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Purpose. Hedyotis diffusa Willd (HDW) is one of the most well-known herbs used in the therapy of cancer. However, the potential mechanisms of its antiangiogenic effects have not been fully explored. Here, we applied a network pharmacology approach to explore the potential mechanisms of HDW against liver cancer angiogenesis (LCA) and used a mouse orthotopic liver cancer model for experimental verification accordingly. Methods. The effective components, primary active compounds, and possible targets in the therapy of LCA were predicted using network pharmacology and bioinformatics. In vivo testing of the pharmacodynamic foundation of HDW in the treatment of LCA was performed. Hepa1-6 cells were implanted in C57BL/6 mice to establish an orthotopic liver cancer model to evaluate the antitumor and antiangiogenesis effects of the drug. Furthermore, protein levels were evaluated by western blotting, immunofluorescence, and immunohistochemistry. Results. We firstly confirmed the therapeutic effect of HDW on LCA and subsequently screened 7 active compounds from HDW according to their pharmacokinetic properties. Network analysis and enrichment analysis indicated that these compounds exhibit antiangiogenic effect by acting on multiple targets and thereby regulating multiple pathways mainly involved in Akt1, IL-6, IL-1β, IL-17, hypoxia inducible factor-1α (HIF-1α), and tumor necrosis factor-α (TNF-α). Importantly, we preliminarily verified the results of the network pharmacology analysis in vivo. Conclusion. Collectively, our work initially explored the therapeutic mechanism of HDW on tumor angiogenesis, which lays an experimental reference for further exploring its pharmacological action and its clinical application.

Gallbladder cancer (GBC) ranks seventh among the gastrointestinal cancers. Messenger RNAs (mRNAs) could regulate the progression of GBC. For the purpose of exploring the targets for GBC treatment, RNA sequencing was used to identify the differential expressed mRNAs between GBC and adjacent tissues. Next, CCK8 assay was used to assess the cell viability, and cell proliferation was investigated by colony formation assay. Flow cytometry was performed to evaluate the cell apoptosis. Protein and mRNA expression were analyzed by western blot and RT-qPCR, respectively. Transwell was performed to evaluate the cell metastasis. GBC-derived exosomes were isolated with ultracentrifugation. To evaluate the function of exosomes in GBC, in vivo model of GBC was constructed. The data revealed IGF2BP3 was identified to be upregulated in GBC, and IGF2BP3 silencing was able to decrease GBC cell proliferation by promoting the apoptosis. The migration and invasion of GBC cells were reduced by IGF2BP3 knockdown. Silencing of IGF2BP3 obviously suppressed the level of p-STAT3 in GBC cells. Meanwhile, GBC cell-derived exosomes notably promoted macrophage M2 polarization via carrying IGF2BP3, and then the polarized macrophages promoted the malignant behavior of GBC cells. Furthermore, exosomes markedly promoted the tumor growth of GBC via promoting macrophage M2 polarization. In summary, knockdown of IGF2BP3 suppressed the malignant behavior of GBC cells. Additionally, knockdown of IGF2BP3 modified tumor microenvironment during the progression of GBC. Thus, these findings might provide a new theoretical basis for exploring a strategies against GBC.

Background Gallbladder cancer (GBC) is the most common biliary tract malignant cancer worldwide. It has been reported that microRNA-575 (miR-575) was involved in the tumorigenesis of many cancers. However, the role of miR-575 during the progression of GBC remains largely unknown. Methods The expression of miR-575 in GBC cells was detected by quantitative real-time polymerase chain reaction. The proliferation of GBC cells was examined by CCK-8 assay and Ki-67 staining. Apoptosis of GBC cells was measured by flow cytometry, and cell invasion was tested by transwell assay. Moreover, protein expressions in GBC cells were evaluated using Western blot. The target gene of miR-575 was predicted using Targetscan and miRDB. Finally, xenograft tumor model was established to verify the function of miR-575 in GBC in vivo. Results Our findings indicated that miR-575 antagonist decreased the proliferation and invasion of GBC cells. In addition, miR-575 antagonist significantly induced apoptosis of GBC cells via inducing G1 arrest. Meanwhile, p27 Kip1 was found to be a direct target of miR-575 with luciferase reporter assay. Moreover, miR-575 antagonist significantly decreased the expressions of CDK1 and cyclin E1 and upregulated the levels of cleaved caspase3 and p27 Kip1 in GBC cells. Finally, miR-575 antagonist notably suppressed GBC tumor growth in vivo. Conclusion Downregulation of miR-575 significantly inhibited the tumorigenesis of GBC via targeting p27 Kip1. Thus, miR-575 might be a potential novel target for the treatment of GBC.

