseases. After the study met its primary endpoint, the Food and Drug Administration approved voretigene neparvovec (Luxturna®) for use in RPE65-associated inherited retinal diseases.Keywords: gene therapy, inherited retinal diseases, Leber congenital amaurosis, Luxturna, RPE65, voretigene neparvovec, retinitis pigmentosa, retina

Yao Yao,* Xuesong Wang,* Jin Gao Department of Anesthesiology, Xiangyang Central Hospital, Affiliated Hospital of Hubei College of Arts and Science, Xiangyang 441021, Hubei, People’s Republic of China*These authors contributed equally to this workCorrespondence: Jin GaoDepartment of Anesthesiology, Xiangyang Central Hospital, Affiliated Hospital of Hubei College of Arts and Science, Jingzhou Street No. 39, Xiangcheng District, Xiangyang 441021, Hubei, People’s Republic of ChinaTel +86 15897965817Email ji84174603@163.comObjective: Widely used in anesthesia, ketamine is reported to induce neurotoxicity in patients. This study aimed to investigate the molecular regulatory mechanism of long non-coding RNA (lncRNA) KCNQ1 opposite strand/antisense transcript 1 (KCNQ1OT1) in ameliorating ketamine-induced neural injury.Materials and Methods: Sprague–Dawley rats were intraperitoneally injected with ketamine to induce neuronal injury. PC-12 cells treated with ketamine were used as the cell model. Ketamine-induced aberrant expression of KCNQ1OT1, miR-206 and brain-derived neurotrophic factor (BDNF) were examined by quantitative real-time polymerase chain reaction (qRT-PCR). The effects of KCNQ1OT1 and miR-206 on ketamine-induced neural injury in PC-12 cells were then examined by MTT and LDH assay. The regulatory relationships between KCNQ1OT1 and miR-206, and miR-206 and BDNF were detected by dual-luciferase reporter assay.Results: Ketamine induced the apoptosis of neurons of the hippocampus in rats, and the apoptosis of PC-12 cells, accompanied by down-regulation of KCNQ1OT1 and BDNF expressions, and up-regulation of miR-206 expression. Overexpression of KCNQ1OT1 enhanced the resistance to apoptosis of PC-12 cells and significantly ameliorated ketamine-induced nerve injury, while transfection of miR-206 had opposite effects. Mechanistically, KCNQ1OT1 could target miR-206 and reduce its expression level, in turn indirectly increase the expression level of BDNF, and play a protective role in neural injury.Conclusion: KCNQ1OT1/miR-206/BDNF axis is demonstrated to be an important regulatory mechanism in regulating ketamine-induced neural injury. Our study helps to clarify the mechanism by which ketamine exerts its toxicological effects and provides clues for the neuroprotection during anesthesia.Keywords: ketamine, neural injury, lncRNA KCNQ1OT1, miR-206, BDNF

Xiaolin Dong,1,2,* Xirui Fan,3,* Xiaoxue He,3 Sijin Chen,3 Weikang Huang,3 Jianpeng Gao,2,3 Yun Huang,2,3 Hui Wang2,3 1Department of Neurology, The Affiliated Yan’An Hospital of Kunming Medical University, Kunming 650051, Yunnan, People’s Republic of China; 2Key Laboratory of Tumor Immunological Prevention and Treatment of Yunnan Province, Kunming 650051, Yunnan, People’s Republic of China; 3Department of Gastroenterology, The Affiliated Yan’An Hospital of Kunming Medical University, Kunming 650051, Yunnan, People’s Republic of China*These authors contributed equally to this workCorrespondence: Yun Huang; Hui WangThe Affiliated Yan’An Hospital of Kunming Medical University, No. 245 Renmin East Road, Kunming 650051, Yunnan, People’s Republic of ChinaEmail 9542287@qq.com; weihool@163.comPurpose: Gastric cancer (GC) is the second leading cause of cancer-related deaths worldwide. tRNA-derived fragments (tRFs) have been identified as potential biomarkers and cancer therapeutic targets. However, the influence of tRFs on GC remains unknown. The key tRFs were researched in vitro function and mechanism.Patients and Methods: Here, differentially expressed tRFs between GC and paracancerous tissues were identified by small RNA sequencing, and the role of key tRF was evaluated in vitro.Results: Eight tRFs were significantly differentially expressed between GC tissues and adjacent tissues: five were significantly upregulated and three were downregulated in GC tissues. The results of target gene prediction and functional enrichment analysis showed that tRFs with different expressions were mainly involved in cell adhesion and connection, cell migration, wingless-type (Wnt), mitogen-activated protein kinase (MAPK), and cancer signaling pathways. Quantitative real-time polymerase chain reaction (qRT-PCR) indicated that the expression of tRF-24-V29K9UV3IU and its target genes (CCND2, FZD3, and VANGL1) in GC tissues and cells was decreased compared with those in the control group. Importantly, overexpression of tRF-24-V29K9UV3IU inhibited cell proliferation, migration and invasion, while promoted cell apoptosis of GC cells.Conclusion: This study suggests that tRF-24-V29K9UV3IU may hinder GC tumor progression by inhibiting cell proliferation, migration, invasion, while promoting cell apoptosis by regulating the Wnt signaling pathways.Keywords: gastric cancer, tRF, invasion and migration, cell apoptosis, tRF-24-V29K9UV3IU

Li-Wei Shi,1,2 Zhi-Bo Zhao,3 Li Zhong,1 Jian Gao,1 Jian-Ping Gong,3 Hang Chen,4 Yu Min,4 Yi-Yin Zhang,5 Zhi Li4 1Department of Gastroenterology, The Second Affiliated Hospital of Chongqing Medical University, Chongqing 400010, People’s Republic of China; 2Department of Gastroenterology, Chongqing People’s Hospital, Chongqing 400010, People’s Republic of China; 3Department of Hepatobiliary Surgery, The Second Affiliated Hospital of Chongqing Medical University, Chongqing 400010, People’s Republic of China; 4Department of Breast & Thyroid Surgery, The Second Affiliated Hospital of Chongqing Medical University, Chongqing 400010, People’s Republic of China; 5Department of Anesthesiology, Chongqing Hospital of Traditional Chinese Medicine, Chongqing 401120, People’s Republic of ChinaCorrespondence: Zhi LiDepartment of Breast & Thyroid Surgery, The Second Affiliated Hospital of Chongqing Medical University, Yuzhong District, Chongqing 400010, People’s Republic of ChinaTel +86 18883147602Email 393884358@qq.comBackground: Rab40b is an evolutionarily conserved Rab GTPase that plays an important role in intracellular trafficking and is closely related to cancer progression. However, the role and potential molecular mechanism of Rab40b in hepatocellular carcinoma (HCC) have not yet been elucidated.Materials and Methods: The expression of Rab40b in HCC tissues and peritumour tissues was tested by qRT-PCR, Western blotting and histological analysis. A Kaplan–Meier survival curve was generated based on the expression of Rab40b in the HCC samples. Cell proliferation assays, wound healing assays, and transwell assays are used to examine the effect of Rab40b on HCC cell growth in vitro. The effect of Rab40b on cell cycle was examined by flow cytometry. A xenograft implantation model was used to assess the effect of Rab40b on the development of HCC cells in vivo.Results: Rab40b protein expression is upregulated in HCC tissues, and this upregulation is associated with high pathological stage and poor prognosis in HCC patients. Rab40b overexpression promotes the proliferation and metastasis of HCC cells by upregulating cyclin D1, cyclin E1 and matrix metalloproteinase 2 (MMP2) through the PI3K/AKT signalling pathway. Conversely, Rab40b inhibitors can significantly inhibit the proliferation and metastasis of HCC cell lines and induce G0/G1 cell cycle arrest and apoptosis. Studies of a nude mouse xenograft model demonstrated that Rab40b knockdown can significantly inhibit the proliferation and progression of HCC tumours in vivo.Conclusion: Overall, this study demonstrates that Rab40b is an important oncoprotein that promotes HCC progression by regulating the expression of cyclin D1, cyclin E1, p21 and MMP2 through the PI3K/AKT signalling pathway.Keywords: Rab40b, HCC, cell cycle, PI3K/AKT, cyclins, MMP2

Qinchen Lu,1– 3,* Jiamin Gao,1– 3,* Shaomei Tang,4,* Zhijian Li,2,3 Xi Wang,2,3 Caiwang Deng,2,3 Jiaxin Hu,1– 3 Yuting Tao,2,3 Qiuyan Wang1– 3 1Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Guangxi Medical University, Nanning 530021, People’s Republic of China; 2Center for Genomic and Personalized Medicine, Guangxi Medical University, Nanning 530021, People’s Republic of China; 3Guangxi Key Laboratory for Genomic and Personalized Medicine, Guangxi Collaborative Innovation Center for Genomic and Personalized Medicine, Nanning 530021, People’s Republic of China; 4Department of Gastroenterology, The First Affiliated Hospital of Guangxi Medical University, Nanning 530021, People’s Republic of China*These authors contributed equally to this workCorrespondence: Qiuyan Wang; Yuting TaoCenter for Genomic and Personalized Medicine, Guangxi Medical University, 22 Shuangyong Road, Nanning 530021, People’s Republic of ChinaTel/Fax +86 7715353342Email qiuyanwang510@yahoo.com; tytxzxh@126.comPurpose: Accumulating evidence indicates that long non-coding RNAs (lncRNAs) play critical roles in the development of many cancer types. However, the changes of lncRNAs expression profiles in hepatocarcinogenesis remain largely unknown. Therefore, the purpose of this study was to identify the clinical significance, oncogenic functions, and potential mechanism of cancer-related lncRNAs in hepatocellular carcinoma (HCC).Materials and Methods: An in vitro hepatocellular carcinoma model was established via oncogene-mediated transformation with a combination of three genetic alterations, including hTERT overexpression, inactivation of P53, and KRAS activation. Changes of biological function and transcriptome profile in these cell lines were determined by colony formation assay, MTT assay, wound-healing scratch assay, xenograft nude mice model, mass cytometry and RNA sequencing (RNA-Seq). Furthermore, 116 HCC tissues and its corresponding normal tumor-adjacent tissues were explored to validate the results of cell lines. Finally, RNA sequencing, single-cell mass cytometry and fluorescence-activated cell sorter were applied to evaluate the potential association between the expression of lncRNA and the stemness of HCC.Results: LncRNA HOXA-AS2 was aberrantly upregulated and it may be involved in the regulation of cancer stem cells during oncogenic transformation. Consistently, lncRNA HOXA-AS2 expression was significantly upregulated in HCC and its higher expression positively correlated with poor prognosis and stem cell-related functions. Moreover, a specific cancer stem cell subpopulation with EPCAM+, C-MYC+ and CK19+ may exist in higher HOXA-AS2 expression HCC patients.Conclusion: LncRNA HOXA-AS2 plays pivotal roles in the occurrence and progression of HCC, which may act as a therapeutic target for prognostic biomarker in hepatocellular carcinoma.Keywords: diagnostic markers, hepatocellular carcinoma, lncRNA HOXA-AS2, multi-omics analysis, oncogenic transformation, stemness

Xiang Li,1,* Qian Zou,1,* Jing Zhang,2 Peng Zhang,2 Xiong Zhou,1 Satya Siva Kishan Yalamarty,3 Xinli Liang,2 Yali Liu,4 Qin Zheng,2 Jianqing Gao5 1State Key Laboratory of Innovative Drug and Efficient Energy-Saving Pharmaceutical Equipment, Jiangxi University of Traditional Chinese Medicine, Nanchang 330006, Jiangxi, People’s Republic of China; 2Key Laboratory of Modern Preparation of TCM, Ministry of Education, Jiangxi University of Traditional Chinese Medicine, Nanchang 330004, Jiangxi, People’s Republic of China; 3Center for Pharmaceutical Biotechnology and Nanomedicine, Northeastern University, Boston, MA 02115, USA; 4College of Science and Technology, Jiangxi University of Traditional Chinese Medicine, Nanchang 330004, Jiangxi, People’s Republic of China; 5Institute of Pharmaceutics, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, Zhejiang, People’s Republic of China*These authors contributed equally to this workCorrespondence: Jing ZhangKey Laboratory of Modern Preparation of TCM, Ministry of Education, Jiangxi University of Traditional Chinese Medicine, Nanchang 330004, Jiangxi, People’s Republic of ChinaTel/Fax +86 791 87118658Email 20101036@jxutcm.edu.cnPurpose: Folic acid and cyclic arginylglycylaspartic acid peptides were introduced to the surface of negatively charged lipid-coated hybrid polydopamine-cysteine cores for the delivery of epirubicin (EPI) (E/PCF-NPs). The combined chemo-photothermal therapy using E/PCF-NPs for triple-negative breast cancer was evaluated.Materials and Methods: The temperature elevation and thermal toxicity of nanoparticles were studied. The morphology and properties of E/PCF-NPs were characterized by transmission electron microscopy, scanning electron microscopy, and atomic force microscopy. Physicochemical properties, including particle size, zeta potential, drug loading, entrapment efficiency (EE%), stability and in vitro release, were determined. The cell viability, reactive oxygen species (ROS) levels, ratios of oxidized nicotinamide adenine dinucleotide to its reduced form (NAD+/NADH), apoptosis assays, and cellular uptake of E/PCF-NPs were determined on 4T1 cells. Pharmacokinetic studies and tissue distributions were performed and detected by an ultra-high performance liquid chromatography/mass spectrometry system. The antitumor effects of E/PCF-NPs under near-infrared (NIR) laser irradiation were also evaluated.Results: The sphere-like morphology of E/PCF-NPs showed a high EE%, uniform size of 106.7 nm, remarkable stability, and highly improved cytotoxicity under NIR laser, when compared to that of photothermal treatment alone. In vitro release of EPI from E/PCF-NPs was pH sensitive, and a greater response was achieved under NIR laser irradiation. Compared to chemotherapy or photothermal treatment alone, the combined treatment in vitro significantly inhibited the survival rate of 4T1 cells to 17.7%, induced ROS generation, and reduced NAD+/NADH significantly. Treatment with E/PCF-NPs under irradiation induced 4T1 cell apoptosis in approximately 93.6% cells. In vitro cellular uptake of E/PCF-NPs was time-dependent. The long-circulating and higher tumor accumulation of E/PCF-NPs resulted in complete ablation of breast tumor tissue through the enhanced photothermal effect by NIR laser irradiation-mediated cell apoptosis.Conclusion: E/PCF-NPs show enhanced anti-cancer effects due to synergistic effects of chemotherapy with photothermal therapy and may be potential therapeutic agents for cancer treatment.Keywords: polydopamine nanoparticles, L-cysteine, epirubicin, pharmacokinetics, triple-negative breast cancer