Colorectal cancer (CRC) is a malignant tumor of the gastrointestinal tract and a leading cause of cancer-associated mortality worldwide. Mex-3 RNA binding family member A (MEX3A) promotes the progression of multiple types of cancer, including ovarian and cervical cancer. However, to the best of our knowledge, the role of MEX3A in CRC is not completely understood. Therefore, the present study aimed to investigate the function of MEX3A in CRC. The mRNA and protein expression levels of MEX3A in CRC cells were analyzed using reverse transcription-quantitative PCR and western blotting, respectively. Cell Counting Kit-8 assays were used to measure cell viability. Cell apoptosis and cell cycle distribution were detected via flow cytometry, and CRC cell invasion was analyzed by performing Transwell assays. Moreover, the mitochondrial membrane potential in CRC cells was measured via JC-1 staining. The results of the present study revealed that the expression levels of MEX3A were upregulated in CRC tissues compared with adjacent healthy tissues. MEX3A knockdown notably inhibited CRC cell viability, and induced apoptosis and mitochondrial injury. In addition, MEX3A knockdown markedly induced G1 phase cell cycle arrest in CRC cells via downregulating CDK2 expression. In conclusion, the findings of the present study suggested that MEX3A knockdown may inhibit the tumorigenesis of CRC cells by regulating CDK2 expression. Therefore, MEX3A may serve as a novel target for CRC treatment.

Purpose: To describe and interpret secular time trends in gallbladder cancer (GBC) incidence, mortality, and diagnostic approach using 37 years of cancer registry data in urban Shanghai. Methods: Data on registration of GBC in urban Shanghai during 1973 and 2009 were collected by the Shanghai Cancer Registry. To describe time trends and to identify specific time points when significant changes occurred, we used joinpoint regression analysis. Results: The age-standardized rates (ASRs) of incidence increased from 1.1/100,000 (1973–1975) to 2.9/100,000 (2006–2009) in men and from 1.7/100,000 (1973–1975) to 3.9/100,000 (2006–2009) in women. ASRs of incidence increased significantly with estimated annual percent changes (EAPCs) of 2.8% in men and 2.5% in women. The mortality trends increased significantly, with EAPCs of 2.8% in men and 2.5% in women. The increasing incidence and mortality rates were primarily observed in men ⩾60 years of age and in women ⩾70 years of age. Notable downward trends in incidence and mortality were identified among women age 60–69 years over the last decade. The percentage of GBC diagnosed by pathology increased steadily over the years while the percentage of GBC diagnosed by imaging, surgery, and biochemistry sharply increased from 1987 onwards. Conclusions: Thirty-seven years of cancer registry data document a tremendous increase in incidence/mortality and a slight decline in incidence/mortality over the last decades for GBC, especially among women, in Shanghai. The development of diagnostic approaches and aging population may play important roles.

Small nucleolar RNA host gene 1 (SNHG1) is critical in the progression of cancers. However, the mechanism by which SNHG1 regulates the progression of colorectal cancer (CRC) remains unclear. Expressions of SNHG1 and miR‐137 in CRC tissues and cell lines were evaluated by quantitative real‐time polymerase chain reaction. A luciferase reporter gene assay was conducted to investigate miR‐137 target. Additionally, RNA pull‐down assay was performed to explore the physical association between miR‐137, SNHG1, and RNA induced silencing complex (RISC). Cell cycling and invasion were examined by flow cytometry (FCM) and transwell assays. The in vivo carcinogenic activity of SNHG1 was examined using murine xenograft models. Expression of RICTOR, serine/threonine kinase 1 (AKT), serum and glucocorticoid‐inducible kinase 1 (SGK1), p70S6K1, and LC3II/LC3I ratio was examined by Western blot analysis. SNHG1 upregulation was observed in CRC tissues and cell lines, which was associated with the lymph node metastasis, advanced TNM stage and poorer prognosis. SNHG1 increased RICTOR level in CRC via sponging miR‐137. In addition, SNHG1 silencing inhibited CRC cell proliferation and migration in vitro and in vivo. SNHG1 regulated RICTOR expression by sponging miR‐137 and promoted tumorgenesis in CRC.

Background: Brief behavioral therapy (BBT-I) for insomnia has been proven to be a simple and effective alternative to cognitive behavioral therapy. However, low adherence limits the application in Chinese primary medical institutions, resulting in delayed or irregular treatment for many patients. This study aimed to explore the efficacy of traditional Chinese medicine supportive treatments on the adherence to behavioral therapy for insomnia in Chinese primary healthcare institutions, with a particular focus on patients who live in regions with weak healthcare systems.Methods: This randomized controlled clinical trial will be conducted in primary medical institutions and will recruit 98 adult participants with insomnia. BBT-I will be used as the base treatment. The participants will be divided into experimental (combined with traditional Chinese medicine supportive treatment n = 49) and control (combined with trazodone treatment, n = 49) groups, and each group will be treated for 4 consecutive weeks. The severity index of insomnia will be used as the main indicator of disease evaluation, with an 8-point reduction in the score considered as effective and a score < 8 considered as cured. The secondary indicators of the disease evaluation will include the Pittsburgh sleep quality index (PSQI), Zung’s self-rating anxiety scale, Zung’s self-rating depression scale, treatment adherence, and adverse event reports. All participants will be followed up at the time of enrollment, 4 weeks after treatment, and 3 months after the end of treatment.Discussion: This clinical trial will provide evidence for the efficacy of traditional Chinese medicine supportive treatment on the adherence to behavioral therapy for insomnia in primary medical institutions. This cheap and accessible model may benefit insomnia patients in medically underserved areas.Trial registration: Chinese Clinical Trial Registry ChiCTR2100042845. Registered on 30 January 2021, dataset: http://www.chictr.org.cn/edit.aspx?pid=65691&htm=4

BackgroundGallbladder cancer (GBC) is a malignant cancer with poor prognosis. Evidences have shown that miRNAs are closely related to the occurrence of GBC; thus, we aimed to explore miRNAs, which plays an important role in the occurrence and development of GBC.MethodsMicroarray analysis was performed to investigate the differentially expressed miRNAs between five non-neoplastic gallbladder tissues (normal tissues) and five gallbladder tumor tissues (tumor tissues). RT-qPCR was performed to detect the level of miR-181b-5p in cells, and CCK-8 was performed to detect cell viability. Then, glucose assay kit or lactic acid assay kit was performed to detect the level of glucose consumption or lactate production. Next, transwell and wound healing assays were used to assess cell migration. In addition, dual-luciferase reporter assay was used to verify the relationship between miR-181b-5p and PDHX. At last, Western blotting was performed to determine the protein level of PDHX.ResultsMicroarray analysis suggested miR-181b-5p was significantly upregulated in GBC tumor tissue. KEGG analysis for the protein targets of miR-181b-5p indicates a close relationship existed between miR-181b-5p and glycolysis. In addition, the level of miR-181b-5p was notably increased in GBC-SD or G415 cells, compared with HIBEpiC cells. GBC cell viability was significantly decreased under hypoxia, and these decreases were exacerbated by miR-181b-5p antagomir. Moreover, glucose consumption or lactate production of GBC cells was significantly upregulated under hypoxia, whereas these increases were completely revered by miR-181b-5p antagomir. Further investigation revealed that PDHX was a direct target of miR-181b-5p.ConclusionIn this study, downregulation of miR-181b-5p inhibits the viability, migration, and glycolysis of GBC by upregulating PDHX under hypoxia. This finding suggested that miR-181b-5p might be considered as a novel therapeutic target for the treatment of GBC.