Jie Gao,1,2,* Xiaohan Chen,1,* Tianjun Ma,1,* Bin He,1 Peng Li,1 Yucheng Zhao,1 Yuejin Ma,1 Jianhua Zhuang,1 You Yin1 1Department of Neurology, Changzheng Hospital, Second Military Medical University, Shanghai 200003, People’s Republic of China; 2Institute of Translational Medicine, Shanghai University, Shanghai 200444, People’s Republic of China*These authors contributed equally to this workCorrespondence: You Yin; Jianhua ZhuangDepartment of Neurology, Changzheng Hospital, Second Military Medical University, Shanghai 200003, People’s Republic of ChinaTel/Fax +86-21-63586818Email yinyou179@163.com; jianhuazh11@126.comPurpose: Alzheimer’s disease (AD) is a neurodegenerative disorder that manifests as abnormal behavior and a progressive decline in memory. Although the pathogenesis of AD is due to the excessive deposition of amyloid β protein (Aβ) outside the neurons in the brain, evidence suggests that tau proteins may be a better target for AD therapy. In neurodegenerative diseases, a decrease in autophagy results in the failure to eliminate abnormally deposited or misfolded proteins. Therefore, induction of autophagy may be an effective way to eliminate tau proteins in the treatment of AD. We investigated the effects of polyethylene glycol (PEG)-ceramide nanomicelles on autophagy and on tau proteins in N2a, a murine neuroblastoma metrocyte cell line.Methods: Ceramide is a sphingolipid bioactive molecule that induces autophagy. PEG-ceramide is a polymer that is composed of the hydrophobic chain of ceramide and the hydrophilic chain of PEG-2000. In this study, we prepared PEG-ceramide nanomicelles that were 10– 20 nm in size and had nearly neutral zeta potential.Results: The results show that PEG-ceramide nanomicelles caused an increase in the LC3-II/LC3-I ratio, while p62 protein levels decreased. Confocal microscopy revealed a significant increase in the number of dots corresponding to autophagosomes and autolysosomes, which indicated autophagic activation. Moreover, PEG-ceramide nanomicelles induced tau degradation in N2a cells through autophagy.Conclusion: In summary, we have confirmed that PEG-ceramide nanomicelles enhanced autophagic flux and degraded overexpressed human tau proteins in N2a cells by regulating the autophagy pathway. Thus, PEG-ceramide nanomicelles show great promise as agents to induce autophagy and degrade tau proteins in the treatment of AD.Keywords: Alzheimer’s disease, autophagosomes, lysosome, nano-carrier, autophagic flux

Dawei Cui,1 Kaijie Wang,2 Yan Liu,2 Junling Gao,3 Jianzhong Cui1,2 1Department of Surgery, Hebei Medical University, Shijiazhuang, Hebei 050017, People’s Republic of China; 2Department of Surgery, Tangshan Gongren Hospital, Tangshan, Hebei, 063000, People’s Republic of China; 3School of Basic Medical Science, North China University of Science and Technology, Tangshan, Hebei 063200, People’s Republic of ChinaCorrespondence: Jianzhong CuiDepartment of Surgery, Hebei Medical University, Shijiazhuang, Hebei 050017, People’s Republic of ChinaEmail jzhcui0001@163.comObjective: Glioma has the highest incidence among the different tumor types within the nervous system, accounting for about 40% of them. Malignant glioma has a high invasion and metastasis rate, which leads to the poor prognosis of patients. By targeting specific genes, microRNAs serve as key regulators in the epithelial–mesenchymal transformation (EMT) process, which could provide new insights into the treatment of glioblastomas (GBM). The detailed molecular role that miR-623 plays in GBM still remains unclear.Materials and Methods: The level of miR-623 in GBM cells was evaluated by RT-PCR. The function of miR-623 overexpression on GBM cell proliferation, migration, and invasion was assessed by MTS, Transwell analysis, and colony formation assay. In addition, a mouse subcutaneous xenograft model was used to study in vivo effects. The binding between miR-623 and TRIM44 was verified by a dual-luciferase reporter assay and the regulatory function of miR-623 on EMT markers was evaluated using Western blot.Results: The expression of miR-623 was repressed in the GBM cancer cell lines. MiR-623 overexpression or TRIM44 knockdown attenuated the proliferation, migration, and invasion of GBM cell lines. TRIM44 could facilitate the reverse suppression of EMT and miR-623 in GBM progression. MiR-623 was found to inhibit TRIM44 expression by directly binding to its 3ʹUTR. In addition, systemic delivery of miR-623 mimic reduced tumor growth and inhibited TRIM44 protein expression in tumor-bearing nude mice. Furthermore, our findings indicated that miR-623 overexpression or TRIM44 down-regulation impeded the proliferation and migratory ability of LN229 and U251MG glioma cells, and miR-623 attenuates TRIM44-induced EMT by directly targeting the 3ʹUTR of TRIM44, which could serve as preliminary research to identify potential therapeutic targets for future treatment of GBM.Conclusion: Overall, microRNA-623 inhibits epithelial–mesenchymal transition to attenuate glioma proliferation by targeting TRIM44.Keywords: GBM, hsa-miR-623, TRIM44, proliferation, EMT

Jian Gao,1,* Qunbang Chen,1,* Yingjia Zhao,2 Ruizhe Hou1 1Department of Neurosurgery, China-Japan Union Hospital of Jilin University, Changchun, Jilin 130033, People’s Republic of China; 2Department of Gynecology and Obstetrics, The Second Hospital of Jilin University, Changchun, Jilin 130000, People’s Republic of China*These authors contributed equally to this workCorrespondence: Ruizhe Hou Department of NeurosurgeryChina-Japan Union Hospital of Jilin University, No. 126 Xiantai Street, Changchun, Jilin 130033, People’s Republic of ChinaEmail houruizhe@yeah.netIntroduction: Colorectal neoplasia differentially expressed (CRNDE) was reported to promote carcinogenesis in several cancers. However, the role of CRNDE in glioblastoma multiforme (GBM) needs to be further explored.Methods: CRNDE expression levels in GBM tissues and cells were explored using real-time quantitative PCR at first. Effects of CRNDE on GBM cell behaviors were detected by conducting in vitro experiments. Interactions of CRNDE, microRNA-337-3p (miR-337-3p), and ELMO domain containing 2 (ELMOD2) were verified by bioinformatics analysis tools and dual-luciferase reporter assay. Expression correlations of CRNDE and ELMOD2 in GBM tissues were analyzed at GEPIA website.Results: CRNDE expression was upregulated in GBM tissues and cells compared with normal counterparts. CRDNE knockdown inhibits proliferation and migration, but promotes apoptosis in GBM cell, while CRNDE overexpression caused opposite effects. Mechanisms exploration indicated CRNDE serves as sponge of miR-337-3p to upregulate ELMOD2 expression. Furthermore, we showed CRNDE and ELMOD2 were positively correlated in GBM tissues.Discussion: In conclusion, our study highlighted the importance of CRNDE/miR-337-3p/ELMOD2 axis in GBM progression and offered novel strategies for GBM treatment.Keywords: CRNDE, miR-337-3p, ELMOD2, glioblastoma multiforme

Junbin Wang1,2 ,* Jin Gao1 ,* Qinnan Chen,1 Weiyan Zou,3 Fen Yang,4 Chenchen Wei,1 Zhaoxia Wang1 1Department of Oncology, The Second Affiliated Hospital of Nanjing Medical University, Nanjing 210011, People’s Republic of China; 2Department of Oncology, The First Affiliated Hospital of Bengbu Medical College, Bengbu 233004, People’s Republic of China; 3Department of Histology and Embryology, Bengbu Medical College, Bengbu 233030, People’s Republic of China; 4Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Nanjing Medical University, Nanjing 211166, People’s Republic of China*These authors contributed equally to this workCorrespondence: Zhaoxia Wang; Chenchen WeiDepartment of Oncology, The Second Affiliated Hospital of Nanjing Medical University, No. 121 Jiangjiayuan Road, Nanjing City, Jiangsu Province 210011, People’s Republic of ChinaEmail zhaoxiawang66@126.com; weichenchen1990@126.comBackground: Long non-coding RNAs (lncRNAs) have been found to contribute to cisplatin resistance in several cancers; however, the role of lncRNA LINC01116 in cisplatin resistance remains unknown in non-small-cell lung cancer. This study aimed to examine the contribution of LINC01116 to cisplatin resistance in lung adenocarcinoma (LAD).Materials and Methods: Cisplatin-resistant A549/DDP cells were generated by treatment with cisplatin by dose escalation. LINC01116 expression was compared between A549 and A549/DDP cells, and between cisplatin-resistant and non-resistant LAD specimens. The cell viability, colony formation, proliferation, migration and invasion were measured using MTT and Transwell assays, and cell apoptosis and cell cycle were detected using flow cytometry. The expression of E-cadherin and Vimentin was quantified. LAD xenografts were modeled in nude mice to investigate the role of LINC01116 on the resistance of LAD to cisplatin.Results: MTT assay measured the IC50 values of 13.49 ± 1.62 and 3.52 ± 1.33 μg/mL for A549/DDP and A549 cells, respectively. LINC01116 was overexpressed in cisplatin-resistant LAD specimens and A549/DDP cells (P < 0.05). Knockdown of LINC01116 inhibited cell viability, proliferation, migration and invasion, promoted apoptosis and enhanced the sensitivity to cisplatin in A549/DDP cells, while LINC01116 overexpression promoted cell viability, proliferation, migration and invasion, inhibited apoptosis and reduced the sensitivity to cisplatin in A549 cells. LINC01116 knockdown resulted in a 2.1-fold increase in E-cadherin expression and a 56% reduction in Vimentin expression in A549/DDP cells, and LINC01116 overexpression resulted in a 45% reduction in E-cadherin expression and a 1.82-fold increase in Vimentin expression in A549 cells.Conclusion: Dysregulation of lncRNA LINC01116 expression results in resistance of LAD to cisplatin via the EMT process. Our findings support the oncogenic role of LINC01116 to promote the development of cisplatin resistance in LAD, and LINC01116 may be a novel predictor of poor response to cisplatin.Keywords: lung adenocarcinoma, cisplatin, chemotherapy resistance, LINC01116, epithelial–mesenchymal transition

Ruosi Yao,1,2 Yu Xie,1 Xiaoyang Sun,1 Menghui Zhang,1 Jian Zhou,1 Linlin Liu,3 Jian Gao,4 Kailin Xu1,2 1Blood Diseases Institute, Xuzhou Medical University, Xuzhou, Jiangsu, People’s Republic of China; 2Department of Hematology, The Affiliated Hospital of Xuzhou Medical University, Xuzhou, Jiangsu, People’s Republic of China; 3College of Medical Imaging, Xuzhou Medical University, Xuzhou, Jiangsu, People’s Republic of China; 4Jiangsu Key Laboratory of New Drug Research and Clinical Pharmacy, Xuzhou Medical University, Xuzhou, Jiangsu, People’s Republic of ChinaCorrespondence: Kailin XuBlood Diseases Institute, Xuzhou Medical University, Xuzhou, Jiangsu, People’s Republic of ChinaEmail lihmd@163.comJian GaoJiangsu Key Laboratory of New Drug Research and Clinical Pharmacy, Xuzhou Medical University, Xuzhou, Jiangsu, People’s Republic of ChinaEmail 18626042662@163.comIntroduction: Multiple myeloma (MM) is an extremely malignant and incurable hematological cancer. Increased expression of the c-Myc oncoprotein is closely associated with shorter overall survival of MM patients, implying that c-Myc is a potential therapeutic target.Main Methods: We identified a potential c-Myc inhibitor 7594– 0037 by structure-based virtual screening from the ChemDiv database. CCK8 assay and flow cytometry were used to detect MM cell viability, cell cycle and apoptosis. Q-PCR and Western blot were used to measure corresponding mRNA and protein expression levels. Protein stability assay measured the stability of c-Myc.Results: Compound 7594– 0037 exhibited stronger anti-proliferative activity against MM cells, and induced MM cell cycle G2 phase arrest and apoptosis. More importantly, compound 7594– 0037 overcame myeloma resistance to bortezomib and exhibited a synergistic effect with bortezomib, resulting in increased MM cell death. The mechanism consists of compound 7594– 0037 facilitating c-Myc protein degradation via decreasing the c-Myc S62 phosphorylation levels mediated by PIM1 kinase. Molecular dynamics simulation with the c-Myc/7594-0037 complex showed that compound 7594– 0037 bound tightly to the N-terminus of c-Myc, and blocked the binding interaction of the two termini of c-Myc, which resulted in c-Myc entering into an unstable state.Conclusion: Overall, our study provides preliminary data for compound 7594– 0037, which can be used as a novel c-Myc inhibitor and is a potential candidate therapeutic drug for multiple myeloma.Keywords: multiple myeloma, c-Myc inhibitor, virtual screening, drug resistance, apoptosis

Jun Gao,1,* Enliang Li,1,* Weiwei Liu,1,* Qingping Yang,2 Chunyan Xie,3 Jiyuan Ai,1 Fan Zhou,1 Wenjun Liao,1 Linquan Wu1 1Department of Hepatobiliary and Pancreatic Surgery, The Second Affiliated Hospital of Nanchang University, Nanchang, Jiangxi, People’s Republic of China; 2Department of Assisted Reproductive, The First Affiliated Hospital of Nanchang University, Nanchang, Jiangxi, People’s Republic of China; 3Jiangxi Medical College, Nanchang University, Nanchang, Jiangxi 330006, People’s Republic of China*These authors contributed equally to this workCorrespondence: Linquan Wu; Wenjun LiaoDepartment of Hepatobiliary and Pancreatic Surgery, The Second Affiliated Hospital of Nanchang University, No. 1, Minde Road, Nanchang, Jiangxi 330006, People’s Republic of ChinaTel +86 791 86311529Fax +86 791 86262262Email wulqnc@163.com; liaowenjun120@sina.comPurpose: This study aimed to investigate the functions of the circular RNA circMYLK (hsa_circ_0002768) in the development of hepatocellular carcinoma (HCC) and to identify the underlying mechanisms of the circMYLK/miR29a/KMT5C axis.Materials and Methods: Quantitative real-time polymerase chain reaction (qRT-PCR) was utilized to explore the expressions of circMYLK, miR-29a and KMT5C in HCC tissues and cells. A potential miRNA (miR-29a) regulated by circMYLK was also explored, and the target relationship between miR-29a and KMT5C was confirmed. FISH, qRT-PCR, Western blotting, and dual-luciferase reporter assays were used to examine the circMYLK/miR29a/KMT5C signaling pathways involved in HCC development. Additionally, HCC cells were implanted into nude mice subcutaneously to test the role of circMYLK in tumor growth.Results: circMYLK was determined to be significantly upregulated in HCC tissues and cells. Suppression of circMYLK repressed HCC cell proliferation, migration, and invasion while increasing apoptosis. In addition, FISH, qRT-PCR, and Western blotting, as well as dual-luciferase reporter assays, revealed that circMYLK could bind to miR-29a. In rescue experiments, miR-29a had the potential to eliminate the inhibitory effect of circMYLK knockdown in HCC. Moreover, miR-29a was found to target the KMT5C gene, which was positively regulated by circMYLK. Finally, a nude mouse tumorigenicity assay showed that injection of circMYLK siRNA into nude mice drastically suppressed xenograft tumor formation in vivo.Conclusion: Our current study demonstrated that circMYLK promotes HCC progression by acting as a competing endogenous RNA of miR-29a, which regulates the downstream oncogene KMT5C.Keywords: hepatocellular carcinoma, circMYLK, miR-29a, KMT5C

Shuo Wu, Liucheng Ding, Hewei Xu, Jie Gao, Yunpeng Shao, Sicong Zhang, Zhongqing Wei Department of Urology, The Second Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu, People’s Republic of ChinaCorrespondence: Zhongqing WeiDepartment of Urology The Second Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu, People’s Republic of ChinaEmail weizq1@163.comPurpose: Long non-coding RNAs (lncRNAs) are involved in the development of various tumors including prostate cancer. The purpose of this study was to explore the function of a natural antisense RNA, IDH1-AS1, exerting potential carcinogenic effects in prostate cancer through a novel molecular mechanism.Materials and Methods: GEPIA and CCLE databases were searched to identify alterations in the expression of IDH1-AS1, which were then verified by RT-qPCR in 20 pairs of matched tumor and normal tissue samples. Subsequently, CCK-8, EdU, and transwell assays were conducted to investigate the carcinogenic effect of IDH1-AS1. RT-qPCR, Western blot, and isocitrate dehydrogenase 1 (IDH1) enzyme activity assays were used to explore the functional relationship between IDH1-AS1 and its sense gene IDH1.Results: IDH1-AS1 expression was found to be significantly increased in prostate cancer tissues and cell lines. IDH1-AS1 knockdown significantly inhibited the proliferation and migration of prostate cancer cells. Interestingly, RT-qPCR and Western blot analyses revealed that IDH1-AS1 did not significantly affect the expression of IDH1 mRNA or protein but was involved in the regulation of IDH1 enzyme activity in prostate cancer cells.Conclusion: Our experiments revealed that the carcinogenic effects of IDH1-AS1 in prostate cancer may depend on a new molecular mechanism, which directly alters IDH1 enzyme activity. Our findings indicate that IDH1-AS1 is a novel candidate target for prostate cancer treatment.Keywords: antisense RNA, divergent transcription, bioinformatics analysis, enzyme activity

Cui Li,1 Hai Liu,2 Qin Niu,3 Jia Gao3 1Department of Pharmacy, The First People’s Hospital of Lianyungang, Lianyungang, Jiangsu, People’s Republic of China; 2Department of Clinical Laboratory, The First People’s Hospital of Lianyungang, Lianyungang, Jiangsu, People’s Republic of China; 3Department of Oncology, The First People’s Hospital of Lianyungang, Lianyungang, Jiangsu, People’s Republic of ChinaCorrespondence: Hai Liu Email sgd889n@163.comBackground: Hypoxia has been shown to induce the malignant progression of cancer, including non-small cell lung cancer (NSCLC). Circular RNA (circRNA) is considered to be an important regulator of cancer progression. However, the role of a newly discovered circRNA, circ_0000376, in the progression of NSCLC is unclear.Methods: The relative expression levels of circ_0000376, miR-1182 and neuro-oncological ventral antigen 2 (NOVA2) were detected via quantitative real-time polymerase chain reaction (qRT-PCR). Glucose consumption and lactate production were determined using Glucose Assay Kit and Lactate Assay Kit, respectively. Moreover, the protein levels of glycolysis markers and NOVA2 were measured using Western blot (WB) analysis. Furthermore, 3-(4, 5-dimethyl-2 thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide (MTT) assay was performed to assess cell viability, and transwell assay was employed to evaluate cell migration and invasion. The interaction between miR-1182 and circ_0000376 or NOVA2 was confirmed by dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay. In addition, animal experiments were conducted to assess the influence of circ_0000376 silencing on NSCLC tumor growth in vivo.Results: Circ_0000376 was upregulated in NSCLC, and its high expression was related to the poor overall survival of NSCLC patients. Hypoxia could enhance circ_0000376 expression and promote the glycolysis, viability, migration, and invasion of NSCLC cells. However, silencing of circ_0000376 could inhibit the glycolysis, viability, migration, and invasion of hypoxia-induced NSCLC cells. Additionally, circ_0000376 could sponge miR-1182, and miR-1182 could target NOVA2. MiR-1182 silencing could reverse the inhibitory effect of circ_0000376 knockdown on NSCLC progression, and NOVA2 overexpression also could reverse the suppressive effect of miR-1182 overexpression on NSCLC progression. Meanwhile, miR-1182 inhibitor could invert the negative regulation effect of circ_0000376 silencing on NOVA2 expression. In addition, circ_0000376 knockdown inhibited the NSCLC tumor growth via regulating the miR-1182 and NOVA2 expression in vivo.Conclusion: Circ_0000376 promoted NSCLC progression by regulating the miR-1182/NOVA2 axis, suggesting that circ_0000376 might be a potential biomarker for NSCLC treatment.Keywords: NSCLC, circ_0000376, miR-1182, NOVA2

Jingyi Dai1 ,* Yingrong Du1 ,* Jianpeng Gao1 ,* Jun Zhao,2 Lin Wang,3 Ying Huang,1 Jiawei Xia,4 Yu Luo,1 Shenghao Li,4 Edward B McNeil5 1Department of Infectious Diseases, Kunming Third People’s Hospital, Kunming, Yunnan, People’s Republic of China; 2School of Public Health and Management, Hubei University of Medicine, Shiyan, Hubei, People’s Republic of China; 3Department of Clinical Laboratory, Kunming Third People’s Hospital, Kunming, Yunnan, People’s Republic of China; 4Department of Critical Care Medicine, Kunming Third People’s Hospital, Kunming, Yunnan, People’s Republic of China; 5Epidemiology Unit, Faculty of Medicine, Prince of Songkla University, Hat Yai, Songkhla, Thailand*These authors contributed equally to this workCorrespondence: Jun ZhaoSchool of Public Health and Management, Hubei University of Medicine, 30 South Renmin Road, Shiyan, Hubei 442000, People’s Republic of ChinaTel +86-719-8891135Fax +86-719-8875336Email stzhao@163.comBackground: The pandemic due to the novel coronavirus disease 2019 (COVID-19) has resulted in an increasing number of patients need to be tested. We aimed to determine if the use of integrated laboratory data can discriminate COVID-19 patients from other pulmonary infection patients.Methods: This retrospective cohort study was conducted at Kunming Third People’s Hospital in China from January 20 to February 28, 2020. Medical records and laboratory data were extracted and combined for COVID-19 and other pulmonary infection patients on admission. A partial least square discriminant analysis (PLS-DA) model was constructed and calibrated to discriminate COVID-19 from other pulmonary infection patients.Results: COVID-19 patients diagnosed and treated in Kunming were balanced in terms of sex and covered all age groups. Most of them were mild cases; only five were severe cases. The first two dimensions of the PLS-DA model could classify COVID-19 and other pulmonary infection patients with an accuracy of 96.6% (95.1% in the cross-validation model). Basophil count, the proportion of basophils, prothrombin time, prothrombin time activity, and international normalized ratio were the five most discriminant biomarkers.Conclusion: Integration of biomarkers can discriminate COVID-19 patients from other pulmonary infections on admission to hospital and thus may be a supplement to nucleic acid tests.Keywords: COVID-19, biomarker, pneumonia, partial least square discriminant analysis

Yujia Huang,1,2,* Qian Feng,3,* Huabo Jiang,1,* Wanding Zhou,1,2 Jinhong Chen,2 Jie Gao,4 Kai Wang,1 Xiaoping Wan,2 Yongsheng Yu1 1Clinical and Translational Research Center, Shanghai First Maternity and Infant Hospital, Tongji University School of Medicine, Shanghai, People’s Republic of China; 2Department of Gynecology, Shanghai First Maternity and Infant Hospital, Tongji University School of Medicine, Shanghai, People’s Republic of China; 3Fujian Provincial Key Laboratory of Advanced Materials Oriented Chemical Engineering, College of Chemistry and Materials Science, Fujian Normal University, Fuzhou, People’s Republic of China; 4Institute of Translational Medicine, Shanghai University, Shanghai, People’s Republic of China*These authors contributed equally to this workCorrespondence: Xiaoping Wan; Yongsheng Yu Email wanxiaoping61@126.com; yongshengyu@tongji.edu.cnPurpose: Besides the tumor cells themselves, solid tumors are comprised of numerous cell types including infiltrating immune cells such as tumor-associated macrophages (TAMs). TAMs are vital stromal components of host immune system and play a critical role in the development of cancer. TAMs can be divided into two subtypes: M1 tumor-suppressive macrophage and M2 tumor-supportive macrophage. To better address the observations of TAMs functional performance, we describe an in vitro system that mimics the populations of TAMs infiltrated into the tumor mass by using our disintegrable supramolecular gelatin (DSG) hydrogels, which are physically crosslinked by host-guest complexations.Materials and Methods: The host–guest interaction was adopted between the aromatic groups of gelatin and the photocrosslinkable acrylated β-cyclodextrins (Ac-β-CDs) to form the DSG hydrogels. The convenient macrophage/endometrial cancer cells heterospheroid 3D model was set up by DSG hydrogels. RT-PCR and Western blot assays were developed to evaluate the efficiencies of inducers on the macrophages. The ELISA and oxygen saturation assays were performed to measure the secretion of VEGF and consumption of oxygen of tumor and/or macrophages, respectively. To determine the antitumor effects of M2 reprogrammed macrophages in vitro and in vivo, migration assay and tumor xenograft model were used, respectively.Results: The host-guest complexations of DSG hydrogels were controllably broken efficiently by soaking into the solution of competitive guest monomers 1-adamantanamine hydrochloride. The DSG hydrogels help IFN-γ reprogram the M2 to M1 and then decrease the tumor/M2 reprogrammed macrophage cells heterospheroid secretion of VEGF and increase the relative oxygen saturation. Significantly, the co-cultural tumor/M2 reprogrammed group from the disintegrated DSG hydrogels reduced the migration of cancer cells in vitro and the tumor growth in vivo.Conclusion: We obtain a TAMs/tumor microenvironment-responsive 3D model based on the novel DSG hydrogels, and will be of utility in cancer therapy and drug discovery.Keywords: supramolecular hydrogel, disintegration, host-guest complexations, tumor mimic model, tumor-associated macrophages, TAMs

Fan Yang,1 Pengzhen Lei,2 Weihui Zeng,3 Jianwu Gao,1 Na Wu1 1Department of Dermatology, Shaanxi Provincial People’s Hospital, Xi’an, Shaanxi 710068, People’s Republic of China; 2Department of Orthopedics, Shaanxi Provincial People’s Hospital, Xi’an, Shaanxi 710068, People’s Republic of China; 3Department of Dermatology, The Second Affiliated Hospital, School of Medicine, Xi’an Jiaotong University, Xi’an, Shaanxi 710048, People’s Republic of ChinaCorrespondence: Fan YangDepartment of Dermatology, Shaanxi Provincial People’s Hospital, No. 256 Youyi West Road, Xi’an, Shaanxi 710068, People’s Republic of ChinaEmail fanfanniu1985@126.comPurpose: Long intergenic non-protein-coding RNA 173 (LINC00173) plays crucial roles in lung cancer. However, the expression and biological functions of LINC00173 in melanoma have not yet been investigated. In this study, we aimed to characterize the involvement of LINC00173 in melanoma and elucidate its mechanisms of action.Materials and Methods: Reverse-transcription quantitative PCR was performed to measure LINC00173 expression in melanoma. A CCK-8 assay, flow cytometry, and migration and invasion assays were applied to examine melanoma cell proliferation, apoptosis, migration, and invasion, respectively. A xenograft tumor experiment was performed to determine the tumorous growth of melanoma cells in vivo.Results: We found that LINC00173 was upregulated in melanoma tissues and cell lines. High LINC00173 expression was closely associated with TNM stage, lymph node metastasis, and shorter overall survival of patients with melanoma. Functional assays revealed that LINC00173 downregulation inhibited melanoma cell proliferation, migration, and invasion and induced apoptosis, suggesting that LINC00173 acts as an oncogenic RNA. LINC00173 knockdown retarded the tumorous growth of melanoma cells in vivo. Mechanistically, LINC00173 increased insulin receptor substrate 4 (IRS4) expression by sponging microRNA-493 (miR-493), thereby acting as a competing endogenous RNA. The effects of LINC00173 knockdown on the malignant phenotype of melanoma cells were reversed by overexpression of IRS4 or knockdown of miR-493.Conclusion: The LINC00173–miR-493–IRS4 pathway regulates melanoma characteristics by increasing the expression of IRS4 via competitive binding of LINC00173 to miR-493, suggesting that this pathway is a potential target for the diagnosis, prognosis, and/or treatment of melanoma.Keywords: LINC00173, miR-493, melanoma, therapeutic target