Background Sheng-Ma-Ge-Gen-Tang (SMGGT; 升麻葛根汤) is a famous prescription of traditional Chinese medicine used against measles of children for many hundreds of years. And its anti-hepatitis B virus (HBV) activity has been justified in clinical, however, the function substances and the mechanisms have not been studied yet. Methods The latent active compounds of Cimicifugae Rhizoma (Shengma)-Radix Puerariae (Gegen) drug pair were searched, and excavated their related targets. Then seek the targets of HBV through three network databases. The drug-disease targets of protein-protein interaction (PPI) data was carried out. The drug-disease targets were enriched by gene ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. Subsequently, anti-HepG2.2.15 cytotoxicity and anti-HBV experiments were performed on the aqueous and ethanol extracts of Shengma-Gegen in vitro to test and verify their anti-HBV activities. Results GO enrichment indicated biological processes (cellular response to lipid, cellular response to organic cyclic compound, etc.), cellular components (vesicle lumen, cytoplasmic vesicle lumen, etc.) and molecular function (kinase binding, steroid binding, etc.). Many cancer-related pathways were enriched in KEGG pathway analysis. Also got some virus and bacteria diseases. A KEGG-targets network showed that RAF1, CCND1, BCL2, and EGFR might be the core targets. Cytotoxicity of Shengma and Gegen to HepG2.2.15 are in proportion to their concentrations. Shengma and Gegen aqueous and ethanol extracts exhibited a curtain extant anti-HBV-DNA activity. Conclusions Shengma-Gegen showed not only anti-HBV activity but also anti-tumor and anti-viral activities, which need to be tested in the future.

Background MicroRNAs (miRNAs) can regulate the post-transcriptional level of gene expression. It has been documented that downregulation of miR-206 is significant in human gastric cancer (GC), whereas its role in GC cell biological behaviors, including proliferation, migration, and invasion, has not been thoroughly investigated. MiR-206 levels have a negative association with lymph node metastasis and tumor invasion, and patients with higher miR-206 expression have better prognoses. Functional studies demonstrated that miR-206 overexpression significantly suppresses GC cell proliferation, migration, and invasion, and induces apoptosis in vitro. Materials and methods MiR-206 and MUC1 were determined by RNA extraction, quantitative real-time polymerase chain reaction, and luciferase reporter gene assays. The viability of GC cells was tested using the Cell Counting Kit 8 assay. Transwell invasion and migration assays detected GC cancer cell proliferation, invasion, and migration. Flow cytometry was applied to analyze apoptotic cells. FACS analysis was applied to detect the mitochondrial membrane potential of cells. Western blotting assay determined protein levels. Results The luciferase reporter gene assay demonstrated that miR-206 might directly bind to the 3'UTR of the MUC1 gene and suppress MUC1 expression. Furthermore, MUCI expression was upregulated and inversely associated with miR-206 levels in GC tissues. More importantly, the miR-206-mediated suppression of proliferation, migration, and invasion, and the induction of apoptosis, were abrogated by MUC1 overexpression. Conclusion Our data demonstrated that miR-206 may exert antitumor activities through inhibiting the expression of MUC1, which may serve as an effective and potential target for GC treatment.

Cyclooxygenase-2 (COX-2) and extracellular signal-regulated kinase 1/2 (ERK1/2) may serve as potential targets in various types of cancer; however, the roles of these proteins in gallbladder carcinoma (GBC) have not been reported previously. In the present study, the expression levels of COX-2 and phospho (p)-ERK1/2 in GBC were examined and the biological activities of celecoxib and PD184161 (specific inhibitors of COX-2 and p-ERK1/2, respectively) on the proliferation, cell cycle and apoptosis of the GBC-SD and NOZ human GBC cell lines were evaluated by a series of in vitro and in vivo studies. COX-2 and p-ERK1/2 protein expression levels were found to be significantly elevated in GBC tissues as well as in GBC-SD and NOZ cells. Treatments with celecoxib and PD184161 significantly inhibited GBC-SD and NOZ cell growth in a concentration-dependent manner, and their combination produced a synergistic inhibitory effect. In addition, celecoxib and PD184161 significantly inhibited tumor growth in xenograft nude mice. Celecoxib treatment led to G1 arrest via the upregulation of p21 and p27 expression in GBC-SD and NOZ cells, whereas PD184161 did not affect cell cycle distribution. The combination of celecoxib and PD184161 was able to promote cell apoptosis by triggering a collapse of mitochondrial membrane potential and activating caspase-3-mediated apoptosis. In conclusion, COX-2 and p-ERK1/2 protein may serve as potential targets for GBC chemotherapy, and the combination of celecoxib and PD184161 could significantly inhibit GBC cell growth, induce cell G1 arrest and trigger cell apoptosis of GBC cells.