Jing Gao,1,* Tanja Törölä,1,* Chuan-Xing Li,2 Steffen Ohlmeier,3 Tuula Toljamo,4 Pentti Nieminen,5 Noboru Hattori,6 Ville Pulkkinen,1 Hiroshi Iwamoto,6 Witold Mazur1 1Heart and Lung Centre, Department of Pulmonary Medicine, University of Helsinki and Helsinki University Hospital, Helsinki, Finland; 2Pulmonomics Group, Respiratory Medicine Unit, Department of Medicine & Centre for Molecular Medicine, Karolinska Institute, Stockholm, Sweden; 3Proteomics Core Facility, Biocentre Oulu, Faculty of Biochemistry and Molecular Medicine, University of Oulu, Oulu, Finland; 4Department of Pulmonary Medicine, Lapland Central Hospital, Rovaniemi, Finland; 5Medical Informatics and Statistics Group, University of Oulu, Oulu, Finland; 6Department of Molecular and Internal Medicine, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan*These authors contributed equally to this workCorrespondence: Jing GaoHeart and Lung Centre, Department of Pulmonary Medicine, University of Helsinki and Helsinki University Hospital, FinlandTel +358417071559Email jing.gao@helsinki.fi, HelsinkiIntroduction: The vitamin D binding protein (VDBP, also known as GC-globulin) and vitamin D deficiency have been associated with chronic obstructive pulmonary disease (COPD). rs7041 and rs4588 are two single nucleotide polymorphisms of the VDBP gene, including three common allelic variants (GC1S, GC1F and GC2). Previous studies primarily assessed the serum levels of vitamin D and VDBP in COPD. However, less is known regarding the impact of the local release of VDBP on COPD lung function. Thus, we examined the association of sputum and plasma VDBP with lung function at baseline and at four years, and examined potential genetic polymorphism interactions.Methods: The baseline levels of sputum VDBP, plasma VDBP and plasma 25-OH vitamin D, as well as the GC rs4588 and rs7041 genotypes, were assessed in a 4-year Finnish follow-up cohort (n = 233) of non-smokers, and smokers with and without COPD. The associations between the VDBP levels and the longitudinal decline of lung function were further analysed.Results: High frequencies of the haplotypes in rs7041/rs4588 were homozygous GC1S/1S (42.5%). Higher sputum VDBP levels in stage I and stage II COPD were observed only in carriers with GC1S/1S genotype when compared with non-smokers (p = 0.034 and p = 0.002, respectively). Genotype multivariate regression analysis indicated that the baseline sputum VDBP and FEV1/FVC ratio at baseline independently predicted FEV1% at follow-up.Discussion and Conclusion: The baseline sputum VDBP expression was elevated in smokers with COPD among individuals with the GC1S/1S genotype, and predicted follow-up airway obstruction. Our results suggest that the GC polymorphism should be considered when exploring the potential of VDBP as a biomarker for COPD.Keywords: vitamin D binding protein, VDBP, genotype, COPD, prospective study, sputum

Yining Xu,1,2,* Teng Yao,2,* Kangmao Huang,2,* Gang Liu,2 Yizhen Huang,2 Jun Gao,2 Huali Ye,2 Shuying Shen,2 Jianjun Ma1,2 1Shaoxing University School of Medicine, Shaoxing, Zhejiang 312000, People’s Republic of China; 2Department of Orthopaedic Surgery, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine & Key Laboratory of Musculoskeletal System Degeneration and Regeneration Translational Research of Zhejiang Province, Hangzhou, Zhejiang, People’s Republic of China*These authors contributed equally to this workCorrespondence: Shuying ShenDepartment of Orthopaedic Surgery, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine & Key Laboratory of Musculoskeletal System Degeneration and Regeneration Translational Research of Zhejiang Province, Hangzhou, Zhejiang 310000, People’s Republic of ChinaTel +0086-057186002200Fax +0086-057186044817Email 11207057@zju.edu.cnJianjun MaShaoxing University School of Medicine, Shaoxing, Zhejiang 312000, People’s Republic of ChinaEmail sealteam@zju.edu.cnPurpose: Prevailing evidences have demonstrated that circular RNAs (circRNAs) are closely associated with various stages of carcinogenesis. However, very few studies have delineated the specific mechanism of association between circRNAs and osteosarcoma (OS). It offers a novel insight that circRNAs can be explored as a potential therapeutic strategy for OS.Materials and Methods: In this study, circTUBGCP3 was chosen from the existing reported circRNA microarray data obtained from OS cell lines and normal bone cells. Subsequently, qRT-PCR was performed to evaluate the expression level of circTUBGCP3 in OS samples and cell lines. Functional assays were conducted to estimate the impact of circTUBGCP3 on human OS cells proliferation, vitality, survivability, and migration. Western blot, luciferase reporter and in vivo tumorigenesis assays were performed to analyze the signaling pathways underlying the interaction of circTUBGCP3, miR-30b, and Vimentin.Results: The data indicate that circTUBGCP3 may act as a sponge of miR-30b that further alters the expression of Vimentin, and promotes the proliferation and metastatic properties of OS cells.Conclusion: circTUBGCP3 serves as a tumor promoter in tumorigenesis by increasing the possibilities of OS initiation and proliferation.Keywords: osteosarcoma, circular RNA, circTUBGCP3, miR-30b, Vimentin

Ying Dong,1 Jing Gao,1 Mengyue Pei,2 Xiaoli Wang,2 Chuangnian Zhang,2 Yingjie Du,1 Yanjun Jiang1 1School of Chemical Engineering and Technology, Hebei University of Technology, Tianjin 300130, People’s Republic of China; 2Tianjin Key Laboratory of Biomaterial Research, Institute of Biomedical Engineering, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin 300192, People’s Republic of ChinaCorrespondence: Jing Gao; Xiaoli Wang Tel +86-22-60204945; +86-22-87890151Email jgao@hebut.edu.cn; wangxl@bme.pumc.edu.cnBackground: Nanocarriers could deliver significantly higher amounts of antigen to antigen-presenting cells (APCs), which have great potential to stimulate humoral and cellular response in cancer immunotherapy. Thereafter, silica solid nanosphere (SiO2) was prepared, and a model antigen (ovalbumin, OVA) was covalently conjugated on the surface of SiO2 to form nanovaccine (OVA@SiO2). And the application of OVA@SiO2 for cancer immunotherapy was evaluated.Materials and Methods: SiO2 solid nanosphere was prepared by the Stöber method, then successively aminated by aminopropyltriethoxysilane and activated with glutaraldehyde. OVA was covalently conjugated on the surface of activated SiO2 to obtain nanovaccine (OVA@SiO2). Dynamic light scattering, scanning electron microscope, and transmission electron microscope were conducted to identify the size distribution, zeta potential and morphology of OVA@SiO2. The OVA loading capacity was investigated by varying glutaraldehyde concentration. The biocompatibility of OVA@SiO2 to DC2.4 and RAW246.7 cells was evaluated by a Cell Counting Kit-8 assay. The uptake of OVA@SiO2 by DC2.4 and its internalization pathway were evaluated in the absence or presence of different inhibitors. The activation and maturation of bone marrow-derived DC cells by OVA@SiO2 were also investigated. Finally, the in vivo transport of OVA@SiO2 and its toxicity to organs were appraised.Results: All results indicated the successful covalent conjugation of OVA on the surface of SiO2. The as-prepared OVA@SiO2 possessed high antigen loading capacity, which had good biocompatibility to APCs and major organs. Besides, OVA@SiO2 facilitated antigen uptake by DC2.4 cells and its cytosolic release. Noteworthily, OVA@SiO2 significantly promoted the maturation of dendritic cells and up-regulation of cytokine secretion by co-administration of adjuvant CpG-ODN.Conclusion: The as-prepared SiO2 shows promising potential for use as an antigen delivery carrier.Keywords: antigen delivery, silica solid sphere, nanovaccine, cancer immunotherapy

Jia Gao,1,* Ziteng Zhang,2,* Hong Su,1 Ling Zong,2 Yan Li1 1Department of Thoracic Surgery, Heze Municipal Hospital, Heze, Shandong 274031, People’s Republic of China; 2Department of Thoracic Surgery, Affiliated Hospital of Jining Medical University, Shandong 272000, People’s Republic of China*These authors contributed equally to this workCorrespondence: Yan LiDepartment of Thoracic Surgery, Heze Municipal Hospital, No. 2888 Caozhou West Road, Heze, Shandong 274031, People’s Republic of ChinaEmail yanli_heze@163.comPurpose: Previous studies have identified the important roles of a long noncoding RNA called FGD5 antisense RNA 1 (FGD5-AS1) in several types of human cancer. Nonetheless, to our knowledge, the expression and functions of FGD5-AS1 in esophageal squamous cell carcinoma (ESCC) have not been clarified. In this study, we aimed to determine the expression status of long noncoding RNA FGD5-AS1 in ESCC, determine its participation in ESCC progression, and uncover the underlying mechanisms.Methods: ESCC tissue samples and paired normal adjacent tissues were collected to quantify FGD5-AS1 expression by reverse-transcription quantitative PCR. The effects of FGD5-AS1 on ESCC cell proliferation, apoptosis, migration, and invasion in vitro as well as tumor growth in vivo were studied using a Cell Counting Kit-8 assay, flow cytometry, Transwell migration and invasion assays, and an in vivo tumor xenograft experiment.Results: FGD5-AS1 was found to be aberrantly upregulated in both ESCC tumors and cell lines compared to the control groups. Increased FGD5-AS1 expression manifested a close association with tumor size, TNM stage, and lymph node metastasis in patients with ESCC. Overall survival of patients with ESCC was shorter in the FGD5-AS1 high-expression group than in the FGD5-AS1 low-expression group. An FGD5-AS1 knockdown markedly attenuated ESCC cell proliferation, migration, and invasion and promoted apoptosis in vitro as well as slowed tumor growth in vivo. Mechanism investigation revealed that FGD5-AS1 can increase SP1 expression by sponging microRNA-383 (miR-383), thus functioning as a competing endogenous RNA. An miR-383 knockdown and recovery of SP1 expression attenuated the inhibition of the malignant characteristics of ESCC cells by the FGD5-AS1 knockdown.Conclusion: Thus, FGD5-AS1 enhances the aggressive phenotype of ESCC cells in vitro and in vivo via the miR-383–SP1 axis, which may represent a novel target for ESCC therapy.Keywords: esophageal squamous cell carcinoma, FGD5 antisense RNA 1, microRNA-383

Rui Xiong, 1 Tao Yin, 2, 3 Jian-long Gao, 2, 3 Yu-feng Yuan 1 1Department of Hepatobiliary and Pancreatic Surgery, Zhongnan Hospital of Wuhan University, Wuhan 430071, Hubei Province, People’s Republic of China; 2Department of Hepatobiliary and Pancreatic Surgery, Hubei Cancer Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430079, Hubei Province, People’s Republic of China; 3Seventh Clinical Medical College, Huazhong University of Science and Technology, Wuhan 430079, Hubei Province, People’s Republic of ChinaCorrespondence: Yu-feng YuanZhongnan Hospital of Wuhan University, No. 169 of Donghu Road, Wuchang District, Wuhan 430071, Hubei Province, People’s Republic of ChinaEmail znyuanyufeng@163.comBackground: Gastric cancer (GC) is the most common malignant tumor of the digestive tract and its molecular mechanism is not clear. HOXD9 plays an important role in tumor progression as transcription factor. In the current study, we explored the role of HOXD9 in GC.Methods: We predicted the expression and potential mechanism of HOXD9 in GC through an online database. The expression of HOXD9 was detected in GC and adjacent tissues, and then we analyzed the relationship between HOXD9 and the prognosis of patients with GC. In vitro, we investigated the effects of HOXD9 on malignant biological behaviors such as proliferation, migration, and invasion of the GC cell line MCG-803. In addition, we have initially studied the underlying mechanism by Western blot.Results: High expression of HOXD9 in GC was predicted by online database prediction and implied poor prognosis. In the clinical sample, we confirmed the above predictions. In vitro, we found that knockdown of HOXD9 could effectively inhibit the proliferation, migration, and invasion of GC cells. In terms of mechanism, HOXD9 may activate the TGF-β/Smad signaling pathway.Conclusion: HOXD9 promotes the malignant biological process of GC, which may be a potential therapeutic target for GC.Keywords: HOXD9, gastric cancer, prognosis, TGF-β/Smad pathway

Gang Wang, 1, 2,* Jinqi Gao, 3,* Jinyan Lv, 1 Xi Chen, 1 Jinyu Wu, 1 Ruoyu Wang, 1, 2 Jianing Jiang 1, 2 1Department of Medical Oncology, The Affiliated Zhongshan Hospital of Dalian University, Dalian, Liaoning Province, People’s Republic of China; 2The Key Laboratory of Biomarker, High-Throughput Screening and Target Translation of Breast and Gastrointestinal Cancer, Dalian, Liaoning Province, People’s Republic of China; 3Department of Intervention, The Second Affiliated Hospital of Dalian Medical University, Dalian, Liaoning Province, People’s Republic of China*These authors contributed equally to this workCorrespondence: Ruoyu Wang; Jianing JiangDepartment of Medical Oncology, The Affiliated Zhongshan Hospital of Dalian University, Dalian, Liaoning Province 116001, People’s Republic of ChinaTel/Fax +86-411-62897370; +86-411-62897233Email wangruoyu1963@163.com; jing_jianing@126.comAbstract: Cabozantinib has been shown to have potent anti-ROS1 activity in many solid malignancies, particularly against those with solvent-front resistance mutations following crizotinib therapy. With regard to the most common CD74-ROS1 fusion, the efficacy of cabozantinib has only been demonstrated in vitro. Therefore, we evaluate the efficacy of cabozantinib in a patient with advanced non-small-cell lung cancer (NSCLC) harboring a CD74-ROS1 fusion in the present study. A 40-year-old female patient presented with 1-month history of cough, white sputum and chest pain. Chest CT scan revealed a consolidation in the middle lobe of the right lung together with multiple cavity lesions spreading in both lungs. Histopathological analysis of biopsy samples from the lesion in the middle lobe of the right lung suggested lung adenocarcinoma. After two lines of chemotherapy and EGFR-TKI therapy, a CD74-ROS1 rearrangement was detected and the patient was administered with cabozantinib for 1.5 years. Since cabozantinib resistance developed, crizotinib therapy was applied and demonstrated clinical effectiveness until now. Together, we report the first case of cabozantinib effectiveness in treating a CD74-ROS1-positive advanced NSCLC patient. Crizotinib remained as an effective therapeutic option following the acquisition of cabozantinib resistance.Keywords: cabozantinib, CD74-ROS1 fusion, NSCLC, crizotinib

In this study, the experimental vapor–liquid equilibria data of the ternary Pb–Sn–Sb alloy system are determined using a new experimental method. The experimental VLE data passed the thermodynamic consistency test (Van Ness test), suggesting that the experimental results are reliable. The activities of the components of Pb–Sn–Sb ternary alloy and those of the corresponding three constituent binaries were calculated using the Wilson equation. The predicted values are in good agreement with the data determined from the experiments, and the average relative deviation and average standard deviation were smaller than ± 4.00% and ± 0.03 for all constituent binaries, respectively, which indicates that the Wilson equation is reliable for calculating the activity of the components of the Pb–Sn–Sb ternary alloy. The VLE data of the Pb– Sn and Sb–Sn binary alloys and Pb–Sn–Sb ternary alloy were calculated based on the VLE theory and the Wilson equation. The calculated VLE data were in good agreement with the data determined from the experiments, indicating that this method is reliable for calculating the VLE of alloy systems. The proposed study offers a valid method for analyzing the composition of products that are dependent on the distillation temperature and system pressure during vacuum distillation, which is of great significance to the experimental design of this process.

Xin Li,1 Jiaqi Gao,1 Zepeng Yu,1 Weihai Jiang,2 Wei Sun,2 Chunyan Yu,1 Jinghui Sun,1 Chunmei Wang,1 Jianguang Chen,1 Shu Jing,2 He Li1 1Department of Pharmacology, College of Pharmacy, Beihua University, Jilin, Jilin 132013, People’s Republic of China; 2Affiliated Hospital of Beihua University, Jilin, Jilin 132011, People’s Republic of ChinaCorrespondence: Shu Jing; He Li Email yitongjingshu@163.com; yitonglh@126.comBackground: Aging is a spontaneous and inevitable phenomenon of biology, which can lead to the gradual deterioration of tissues and organs. One of the age-related deterioration processes is immunosenescence, which leads to changes in the function of immune systems, including immune cells and associated cytokines. A proper modulation of immune responses can improve the age-related immunosenescence process and then reach healthy aging. Schisandra sphenanthera, a traditional Chinese medicine, has been used as both a medicine and a nutritional supplement for thousands of years. Anwulignan, a monomer compound of Schisandra sphenanthera lignans, has been reported to possess an immunomodulatory effect. Therefore, this study was designed to further explore whether Anwulignan could also modulate the immune functions in aging model mice and the underlying mechanism.Methods: D-galactose (D-gal) is often used as an inducer of immunosenescence in animals. In this study, a mice model was created by subcutaneous D-gal (220 mg kg− 1) for successive 42 days. Then, the blood and spleen tissue samples were taken for the analysis and observation of cytokine levels, immunoglobulin levels, leukocyte numbers, and the phagocytic activity of macrophages, as well as the histological changes, the proliferation ability of lymphocytes, and the biochemical parameters in the spleen tissue.Results: Anwulignan significantly increased the serum levels of IL-2, IL-4, IFN-γ, lgG, lgM, and lgA, decreased the content of TNF-α and IL-6 in the aging mice, and increased the blood leukocyte number, the phagocytic activity, the lymphocyte proliferation, and the spleen index in vitro. Anwulignan also significantly increased the activities of SOD and GSH-Px, decreased the contents of MDA and 8-OHdG in the spleen tissue, up-regulated the expressions of Nrf2, HO-1, and Bcl2, down-regulated the expressions of Keap1, Caspase-3, and Bax in the spleen cells, and reduced the apoptosis of spleen lymphocytes.Conclusion: Anwulignan can restore the immune function that is declined in D-gal-induced aging mice partly related to its antioxidant capacity by activating the Nrf2/ARE pathway and downstream enzymes, as well as its anti-apoptotic effect by regulating Caspase-3 and the ratio of Bcl2 to Bax in the spleen.Keywords: Anwulignan, immunosenescence, antioxidation, anti-apoptosis

Jianbo Gao, 1 Bing Xia, 1 Shengyou Li, 1 Liangliang Huang, 2 Teng Ma, 1 Xiaowei Shi, 1 Kai Luo, 3 Yujie Yang, 1 Laihe Zhao, 1 Hao Zhang, 4 Beier Luo, 5 Jinghui Huang 1 1Department of Orthopaedics, Xijing Hospital, Fourth Military Medical University, Xi’an, People’s Republic of China; 2Department of Orthopaedics, The General Hospital of Central Theater Command of People’s Liberation Army, Wuhan, People’s Republic of China; 3Department of Orthopaedics, The 985th Hospital of the PLA Joint Logistics Support Force, Taiyuan, People’s Republic of China; 4Department of Spinal Surgery, People’s Hospital of Longhua District, Shenzhen, People’s Republic of China; 5Department of Orthopaedics, Changhai Hospital, Second Military Medical University, Shanghai, People’s Republic of China*These authors contributed equally to this workCorrespondence: Beier LuoDepartment of Orthopaedics, Changhai Hospital, Second Military Medical University, 800 Xiangyin Road, Shanghai 200433, People’s Republic of ChinaTel/Fax +862131161700Email bear_luo@126.comJinghui HuangDepartment of Orthopaedics, Xijing Hospital, Fourth Military Medical University, 169 Changle West Road, Xi’an, Shaanxi 710032, People’s Republic of ChinaTel/Fax +862984775275Email huangjh@fmmu.edu.cnPurpose: The clinical outcome of spinal cord injury is usually poor due to the lack of axonal regeneration and glia scar formation. As one of the most classical supporting cells in neural regeneration, Schwann cells (SCs) provide bioactive substrates for axonal migration and release molecules that regulate axonal growth. However, the effect of SC transplantation is limited by their poor migration capacity in the astrocyte-rich central nervous system.Methods: In this study, we first magnetofected SCs with chondroitinase ABC-polyethylenimine functionalized superparamagnetic iron oxide nanoparticles (ChABC/PEI-SPIONs) to induce overexpression of ChABC for the removal of chondroitin sulfate proteoglycans. These are inhibitory factors and forming a dense scar that acts as a barrier to the regenerating axons. In vitro, we observed the migration of SCs in the region of astrocytes after the application of a stable external magnetic field.Results: We found that magnetofection with ChABC/PEI-SPIONs significantly up-regulated the expression of ChABC in SCs. Under the driven effect of the directional magnetic field (MF), the migration of magnetofected SCs was enhanced in the direction of the magnetic force. The number of SCs with ChABC/PEI-SPIONs migrated and the distance of migration into the astrocyte region was significantly increased. The number of SCs with ChABC/PEI-SPIONs that migrated into the astrocyte region was 11.6- and 4.6-fold higher than those observed for the intact control and non-MF groups, respectively. Furthermore, it was found that SCs with ChABC/PEI-SPIONs were in close contact with astrocytes and no longer formed boundaries in the presence of MF.Conclusion: The mobility of the SCs with ChABC/PEI-SPIONs was enhanced along the axis of MF, holding the potential to promote nerve regeneration by providing a bioactive microenvironment and relieving glial obstruction to axonal regeneration in the treatment of spinal cord injury.Keywords: Schwann cells, astrocytes, magnetic field, superparamagnetic iron oxide nanoparticles, spinal cord injury, cell orientation

Xiao Luo, 1, 2,* Zhangya He, 1, 3,* Xiaomin Sun, 1, 3 Xinqian Gu, 1, 3 Wanyu Zhang, 1, 3 Jiayi Gao, 1, 3 Xiaomin Li, 1, 3 Ru Jia, 4 Junxiang Wei, 5 Yan Yu, 1, 3 Xiaoqin Luo 1, 3 1Department of Nutrition and Food Safety, School of Public Health, Xi’an Jiaotong University, Xi’an 710061, People’s Republic of China; 2Department of Physiology and Pathophysiology, School of Basic Medical Sciences, Xi’an Jiaotong University Health Science Center, Xi’an 710061, People’s Republic of China; 3Nutrition and Food Safety Engineering Research Center of Shaanxi Province, School of Medicine, Xi’an Jiaotong University, Xi’an 710061, People’s Republic of China; 4Department of Prosthodontics, Stomatological Hospital, College of Stomatology, Xi’an Jiaotong University, Xi’an 710061, People’s Republic of China; 5Department of Epidemiology and Health Statistics, School of Public Health, Xi’an Jiaotong University, Xi’an 710061, People’s Republic of China *These authors contributed equally to this workCorrespondence: Xiaoqin LuoDepartment of Nutrition and Food Safety, School of Public Health, Xi’an Jiaotong University, Xi’an 710061, People’s Republic of ChinaTel/Fax +86-29-82655111Email luoxiaoqin2012@mail.xjtu.edu.cnAim: Docosahexaenoic acid (DHA; C22; n-3) shows beneficial effects on Non-alcoholic fatty liver disease (NAFLD). Deacetylase Sirtuin1 (Sirt1) was reported to increase energy metabolism and decrease lipogenesis. Here, we investigated whether DHA plays a role in protecting against hepatic steatosis via Sirt1.Main Methods: Both in vivo and in vitro hepatic steatosis models were used: diet-induced obesity (DIO) model (middle-aged C57BL/6 mice fed a high-fat diet (HFD)) and palmitic acid (PA)-induced lipid accumulation cell model (HepG2 cells).Key Findings: In DIO mice, treatment with DHA (gavage supplementation) for 8 weeks not only inhibited the lipid accumulation, but also increased fatty acids (FA) oxidation and induced triglyceride export in liver. These changes were accompanied by attenuation of inflammation. Moreover, DHA reversed the HFD-induced reduction of Sirt1 in liver. Interestingly, the beneficial effects of DHA were reversed by lentivirus-mediated Sirt1 knockdown, accompanied with increased expression of markers of lipogenesis, inflammation and reduced FA oxidation. In HepG2 cells, DHA prevented the accumulation of PA-induced lipid droplets, the decrease of FA oxidation and the reduction of Sirt1 level. Inhibition of Sirt1 by sirtinol partially reversed the beneficial effects of DHA on PA-treated cells.Significance: DHA alleviated hepatic steatosis and reduced inflammation of liver in obese middle-aged mice by mechanisms involving Sirt1 activation.Keywords: non-alcoholic fatty liver disease, steatosis, docosahexaenoic acid, high-fat diet, Sirt1

Jie Gao,1,2,* Junjie Liu,1,3,* Fangyuan Xie,4,* Ying Lu,3 Chuan Yin,5 Xian Shen1 1Department of Gastrointestinal Surgery, The Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University, Zhejiang, People’s Republic of China; 2Scientific Research Center, The Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University, Zhejiang, People’s Republic of China; 3Department of Pharmaceutical Sciences, Second Military Medical University, Shanghai, People’s Republic of China; 4Department of Pharmacy, Shanghai Eastern Hepatobiliary Surgery Hospital, Shanghai, People’s Republic of China; 5Department of Gastroenterology, Changzheng Hospital, Second Military Medical University, Shanghai, People’s Republic of China*These authors contributed equally to this workCorrespondence: Jie Gao; Xian ShenDepartment of Gastrointestinal Surgery, The Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University, 109 Xueyuan W Road, Lucheng, Wenzhou, Zhejiang 325000, People’s Republic of ChinaTel +86-0577-88816381; 86-0577-88002709Fax +86-0577-88816381Email gaojiehighclea@163.com; shenxian1963@sina.comPurpose: Conventional chemotherapy is hampered by the presence of breast cancer stem cells (BCSCs). It is crucial to eradicating both the bulky breast cancer cells and BCSCs, using a combination of conventional chemotherapy and anti-CSCs drugs. However, the synergistic ratio of drug combinations cannot be easily maintained in vivo. In our previous studies, we demonstrated that the simultaneous delivery of two drugs via nanoliposomes could maintain the synergistic drug ratio for 12 h in vivo. However, nanoliposomes have the disadvantage of quick drug release, which makes it difficult to maintain the synergistic drug ratio for a long time. Herein, we developed a co-delivery system for docetaxel (DTX)—a first-line chemotherapy drug for breast cancer—and salinomycin (SAL)—an anti-BCSCs drug—in rigid nanoparticles constituted of polylactide-co-glycolide/D-alpha-tocopherol polyethylene glycol 1000 succinate (PLGA/TPGS).Methods: Nanoparticles loaded with SAL and DTX at the optimized ratio (NSD) were prepared by the nanoprecipitation method. The characterization, cellular uptake, and cytotoxicity of nanoparticles were investigated in vitro, and the pharmacokinetics, tissue distribution, antitumor and anti-CSCs activity of nanoparticles were evaluated in vivo.Results: We demonstrated that a SAL/DTX molar ratio of 1:1 was synergistic in MCF-7 cells and MCF-7-MS. Moreover, the enhanced internalization of nanoparticles was observed in MCF-7 cells and MCF-7-MS. Furthermore, the cytotoxicity of NSD against both MCF-7 cells and MCF-7-MS was stronger than the cytotoxicity of any single treatment in vitro. Significantly, NSD could prolong the circulation time and maintain the synergistic ratio of SAL to DTX in vivo for 24 h, thus exhibiting superior tumor targeting and anti-tumor activity compared to other treatments.Conclusion: Co-encapsulation of SAL and DTX in PLGA/TPGS nanoparticles could maintain the synergistic ratio of drugs in vivo in a better manner; thus, providing a promising strategy for synergistic inhibition of breast cancer.Keywords: combined therapy, breast cancer stem cells, docetaxel, salinomycin, nanoparticles