Background Tissue factor pathway inhibitor 2 (TFPI-2) has been recently identified as a tumor suppressor gene in several human cancers, whereas its role in thyroid cancer has been unclear. Methods The TFPI-2 protein level in thyroid cancer tissues and cell lines (8305C and B-CPAP) were examined using immunohistochemistry and immunoblotting. The TFPI-2 promoter methylation was examined using methylation-specific polymerase chain reaction (MSP). Lentivirus containing TFPI-2 cDNA (Lenti-TFPI-2) was constructed to elevate TFPI-2 expression in 8305C and B-CPAP cells. The effects of Lenti-TFPI-2 on cell proliferation in vitro and in vivo were evaluated by MTT assay and mouse xenograft model. Annexin V/PI double staining assay was performed to detect the effect of Lenti-TFPI-2 on cell apoptosis. Results TFPI-2 protein level were decreased in cancer tissues and lymph node metastasis, and TFPI-2 protein level is positively associated with survival time. The promoter of TFPI-2 is hypermethylated in cancer tissues. TFPI-2 mRNA and protein levels were abundant in normal human thyroid follicular cell line Nthy-ori 3-1 cells, whereas they were decreased in 8305C and B-CPAP cells. pcDNA-TFPI-2 elevated TFPI-2 mRNA and protein in 8305C and B-CPAP cells. TFPI-2 overexpression suppressed proliferation and induced apoptosis of 8305C and B-CPAP cells. Conclusions TFPI-2 inactivation may play a role in thyroid cancer tumorigenesis and development. TFPI-2 overexpression suppressed cell proliferation through induction of cell apoptosis, suggesting that TFPI-2 may serve as a novel and effective target for thyroid cancer therapy.

Trastuzumab has led to a marked improvement in the outcomes of patients with human epidermal growth factor receptor 2 (HER‑2)‑positive breast cancer. However, the effects of trastuzumab on HER‑2‑positive breast cancer are limited by the emergence of its cardiotoxicside effects. MicroRNA (miR)‑135b‑5p has been shown to inhibit tumor metastasis in breast cancer. The present study aimed to explore the effects of miR‑135b‑5p overexpression on the efficacy of trastuzumab in HER‑2‑positive breast cancer. Reverse transcription‑quantitative PCR was performed to detect the levels of miR‑135b‑5p. Cell viability was evaluated with a Cell Counting Kit‑8 assay. Annexin V/propidium iodide staining was employed to detect the number of apoptotic cells. Flow cytometry assay was performed to investigate the cell cycle. Western blotting was used to detect the expression levels of Bax, cleaved caspase‑3, Bcl‑2, cyclin D2, p27Kip1 and cyclin E1. Cell migration and invasion were detected by Transwell assay. Luciferase assays were conducted to identify the target gene of miR‑135b‑5p. In addition, an in vivo tumor xenograft model was established. miR‑135b‑5p agomir significantly enhanced the anti‑proliferative effect of trastuzumab on HER‑2‑positive breast cancer cells via the induction of apoptosis, whereas the anti‑metastatic effect of trastuzumab was enhanced by miR‑135b‑5p agomir treatment. Subsequently, luciferase assays indicated that cyclin D2 was the direct target of miR‑135b‑5p, whereas overexpression of the latter arrested cell cycleduring the G0/G1 phase. Moreover, miR‑135b‑5p agomir notably increased the antitumor effect of trastuzumab in vivo. The data demonstrated that miR‑135b‑5p sensitized HER‑2‑positive breast cancer cells to trastuzumab in vitro and in vivo by directly binding to cyclin D2. These results suggested that the combination of miR‑135b‑5p with trastuzumab may be a therapeutic strategy for patients with HER‑2‑positive breast cancer.

Additional file 1: Figure S1. Multi-factor dimensionality reduction (MDR) model was established to assess the interactive effect of single nucleotide polymorphism rs12982687 (C > T) and environmental exposures on colorectal cancer (CRC) risk. (A) The rs12982687 could interact with smoking and alcohol to promote risk of CRC. The grayer the boxes, the more CRC risk. Within each box, bars on the left and on the right, respectively, represented case group and control group. The height of each bar was representative of sample size. (B) The hierarchical interaction graph was reflective of interaction degree among rs12982687, smoking and alcohol drinking. (C) The interaction dendrogram symbolized strength of synergy interaction.

Additional file 2: Table S1. Polymerase chain reaction amplification primer sequences.

Additional file 3: Table S2. Stratified analyses between rs12982687 and rs11085996 of UCA1 and susceptibility to colorectal cancer.

Additional file 6: Detailed results of stratified analyses regarding association of candidate SNPs in UCA1 with susceptibility to CRC and evaluating the interactive contribution of candidate SNPs in UCA1 and smoking to CRC risk by establishing GEI and MDR models.

Additional file 5: Table S4. KEGG pathways shared by genes targeted by miR-873-5p, miR-1207-5p and miR-584.

Additional file 4: Table S3. The optimal multifactor dimensionality reduction model for predicting onset of colorectal cancer.

Background Gallbladder cancer (GBC) ranks fifth in the most common malignancy of the gastrointestinal tract worldwide. It is reported many small nucleolar RNAs (SNORNs) could regulate the progression of GBC. To identify potential therapeutic targets for GBC, we conducted microarray analysis in GBC tissues and adjacent normal tissues. We found that SNORA21 was downregulated most in gallbladder tumor samples. Therefore, this research aimed to investigate the role of SNORA21 during the tumorigenesis of GBC. Methods The differential expression of SNORNs between GBC tissues and para-carcinoma tissues were examined by microarray analysis and that were confirmed by qRT-PCR. Cell proliferation was tested by CCK-8 and immunofluorescence. Cell apoptosis and cell cycle in GBC were detected by flow cytometry. Expression of proteins in GBC cells was measured by Western-blot. Transwell assay was used for testing the cell migration and invasion. Xenograft tumor model was established to verify the effect of SNORA21 overexpression on GBC in vivo. Results The results revealed that SNORA21 overexpression inhibited the proliferation, migration and invasion of GBC cells. Moreover, overexpression of SNORA21 significantly increased the expression of E-cadherin and decreased the levels of N-cadherin and vimentin. Meanwhile, overexpression of SNORA21 significantly induced apoptosis and G1 arrest of GBC cells. Finally, SNORA21 overexpression significantly suppressed the growth of gallbladder tumors in vivo. Conclusion Overexpression of SNORA21 significantly suppressed the tumorigenesis of GBC in vitro and in vivo, which may serve as a potential novel target for the treatment of GBC.

Background This investigation was arranged to elucidate whether single nucleotide polymorphisms (SNPs) of lncRNA UCA1 was implicated in elevating colorectal cancer (CRC) risk by interacting with environmental exposures. Methods LncRNASNP database was firstly adopted to predict SNPs that possibly affected binding of UCA1 with miRNAs and then the interactive effect of SNPs and environmental exposure on CRC risk was evaluated by recurring to type 2 gene-environment interactions (GEI) model. Besides, MTT assay, colony formation assay, transwell assay and wound healing assay were performed to assess the activity of CRC cell lines which carried distinct genotypes of specific SNPs. The impact of nicotine on activity of CRC cells was also appraised. Results SNP rs12982687 of UCA1 intervened in the binding capacity of UCA1 with several miRNAs, especially miR-873-5p. MiRNAs regulated by UCA1, as predicted by mirPath software, shared genes that were enriched in HIF1 signaling pathway. Moreover, homozygote TT of rs12982687 reduced CRC risk among smokers, and CRC cells that carried rs12982687 (CC) displayed strong migration and invasion. By contrast, miR-873-5p mimic, which reduced UCA1 expression, delayed metastasis of CRC cells (all P P Conclusions SNP rs12982687 was involved in smoking-triggered CRC progression, given its influence on UCA1's binding with miR-873-5p and HIF-1 signaling.