Benfan Zhu,1,* Jie Gao,1–4,* Yeling Ouyang,3,4 Zhiqiang Hu,3,4 Xiangdong Chen3,4 1Department of Pain, The First Affiliated Hospital of Anhui Medical University, Hefei 230022, People’s Republic of China; 2Department of Anesthesiology, The First Affiliated Hospital of Anhui Medical University, Hefei 230022, People’s Republic of China; 3Department of Anesthesiology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, People’s Republic of China; 4Institute of Anesthesiology and Critical Care Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, People’s Republic of China*These authors contributed equally to this workCorrespondence: Xiangdong ChenDepartment of Anesthesiology, Union Hospital, No.1277 Jiefang Avenue, Wuhan, Hubei province 43022, People’s Republic of ChinaTel +86-15071096621Fax +86-27-85351633Email xiangdongchen@yeah.netBackground: The emerging role of inflammation in the initiation and maintenance of neuropathic pain has been confirmed. Previous studies have reported that miR138 has neuroprotective and anti-inflammatory effects in animal models of spinal cord injury and in human coronary artery endothelial cell injury, while its effect on neuropathic pain is still not known. As the mechanism of neuropathic pain remains unclear, we investigated whether miR138 is involved in the development of neuropathic pain and the role of miR138 in the modulation of inflammation in the spinal cord in a mouse model of neuropathic pain induced by spared sciatic nerve injury (SNI).Materials and methods: Firstly, the expression of miR138 in spinal cord was evaluated on days 1, 3, 5, 7, 9 and 14 after SNI. And then, LV-miR-control and LV-miR138 were intrathecally injected 1 week before the surgery followed by investigation of the expression of miR138, mechanical allodynia and thermal hyperalgesia on day 1, 3, 5, 7, 9, 14 after SNI. Ipsilateral L4-L6 spinal cord tissue was harvested on day 14 post-SNI and detected by Western blotting, enzyme-linked immunosorbent assay or immunohischemistry.Results: We observed decreased expression of miR138 and increased expression of proinflammatory cytokines, along with activated microglia, astrocytes and nuclear factor-κВ (NF-κВ), in the spinal cord dorsal horn after SNI. Moreover, the intrathecal upregulation of miR138 significantly alleviated SNI-induced mechanical allodynia and thermal hyperalgesia, downregulated the production of proinflammatory cytokines, and deactivated microglia, astrocytes and NF-κВ.Conclusion: The results indicate that miR138 contributes to the development of neuropathic pain and that the overexpression of miR138 alleviates pain hypersensitivity by inhibiting proinflammatory cytokine production and glial activation, which suggests a novel target for reducing neuropathic pain.Keywords: neuropathic pain, miR138, spared sciatic nerve injury, NF-κ&#x0412

Abstract Background Takotsubo syndrome (TTS), primarily an acute myocardial inflammatory condition engendered by catecholamine exposure, is associated with similar long-term mortality rates to those of patients with acute myocardial infarction. However, there is increasing evidence of a nexus between TTS and underlying malignancies:- many patients have antecedent cancer (A/Ca), while incremental risk of late cancer-related death has also been reported. Purpose To evaluate potential interactions between A/Ca among TTS patients and both early and late clinical course. Methods Three hundred forty-six consecutive TTS patients [aged 69 ± 13 (SD) years, males: 8.2%] were prospectively followed up for a median duration of 4.1 (IQR 2.2–6.4) years. Associations between A/Ca and severity of acute attacks, in-hospital complications and long-term death rates were sought utilising univariate analyses followed by multiple logistic regression analysis. Results A/Ca (present in 16.8% of patients) was associated with (i) greater elevation of hs-CRP and NT-proBNP concentrations (p = 0.01 and 0.04, respectively), (ii) more complicated in-hospital clinical course, with major adverse cardiac events (MACE) in 30.9% of patients, compared to 18.2% in non-A/Ca patients (p = 0.04). Long-term all-cause mortality rate was also greater [hazard ratio (HR) = 2.4, p = 0.0001] in A/Ca patients, with an excess cardiovascular (CVS) fatality rate (HR = 3.1, p = 0.001). On multivariate analysis, male gender, peak plasma concentrations of normetanephrine and hs-CRP, early arrhythmias and development of shock, but not A/Ca per se, were all independently associated with increased long-term mortality rate. Furthermore, patients discharged on β-adrenoceptor antagonists (βBl) or angiotensin converting enzyme inhibitors/ angiotensin receptor blockers (ACEi/ARB) had lower long-term mortality rates (β = − 0.2, p = 0.01; β = − 0.14, p = 0.05, respectively). Conclusions (1) A/Ca is associated with greater clinical severity of initial TTS attacks and substantially greater long-term CVS-related as well as all-cause mortality. (2) Post-discharge therapy with either βBl or ACEi/ARB is associated with reductions in long-term mortality rates. Overall, the current data suggest operation of substantial interactions between neoplasia and TTS, both at the level of pathogenesis and of outcomes.

Yuzhu Ma,1,* Daolu Yuwen,2,* Jingwei Chen,1 Bingfeng Zheng,1 Jian Gao,1 Minmin Fan,1 Wenwen Xue,1 Yixuan Wang,1 Wuhao Li,1 Yongqian Shu,3 Qiang Xu,1 Yan Shen1 1State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing 210093, People’s Republic of China; 2Department of Oncology, Nanjing First Hospital, Nanjing Medical University, Nanjing 210006, People’s Republic of China; 3Department of Clinical Oncology, The First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, People’s Republic of China*These authors contributed equally to this workCorrespondence: Qiang Xu; Yan ShenState Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, 22 Han Kou Road, Nanjing 210093, People’s Republic of ChinaTel/Fax +86 25 8968 7620Email molpharm@163.com; shenyan@nju.edu.cnIntroduction: Exosomes are important mediators of intercellular communication. Previously, we characterized circulating exosomal miR-425-3p as a non-invasive prognostic marker for predicting clinical response to platinum-based chemotherapy in patients with non-small cell lung cancer (NSCLC).Methods: Circulating exosomal miR-425-3p was validated by qRT-PCR in paired serum samples from NSCLC patients during the course of platinum-based chemotherapy. Cell coculture was performed to examine the effects of exosomal miR-425-3p on the sensitivity of recipient A549 cells to cisplatin. Using bioinformatics, ChIP and luciferase reporter assays, the transcription factor essential for miR-425-3p expression was identified. Autophagic activity in the recipient cells was determined by Western blot and fluorescence microscopy.Results: Higher levels of exosomal miR-425-3p were found in serum samples from the patients in tolerance versus those at baseline. An upward trend in the expression of circulating exosomal miR-425-3p was revealed during chemotherapy. Furthermore, the expression of exosomal miR-425-3p could be induced by cisplatin in NSCLC cells. Exosomes isolated from either cisplatin-treated or cisplatin-resistant NSCLC cells conferred chemoresistance to sensitive A549 cells in a miR-425-3p-dependent manner. Cisplatin-induced c-Myc was found to directly bind the miR-425-3p promoter and transactivated its expression. Exosomal miR-425-3p facilitated autophagic activation in the recipient cells by targeting AKT1, eventually leading to chemoresistance.Discussion: Our results suggest that apart from a prognostic marker of treatment response, exosomal miR-425-3p might be a potential dynamic biomarker to tailor cisplatin resistance in NSCLC patients during the treatment and represent a promising therapeutic target for therapy-resistant NSCLC.Keywords: exosomes, miR-425-3p, NSCLC, chemoresistance, platinum-based chemotherapy, autophagy

Chengwu Gong,1,* Jiyuan Ai,1,* Yun Fan,2 Jun Gao,1 Weiwei Liu,1 Qian Feng,3 Wenjun Liao,1 Linquan Wu1 1Department of General Surgery, The Second Affiliated Hospital of Nanchang University, Nanchang 330006, People’s Republic of China; 2Department of Neurology, Tongji Hospital, Huazhong University of Science and Technology, Wuhan 430000, People’s Republic of China; 3Department of Emergency Medicine, The Second Affiliated Hospital of Nanchang University, Nanchang 330006, People’s Republic of China*These authors contributed equally to this workCorrespondence: Linquan Wu; Wenjun LiaoDepartment of General Surgery, The Second Affiliated Hospital of Nanchang University, No. 1, Minde Road, Nanchang 330006, People’s Republic of ChinaTel +86 791 86311529Fax +86 791 86262262Email wulqnc@163.com; liaowenjun120@163.comPurpose: Studies show that high expression of non-SMC condensin I complex subunit G (NCAPG) is associated with many tumors. In this study, we explore the mechanism by which NCAPG promotes proliferation in hepatocellular carcinoma (HCC).Patients and methods: Liver cancer and paracancerous tissue specimens of 90 HCC patients were collected, and expression levels of NCAPG in these tissues and cell lines were evaluated by Western blotting and immunohistochemistry. HCC cells were transfected with siRNAs and plasmids, and pathway activators or inhibitors were added. The 5-ethynyl-2ʹ-deoxyuridine (EdU) proliferation assay was used to measure cell proliferation. Flow cytometry was used to evaluate cell apoptosis. Western blot assays were performed as a standard procedure to detect total protein expression. Treated HCC cells were subcutaneously injected into nude mice.Results: Analysis using the Oncomine database showed that NCAPG was upregulated in HCC and immunohistochemistry and Western blot assays showed it was upregulated in both HCC tissues and HCC cell lines. The overexpression of NCAPG could promote HCC cell proliferation and reduce HCC cell apoptosis. More importantly, RNA-sequencing analysis predicted that NCAPG plays a role in the HCC via PI3K-AKT signaling pathway. The PI3K/AKT/FOXO4 pathway was aberrantly activated, and the expressions of apoptosis-related protein were altered when NCAPG was overexpressed or silenced both in vitro and in vivo. LY294002, a PI3K inhibitor, could eliminate the NCAPG role of promoting HCC cell proliferation and reducing HCC cell apoptosis, while 740Y-P, a PI3K activator, contributed to the opposite effect.Conclusion: NCAPG functions as an oncogene in HCC and plays a role in promoting cell proliferation and antiapoptosis through activating the PI3K/AKT/FOXO4 pathway.Keywords: NCAPG, hepatocellular carcinoma, PI3K/AKT, FOXO4, proliferation

Shuzhi Zang,1 Shasha Zhao,2 Xinyuan Gao,1 Yunxia Li,3 Chunlei Zhong,3 Jianlian Gao4 1Respiratory Ward 1, The First Affiliated Hospital of Xinxiang Medical University, Weihui, Henan 453100, People’s Republic of China; 2Respiratory Intensive Care Unit, The First Affiliated Hospital of Xinxiang Medical University, Weihui, Henan 453100, People’s Republic of China; 3Respiratory Ward 2, The First Affiliated Hospital of Xinxiang Medical University, Weihui, Henan 453100, People’s Republic of China; 4Clinical Pharmacy, The First Affiliated Hospital of Xinxiang Medical University, Weihui, Henan 453100, People’s Republic of ChinaCorrespondence: Shuzhi ZangRespiratory Ward 1, The First Affiliated Hospital of Xinxiang Medical University, No. 88, Jiankang Road, Weihui City, Henan Province 453100, People’s Republic of ChinaEmail zangshuzhi01@126.comBackground: Dysregulation of microRNAs has been reported to be responsible for drug resistance of cancers. However, the association between aberrant expression of miR-26b and cisplatin resistance in non-small cell lung cancer (NSCLC) remains unclear.Methods: PC9 and A549 were used to establish the cisplatin resistance models on NSCLC. Expression of miR-26b in cisplatin-resistant PC9 and A549 cells (PC9/R and A549/R) was detected by quantitative real-time PCR assays. Drug sensitivity and mitochondrial apoptosis were detected by Cell Counting Kit-8 assay and flow cytometry assay, respectively. The target relationship between miR-26b and tafazzin (TAZ) was validated by dual-luciferase reporter assay.Results: Obvious downregulation of miR-26b was observed in PC9/R and A549/R cells. Restoration of miR-26b partially reversed the cisplatin resistance of PC9/R and A549/R cells. Expression of TAZ was increased in PC9/R and A549/R cells compared to the parental PC9 and A549 cells. Results of dual-luciferase reporter assays verified that TAZ was targeted by miR-26b. We showed that restoration of miR-26b expression inhibited the TAZ expression and thus expanded the mitochondrial pathway of apoptosis induced by cisplatin in PC9/R and A549/R cells.Conclusion: Restoration of miR-26b expression partially reverses the cisplatin resistance of NSCLC by targeting TAZ. miR-26b/TAZ axis may represent a potential strategy to reverse the cisplatin in NSCLC.Keywords: NSCLC, cisplatin, resistance, miR-26b, TAZ

Rui Xiong, Jian-long Gao, Tao YinDepartment of Hepatobiliary and Pancreatic Surgery, Hubei Cancer Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430071, People’s Republic of ChinaCorrespondence: Tao YinDepartment of Hepatobiliary and Pancreatic Surgery, Hubei Cancer Hospital, Tongji Medical College, Huazhong University of Science and Technology, No.116 of Zhuodaoquan South Road, Hongshan District, Wuhan, Hubei 430071, People’s Republic of ChinaTel +86 0 278 767 0138Email yintao299@163.comBackground: GTPase-activating protein SH3 domain-binding protein 1 (G3BP1) is involved in various biological functions, including cell growth, metastasis, differentiation, apoptosis, and RNA metabolism. In current study, we aimed to investigate the effect of G3BP1 on gastric cancer (GC).Methods: The expression of G3BP1 in GC tissues and cell lines was assessed by immunohistochemistry and Western blotting. Correlations of G3BP1 expression with clinicopathological and prognosis of GC patients were evaluated. The functions of G3BP1 in regulating proliferation, migration and invasion of GC cell were investigated using small interfering RNA (siRNA) strategies. Preliminary exploration of its underlying mechanism using Western blotting.Results: G3BP1 expression was upregulated in GC tissues compared with adjacent tissues, and the higher G3BP1 expression was correlated with poor prognosis. G3BP1 knockdown decreased GC cell proliferation, migration and invasion. Mechanistically, silencing of G3BP1 inhibits the activation of the transforming growth factor (TGF)-β/Smad signaling pathway in GC cells.Conclusion: G3BP1 plays an important role in the progression of GC as an oncogene and may become a new therapeutic target.Keywords: gastric cancer, G3BP1, prognosis, TGF-β/Smad signaling pathway

Li Xu, Xu Yang, Shan Ke, Xue-mei Ding, Shao-hong Wang, Jun Gao, Wen-bing SunDepartment of Hepatobiliary Surgery, Beijing Chao-yang Hospital Affiliated with Capital Medical University, Beijing 100043, People’s Republic of ChinaCorrespondence: Jun Gao; Wen-bing SunDepartment of Hepatobiliary Surgery, Beijing Chao-yang Hospital Affiliated with Capital Medical University, No. 5 Jingyuan Street, Beijing 100043, People’s Republic of ChinaFax +86 105 171 8017Email gaojun8430@163.com sunwenbing@bjcyh.comAbstract: Hepatic sclerosing hemangioma is a rare benign disease that occurs in association with hepatic cavernous hemangioma degeneration and sclerosis. Recent studies have shown that radiofrequency (RF) ablation is an alternative treatment for hepatic cavernous hemangiomas, even for large hemangiomas (≥10 cm). However, RF ablation might not be suitable to treat large sclerosing hemangiomas. We herein report the successful surgical removal of a large hepatic sclerosing hemangioma after RF ablation treatment failure in a 65-year-old man. In conclusion, we suggest that resection should be chosen as a first-line therapy for the disease.Keywords: resection, radiofrequency ablation, hepatic cavernous hemangiomas, hepatic sclerosing hemangioma

Leisheng Wang,1,* He En,2,* Lei Yang,3 Yanbing Zhang,2 Baisheng Sun,4 Jianjiang Gao51Department of Orthopedics, Yantaishan Hospital, Yantai, Shandong Province 264000, People’s Republic of China; 2Department of Outpatient, The 81st Group Army Hospital of Chinese People’s Liberation Army, Zhangjiakou City, Hebei Province, People’s Republic of China; 3Department of Outpatient, The 80th Group Army Hospital of Chinese People’s Liberation Army (formerly the 89th Hospital of the People’s Liberation Army), Weifang City, Shandong Province, People’s Republic of China; 4Department of Emergency, The Fifth Medical Center of the General Hospital of the Chinese People’s Liberation Army (formerly the 307th Hospital of the People’s Liberation Army), Beijing 100071, People’s Republic of China; 5Department of Emergency, Haiyang People’s Hospital, Haiyang, Shandong 265100, People’s Republic of ChinaCorrespondence: Jianjiang GaoDepartment of Emergency, Haiyang People’s Hospital, No. 73 Haiyang Road, Haiyang, Shandong Province 265100, People’s Republic of ChinaTel +86 535 322 6638Fax +86 535 322 6815Email jianjiang_gao@sohu.com*These authors contributed equally to this workBackground: Osteosarcoma (OSA), the most common primary bone malignancy, is characterized by a wide spectrum of complicated pathologies and frequent distal metastasis and causes death in adolescents and young adults worldwide. Antitumor drug treatment strategies include various cytotoxic chemotherapy drugs, while molecular targeted therapy for OSA is currently less used. The present work revealed the role played by the miR-596/Survivin axis in affecting the sensitivity of OSA cells to anlotinib, a novel molecular targeting agent.Methods: By virtual screening, we found that miR-596 might target Survivin by using an online tool (miRDB). RNA levels of miR-596 and Survivin in clinical specimens were examined with qPCR. The effect of miR-596 on anlotinib’s antitumor effect was examined with MTT experiments, the subcutaneous tumor model, or the intramuscular tumor model.Results: Overexpression of miR-596 via lentiviral particles repressed the protein level of Survivin in U2OS cells. Transfection of miR-596 enhanced the antitumor effect of anlotinib on U2OS cells or five cell lines derived from OSA patients.Conclusion: miR-596 targets Survivin and enhances the antitumor effect of anlotinib on OSA cells.Keywords: osteosarcoma cell, microRNAs, Survivin, molecular targeting agents, anlotinib

Lu Xie,1 Jie Xu,1 Sen Dong,1 Jian Gao,2 Xiaodong Tang,1 Taiqiang Yan,1 Rongli Yang,1 Wei Guo11Musculoskeletal Tumor Center, Peking University People’s Hospital, Beijing, People’s Republic of China; 2Catheterization Room & Radiology Department, Peking University People’s Hospital, Beijing, People’s Republic of ChinaPurpose: We intend to analyze the gain and loss from transcatheter intra-arterial (IA) limb infusion of cisplatin for extremity osteosarcoma in the past six years.Patients and methods: Between December 2009 and August 2014, a total of 99 patients were analyzed for efficiency and followed up for long-term survival. Based on the different administration methods of cisplatin, we divided them into the following two cohorts: IA infusion of cisplatin (n=48) and intravenous (IV) infusion of cisplatin (n=51). Except for cisplatin, all the other drugs were given intravenously. Cisplatin was given intra-arterially with an infusion time of 3 hrs or 6 hrs using a pump, whereas historical controls received IV infusion of cisplatin within 60 mins. Tumor neovascularity (TNV) was analyzed before infusion, and subsequent arteriograms were compared with the baseline to determine percent changes. Definitive surgery with intended wide resection and postoperative pathological evaluation were performed in all these patients.Results: No local or overall survival benefit was found in the patients preoperatively treated with IA infusion of cisplatin compared with IV infusion (P=0.336 and 0.173, respectively). Furthermore, serial arteriography was used to predict a good histologic response with an accuracy of 73.1% and a sensitivity of 100%. There were sporadic cases with the telangiectatic subtype, which did not respond very well to IV chemotherapy, but later, the tumor obviously shrank after IA infusion of cisplatin. Our study also showed that the rates of the complication of skin and muscle necrosis were not so low as reported.Conclusion: We did not observe any survival advantage of chemotherapy using IA infusion in osteosarcoma of the extremities. Arteriography for TNV can be used to predict the tumor histologic response. Malposition of the catheter might severely increase the complication of skin or muscle necrosis.Keywords: intra-arterial chemotherapy, tumor neovascularity, telangiectatic osteosarcoma, complications

Xiao-Hui Wang,1 Hai-Ying Wu,2 Jian Gao,1 Xu-Hui Wang,1 Tian-Hui Gao,3 Shu-Feng Zhang11Department of Pediatric Surgery, People’s Hospital of Zhengzhou University (Henan Provincial People’s Hospital), Zhengzhou 450003, People’s Republic of China; 2Department of Obstetrics, People’s Hospital of Zhengzhou University (Henan Provincial People’s Hospital), Zhengzhou 450003, People’s Republic of China; 3Department of Medical Oncology, People’s Hospital of Zhengzhou University (Henan Provincial People’s Hospital), Zhengzhou 450003, People’s Republic of ChinaBackground: Neuroblastoma (NB) displays the most heterogeneity in clinical manifestation. The insulin-like growth factor 1 receptor (IGF1R) has long been recognized for its role in tumourigenesis and growth. The IGF/IGF1R pathway is important in maintaining cell survival. It is reported that IGF1R participates in the occurrence of NB, but the mechanism is still unclear.Methods: Human NB cell lines IMR-32 and SH-SY5Y were recruited in this study. IGF1R was knocked down by transfection with short hairpin RNA. Signal transducer and activator of transcription 3 (STAT3) expression was inhibited by Cryptotanshinone treatment. Cell proliferation, migration, and invasion were determined by MTT assay, wound healing assay, and cell invasion assay, respectively. The cancer stem cell properties were characterized by tumour sphere formation assay and colony formation assay. The mRNA and protein expression levels of related proteins were detected by RT-PCR and Western blot, respectively.Results: The knockdown of IGF1R inhibits NB cell tumourigenesis and the epithelial-mesenchymal transition (EMT) of NB cells. Additionally, IGF1R was found to stimulate cancer stem cell-like properties in NPC cells. The knockdown of IGF1R significantly reduced the phosphorylation of AKT, and STAT3, indicating that the activation of the AKT and STAT3 pathways was inhibited by IGF1R knockdown. Furthermore, IGF1R was demonstrated to stimulate cancer stem cell-like properties in NB cells via the regulation of the STAT3/AKT axis.Conclusion: IGF1R promotes cancer stem cell properties to facilitate EMT in neuroblastoma via the STAT3/AKT axis.Keywords: IGF1R, neuroblastoma, epithelial mesenchymal transition, stemness, STAT3, AKT

Borong Chen,1* Zhipeng Zhu,1* Lulu Li,1* Weipeng Ye,2 Junjie Zeng,1 Jin Gao,1 Shengjie Wang,1 Liang Zhang,1 Zhengjie Huang1,21Department of Gastrointestinal Surgery, Xiamen Cancer Hospital, The First Affiliated Hospital of Xiamen University, Xiamen, Fujian 361003, People’s Republic of China; 2Department of Clinical Medicine, Fujian Medical University, Fuzhou, Fujian 350004, People’s Republic of China*These authors contributed equally to this work Objective: Using the gastric cancer cell line SGC7901, we constructed a cell line that overexpressed octamer-binding protein 4 (Oct4) and SRY-box 2 (Sox2) to explore the stem cell oncological and biological characteristics of these cells and to elucidate the mechanisms of Oct4 and Sox2 in cancer.Methods: A lentiviral vector containing the Sox2 gene was constructed and transfected into a gastric cancer cell line overexpressing Oct4 (SGC7901-Oct4) to obtain a stably transfected cell line (SGC7901-Oct4-Sox2). Oct4 and Sox2 expression was detected by RT-PCR and Western blotting. The proliferation, drug resistance, migration, and invasion abilities of the cells were assessed using in vitro (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium), drug resistance, scratch-wound migration, transwell migration, transwell invasion, and spherical clone formation assays, and their tumorigenic ability was assessed using a tumor formation experiment in mice.Results: Compared with the control group, the expression of Oct4, Sox2, CD44, and E-cadherin was significantly higher in the group that overexpressed Oct4 and Sox2, while the expression of c-Myc and Klf4 did not significantly change. The proliferation, drug resistance, migration, and invasion abilities were significantly enhanced in the overexpression group, and the tumorigenic ability in mice was also significantly enhanced, with significantly increased tumor size and weight.Conclusion: The proliferation, drug resistance, migration, invasion, and tumorigenic abilities of SGC7901 cells overexpressing Oct4 and Sox2 were significantly improved. Oct4 and Sox2 play important roles in the proliferation, migration, invasion, and tumorigenicity of gastric cancer cells, and the two genes may be synergistic to a certain degree.Keywords: stomach cancer SGC7901, Oct4, Sox2, stem cells

Ziran Zhang,1 Wei Zhao,2 Xiaoting Lin,1 Jing Gao,1 Zhiqian Zhang,2 Lin Shen11Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education/Beijing), Department of Gastrointestinal Oncology, Peking University Cancer Hospital and Institute, Beijing, People’s Republic of China; 2Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Department of Cell Biology, Peking University Cancer Hospital and Institute, Beijing, People’s Republic of ChinaPurpose: Cancer stem cells (CSCs) are a subpopulation of cancer cells with self-renewal property and responsible for tumor malignancy, progression and drug resistance. Researches on CSC-specific markers in gastric cancer remain limited. Our current study explored the expression of voltage-dependent calcium channel α2δ1 subunit and the potential of using α2δ1 as a CSC marker in gastric cancer. We also compared the specificity of α2δ1 and CD44 in identifying gastric cancer stem cells (GCSCs).Materials and methods: Expression of α2δ1 was analyzed in gastric cancer cell lines, patient-derived xenograft (PDX) models and clinical samples of malignant ascites of gastric cancer patients. α2δ1+, gastric cancer cells were isolated from gastric cancer cell lines. CSC properties of α2δ1+, gastric cancer cells were then verified by subsequent tests both in vitro and in vivo.Results: The expression level of α2δ1 was found to differ drastically among gastric cancer cell lines, PDX models and clinical samples of malignant ascites. α2δ1+, gastric cancer cells sorted from HGC-27 and SGC-7901 cell lines demonstrated significant self-renewal properties, including tumorigenic capacity, sphere-formation capacity and asymmetric differentiation potential. Knockdown of α2δ1 in α2δ1+, HGC-27 significantly inhibited CSC properties and rendered HGC-27 more sensitive to chemotherapy. Flow cytometry showed that α2δ1+, gastric cancer cells accounted for a small fraction of CD44+, gastric cancer cells. Isolated CD44+,α2δ1+, HGC-27 cells displayed more significant tumorigenic capacity and sphere-forming capacity compared with their CD44+,α2δ1−, counterparts.Conclusion: α2δ1+, gastric cancer cells possessed CSC properties. α2δ1 could be a proper marker in identifying GCSCs with superior specificity than CD44. The combination of α2δ1 and CD44 could be used to identify GCSCs with improved accuracy. Knockdown of α2δ1 combined with chemotherapy displayed higher therapeutic efficacy on gastric cancer cells, suggesting that α2δ1 could be a potential target for anticancer treatment.Keywords: α2δ1, gastric cancer, cancer stem cell, HGC-27