// Yiyu Qin 1, 2, 3, 4, * , Li Meng 2, * , Yang Fu 5 , Zhiwei Quan 3 , Mingzhe Ma 3 , Mingzhe Weng 3 , Zhengdong Zhang 4 , Cuixiang Gao 2 , Xinghua Shi 2 , Koulan Han 1 1 Clinical College, Yancheng Institute of Health Sciences, Yancheng, Jiangsu 224000, China 2 Research Centre of Biomedical Technology, Yancheng Institute of Health Sciences, Yancheng, Jiangsu 224000, China 3 Department of General Surgery, Xinhua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai 200092, China 4 Department of Environmental Genomics, Jiangsu Key Laboratory of Cancer Biomarkers, Prevention and Treatment, Collaborative Innovation Center For Cancer Personalized Medicine, Nanjing Medical University, Nanjing 210029, China 5 Department of Gastrointestinal Surgery, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan 450052, China * These authors have contributed equally to this work Correspondence to: Yiyu Qin, email: qyy128@163.com Zhengdong Zhang, email: zdzhang@njmu.edu.cn Koulan Han, email: hkl414@163.com Keywords: gallbladder cancer, SNORA74B, PHLPP, snoRNA, AKT Received: October 04, 2016 Accepted: January 08, 2017 Published: February 13, 2017 ABSTRACT Small nucleolar RNAs (snoRNAs) have been implicated in the development of many cancers. We therefore examined the differential expression of snoRNAs between gallbladder cancer (GBC) tissues and matched adjacent non-tumor tissues using expression microarray analysis with confirmation by quantitative real-time PCR (qRT-PCR). Western blot analysis showed that SNORA74B levels were higher in GBC than non-tumor tissues. SNORA74B expression was positively associated with local invasion, advanced TNM stage, CA19-9 level, and Ki67 expression in patients with GBC, while it was negatively associated with expression of PHLPP, an endogenous Akt inhibitor. Moreover, SNORA74B expression was prognostic for overall survival (OS) and disease-free survival (DFS). Functional studies revealed that silencing SNORA74B in GBC cells using sh-SNORA74B suppressed cell proliferation, induced G1 arrest, and promoted apoptosis. Preliminary molecular investigation revealed that SNORA74B silencing inhibited activation of the AKT/mTOR signaling pathway, while increasing PHLPP expression. PHLPP depletion using shRNA abrogated sh-SNORA74B suppression of GBC cell proliferation, indicating that the antitumor effects of SNORA74B silencing were mediated by PHLPP. These findings define the important role of SNORA74B in cell proliferation, cell cycle, and apoptosis of GBC, and suggest that it may serve as a novel target for GBC treatment.