Jiyuan Ai,* Chengwu Gong,* Junjun Wu, Jun Gao, Weiwei Liu, Wenjun Liao, Linquan WuDepartment of Hepatobiliary Surgery, The Second Affiliated Hospital of Nanchang University, Nanchang, Jiangxi, People’s Republic of China*These authors contributed equally to this workPurpose: Numerous studies have shown that the expression of microRNA-181c (miR-181c) is inhibited in various cancers, which suggests that it has a cancer suppressive effect. In the current study, we evaluated the regulation and characteristics of miR-181c in human hepatocellular carcinoma (HCC).Materials and methods: Samples of tumor tissues and adjacent non-tumor tissues were collected from 52 patients with HCC, and expression levels of miR-181c in these samples were investigated via quantitative real-time polymerase chain reaction. HCC cell migration and invasion were investigated via wound healing assays and transwell assays. HCC cell apoptosis rates were assessed via flow cytometry, and HCC proliferation was assessed via 5-ethynyl-20-deoxyuridine assays. In vivo tumors were initiated by subcutaneously inoculating HCC cells into nude mice. And various biomarkers were investigated via western blotting.Results: In microarray datasets and tumor tissues, significant downregulation of miR-181c was apparent compared with non-tumorous adjacent tissues. Expression of miR-181c in HCC cells was also significantly lower than it was in normal human liver cells. miR-181c regulated the migration, invasion, apoptosis, and proliferation of HCC cell lines in vitro, and tumor development in vivo. Observations also suggest that miR-181c regulates NCAPG in HCC cells, and its expression affects cellular invasion, migration, proliferation, and apoptosis. There was a negative correlation between miR-181c expression and NCAPG in HCC tissue samples.Conclusion: miR-181c exhibits tumor-suppression via the regulation of NCAPG levels.Keywords: miR‑181c, NCAPG, hepatocellular carcinoma, biological function

Kaidi Yang,1,2 Jian Gao,3 Mao Luo1,4,5 1Key Laboratory of Medical Electrophysiology of Ministry of Education, Collaborative Innovation Center for Prevention and Treatment of Cardiovascular Disease of Sichuan Province, Southwest Medical University, Luzhou, China; 2Key Laboratory of Tumor Immunology, The First Affiliated Hospital, Army Medical University, Chongqing, China; 3Department of Life Sciences and Technology, Yangtze Normal University, Chongqing, China; 4Drug Discovery Research Center, Southwest Medical University, Luzhou, Sichuan, China; 5Laboratory for Cardiovascular Pharmacology, Department of Pharmacology, School of Pharmacy, Southwest Medical University, Luzhou, Sichuan, China Background: Basal-like breast cancer (BLBC) is the most aggressive subtype of breast cancer (BC) and links to poor outcomes. As the molecular mechanism of BLBC has not yet been completely discovered, identification of key pathways and hub genes of this disease is an important way for providing new insights into exploring the mechanisms of BLBC initiation and progression.Objective: The aim of this study was to identify potential gene signatures of the development and progression of the BLBC via bioinformatics analysis. Methods and results: The differential expressed genes (DEGs) including 40 up-regulated and 21 down-regulated DEGs were identified between GSE25066 and GSE21422 microarrays, and these DEGs were significantly enriched in the terms related to oncogenic or suppressive roles in BLBC progression. In addition, KEGG pathway and GSEA (Gene Set Enrichment Analysis) enrichment analyses were performed for DEGs between the basal type and non-basal-type breast cancer from GSE25066 microarray. These DEGs were enriched in pathways such as cell cycle, cytokine-cytokine receptor interaction, chemokine signaling pathway, central carbon metabolism signaling and TNF signaling pathway. Moreover, the protein-protein interaction (PPI) network was constructed with those 61 DEGs using the Cytoscape software, and the biological significance of putative modules was established using MCODE. The module 1 was found to be closely related with a term of mitosis regulation and enriched in cell cycle pathway, and thus confirmed the pathological characteristic of BLBC with a high mitotic index. Furthermore, prediction values of the top 10 hub genes such as CCNB2, BUB1, NDC80, CENPE, KIF2C, TOP2A, MELK, TPX2, CKS2 and KIF20A were validated using Oncomine and Kaplan-Meier plotter. Conclusion: Our results suggest the intriguing possibility that the hub genes and modules in the PPI network contributed to in-depth knowledge about the molecular mechanism of BLBC, paving a way for more accurate discovery of potential treatment targets for BLBC patients. Keywords: basal-like breast cancer, bioinformatics, differentially expressed genes, hub genes, molecular mechanism

Dongshao Chen,1,* Xiaoting Lin,1,* Cheng Zhang,1 Guo An,2 Zhongwu Li,3 Bin Dong,3 Lin Shen,1 Jing Gao,1 Xiaotian Zhang1 1Department of Gastrointestinal Oncology; 2Department of Laboratory Animal; 3Department of Pathology, Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education, Beijing), Peking University Cancer Hospital and Institute, Beijing 100142, China *These authors contributed equally to this work Background: Characterized by elevated AFP levels in serum, AFP-producing gastric cancer (APGC) is a very special type of gastric cancer (GC) that is difficult to treat and has poor prognosis. However, little is known about the role of AFP in GC, which was investigated in this study with in vitro and in vivo experiments. Methods: APGC cells were established with lentivirus infection and validated by PCR assay and ELISA in HCG27 and AGS cells. Cell growth, migration, and invasion were determined by CCK8, transwell assays, and animal experiments. RNA sequencing, Western blot, dual-luciferase-reporter assays, and RNA interference were employed to understand mechanisms underlying AFP activity, followed by therapeutic investigations for APGC. Results: APGC cells featured significantly increased AFP levels in cellular supernatants. AFP potentiated growth and aggression in GC cell lines and their derived xenografts. Wnt-signaling activation was responsible for AFP function, indicated by decreased Axin 1 and pGSK3β, followed by cascade activation of β-catenin, downstream transcription factors TCF1/TCF7, and the target gene – c-Myc. Wnt-signaling blockade by Axin 1 rescue or pathway inhibitor XAV939 reversed AFP function, suggesting the potential therapeutic value of APGC. Conclusion: AFP played a critical role in APGC through activating Wnt signaling, and targeting Wnt pathways might be a promising strategy against APGC. Keywords: alpha-fetoprotein, AFP, AFP-producing gastric cancer, APGC, Axin 1, Wnt signaling, Wnt-signaling inhibitor

Yanxiang Xue,1,2 Xiaofang Hong,3 Jie Gao,1,2 Renze Shen,3 Zhanchao Ye3 1Department of Stomatology, The Liwan Hospital of The Third Affiliated Hospital of Guangzhou Medical University, Guangzhou 510000, China; 2Department of Stomatology, Southern Medical University Guangzhou, Guangzhou 510515, China; 3Department of Stomatology, Zhongshan Hospital of Xiamen University, Medical College of Xiamen University, Xiamen University, Xiamen 361000, China Objective: This study aims to produce nanoparticles of chitosan (CS), poly(lactic-co-glycolic acid) (PLGA), and silver and investigate the optimal composite ratio of these three materials for periodontal tissue regeneration.Methods: PLGA nanoparticles (nPLGA), CS nanoparticles (nCS), and silver nanoparticles (nAg) were prepared. The antibacterial properties of single nanoparticles and their effects on the proliferation and mineralization of periodontal membrane cells were investigated. Different ratios of nPLGA and nCS were combined, the proliferation and mineralization of periodontal membrane cells were investigated, and based on the results, the optimal ratio was determined. Finally, nPLGA and nCS in optimal ratio were combined with nAg, and the effects of the complex of these three materials on the proliferation and mineralization of periodontal membrane cells were investigated and tested in animals.Results: The single nanoparticles were found to have no cytotoxicity and were able to promote cell mineralization. nCS and nAg in low concentrations showed antibacterial activity; however, nAg inhibited cell proliferation. The nPLGA and nCS complex in 3:7 ratio contributed to cell mineralization and had no cytotoxicity. nPLGA/nCS/nAg complex, which had the optimal proportion of the three materials, showed no cytotoxicity and contributed to cell mineralization.Conclusion: nPLGA/nCS/nAg complex had no cytotoxicity and contributed to cell mineralization. The 3:7 ratio of nPLGA/nCS and 50 µg/mL nAg were found as the optimal proportion of the three materials. Keywords: nanoparticles, bone regeneration, periodontitis

Xinxin Wang,1 Diangang Chen,1 Jianbao Gao,1 Haixia Long,1 Haoran Zha,2 Anmei Zhang,1 Chi Shu,1 Li Zhou,1 Fei Yang,1 Bo Zhu,1 Wei Wu3 1Institute of Cancer, Xinqiao Hospital, Army Medical University, Chongqing, China; 2Department of Oncology, The General Hospital of the People’s Liberation Army Rocket Force, Beijing, China; 3Department of Cardiothoracic Surgery, Southwest Hospital, Army Medical University, Chongqing, China Purpose: Centromere protein U (CENPU) abnormally exhibits high expression in various types of human tumor tissues and participates in tumor progression; however, its expression pattern and biological function in lung cancer have not yet been elucidated. In the present study, we explored the clinical significance and biological function of CENPU in lung cancer.Materials and methods: The Cancer Genome Atlas (TCGA) data analyses, quantitative real-time PCR (RT-PCR), and Western blotting were performed to quantify CENPU and FOXM1 expression in non-small-cell lung cancer (NSCLC) samples. Survival data were obtained from Kaplan–Meier plotter or PROGgene V2 prognostic database. The function of CENPU in lung cancer cell proliferation was determined using 5-ethynyl-2′-deoxyuridine (EdU), Cell Counting Kit-8 (CCK-8), and cell cycle assays, and the underlying mechanism was determined through bioinformatic analyses and validated by in vitro siRNA or plasmid transfection experiments.Results: CENPU was abnormally overexpressed in NSCLC samples compared with matched paired normal tissues. Higher expression of CENPU predicted worse overall survival (OS) and relapse-free survival (RFS) in NSCLC patients. Knockdown of CENPU expression by siRNA significantly inhibited proliferation and delayed cell cycle progression of lung cancer cells. To figure out the mechanism, bioinformatic analyses were performed and the results showed that the transcription factor, FOXM1, positively correlated with CENPU. Further in vitro experiments indicated that FOXM1 was the possible downstream transcription factor of CENPU as the knockdown of CENPU led to lower expression of FOXM1 and the overexpression of FOXM1 significantly reversed the inhibition of proliferation caused by CENPU knockdown. Furthermore, FOXM1 was highly expressed in NSCLC. The knockdown of FOXM1 also attenuated proliferation and induced G1 arrest in lung cancer cells.Conclusion: CENPU was highly expressed in NSCLC tissues, wherein it promoted lung cancer cell proliferation via the transcription factor, FOXM1, which could be a potential target for therapeutic strategies. Keywords: CENPU, NSCLC, proliferation, FOXM1

Jun Gao,1,2 Kang Zeng,1 Yi Liu,3 Lin Gao,4 Lishi Liu1 1Department of Dermatology, Nanfang Hospital, Southern Medical University, Guangzhou, China; 2Department of Dermatology, Liuzhou Worker’s Hospital, Liuzhou, China; 3Department of Hand and Foot Surgery, Liuzhou Worker’s Hospital, Liuzhou, China; 4Department of Clinical Medical Research Center, The 2nd Clinical Medicine College (Shenzhen People’s Hospital) of Jinan University, Shenzhen, China Introduction: Melanoma has been reported as the most common malignancy in skin cancer. The small nucleolar RNA host gene 5 (SNHG5), an lncRNA, has been proven as a vital regulator in several types of carcinoma. This study was designed to investigate the detailed roles and possible mechanisms of SNHG5 in melanoma progression. Methods: Quantitative real-time PCR (qRT-PCR) analysis was conducted to detect the expression levels of SNHG5, miR-26a-5p and transient receptor potential, canonical 3 (TRPC3) mRNA in melanoma tissues and cells. CCK-8 assay was used to measure the cell viability. Flow cytometry assays were performed to determine the cell cycle distribution and apoptosis. The invasive ability was assessed by a 24-well Transwell insert. Western blot analysis was employed to evaluate the protein expression of TRPC3. Dual luciferase reporter assay, RNA immunoprecipitation (RIP) assay, and RNA pull-down assay were applied to identify the interactions among SNHG5, miR-26a-5p and TRPC3.Results: The results showed that SNHG5 expression was increased in melanoma tumor tissues and cell lines. Higher SNHG5 expression was correlated with advanced pathogenic status. Moreover, SNHG5 could serve as a molecular sponge of miR-26a-5p. SNHG5 downregulation repressed proliferation, promoted apoptosis, and decreased invasion in melanoma cells, while these effects were greatly counteracted by miR-26a-5p inhibitor. Furthermore, miR-26a-5p directly targeted TRPC3 to suppress its expression, and this effect was aggravated following SNHG5 downregulation. Also, TRPC3 depletion exerted similar tumor-suppressive functions as SNHG5 knockdown. Conclusion: SNHG5 promoted melanoma development by inhibiting miR-26a-5p and facilitating TRPC3 expression, highlighting the potential of SNHG5 as a novel target therapy for melanoma. Keywords: lncRNA, SNHG5, miR-26a-5p, TRPC3, cutaneum carcinoma