// Mingdi Zhang 1, 2 , Wei Gong 1 , Bin Zuo 3 , Bingfeng Chu 1 , Zhaohui Tang 1 , Yong Zhang 1 , Yong Yang 1 , Di Zhou 1 , Mingzhe Weng 1 , Yiyu Qin 1 , Mingzhe Ma 1 , Alex Jiang 4 , Fei Ma 5 , Zhiwei Quan 1 1 Department of General Surgery, Xinhua Hospital, Shanghai Jiaotong University, School of Medicine, Shanghai, 200092, China 2 Department of Anesthesiology and Surgical Intensive Care Unit, Xinhua Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, 200092, China 3 Department of Orthopedic Surgery, Xinhua Hospital, Shanghai Jiaotong University, School of Medicine, Shanghai, 200092, China 4 Schulich School of Medicine and Dentistry, Western Ontario University, London, ON N6A 3K6, Canada 5 Department of Oncology, Xinhua Hospital, Shanghai Jiaotong University, School of Medicine, Shanghai, 200092, China Correspondence to: Zhiwei Quan, email: zhiwquan@163.com Fei Ma, email: irismafei@126.com Keywords: gallbladder cancer, microRNAs, epithelial–mesenchymal transition, twist, interleukin-6 Received: January 23, 2016 Accepted: September 25, 2016 Published: October 15, 2016 ABSTRACT Cytokine is a key molecular link between chronic inflammation and gallbladder cancer (GBC) progression. The potential mechanism of cytokine-associated modulation of microRNAs (miRNAs) expression in GBC progression is not fully understood. In this study, we investigated the biological effects and prognostic significance of interleukin-6 (IL-6) -induced miRNAs in the development of GBC. We identify that inflammatory cytokine, IL-6 promotes proliferation, migration, invasion and epithelial-mesenchymal transition (EMT) of GBC both in vitro and in vivo . Among all the changed miRNAs in miRNA profiling, miR-33a expression was significantly decreased in IL-6 treated GBC cell lines, as well as in GBC tissues compared with case-matched normal tissues and cholecystitis tissues. In turn, miR-33a suppresses IL-6−induced tumor metastasis by directly binding Twist which was identified as an EMT marker. High expression of miR-33a suppressed xenograft tumor growth and dissemination in nude mice. The downregulation of miR-33a was closely associated with advanced clinical stage, lymph node metastasis, and poor clinical outcomes in patients with GBC. miR-33a acts as a tumor suppressor miRNA in GBC progression and may be considered for the development of potential therapeutics against GBC.

Increasing evidence has revealed a correlation between chronic inflammation and gallbladder cancer (GBC). However, the underlying molecular mechanisms remain to be elucidated. In the present study, secretion of interleukin (IL)-1β was examined in tissues of GBC, chronic cholecystitis and normal gallbladder, as well as in the supernatant of GBC-SD, SGC996 and HIBEpiC cells. The effect of IL-1β on the proliferation and migration of GBC cell lines was also evaluated. In addition, the role of Twist in IL-1β-induced proliferation of GBC cells was also studied. It was observed that the level of IL-1β protein in normal gallbladder tissue was low, while it was significantly increased in GBC and chronic cholecystitis tissues. The level of IL-1β protein and mRNA in GBC-SD and SGC996 cells was markedly higher than those in HIBEpiC cells. Exogenous IL-1β promoted the proliferation of GBC-SD and SGC996 cells in vitro and in vivo, and also promoted migration in vitro. The level of Twist protein was significantly increased following treatment with exogenous IL-1β. In addition, gene silencing of Twist blocked IL-1β-induced proliferation and migration of GBC-SD and SGC996 cells. Taken together, these results indicate that IL-1β promotes proliferation and migration of GBC cells via Twist activation.

Sijunzi decoction (SJZD) is a traditional Chinese herbal medicine. Previous studies have indicated that SJZD exhibits antitumor activity. However, the underlying molecular mechanism has not been fully elucidated. To explore the antitumor mechanism of SJZD, the effects of serum from rats treated with SJZD on the proliferation of MKN-28 and HGC-27 gastric carcinoma cell lines were systematically investigated. It was found that SJZD-treated rat serum significantly inhibited the growth of MKN-28 and HGC-27 cells in vitro. The results obtained from a colony formation assay showed that SJZD-treated rat serum decreased the colony formation ability of MKN-28 and HGC-27 cells. The apoptosis rate in MKN-28 and HGC-27 cells was also increased following treatment with SJZD-treated rat serum. Flow cytometry with cell sorting revealed the presence of side population (SP) cells in MKN-28 and HGC-27 cells though Hoechst 33342 staining, and verapamil reduced the SP percentage. Further analysis showed that SJZD-treated rat serum promoted the apoptosis of SP cells in MKN-28 and HGC-27 cell lines by upregulating Bax, caspase-3 and PARP and downregulating bcl-2. These data revealed the therapeutic effect of SJZD-treated rat serum on gastric carcinoma. Following the preliminary identification of the inhibitory effect on the growth of gastric cancer cells in vitro, the growth inhibitory effect of SJZD-treated rat serum on SP cells was confirmed, and this inhibition particularly involved the induction of cell apoptosis.

The present study aimed to investigate the expression of interleukin‑6 (IL‑6) in gallbladder cancer (GBC) tissues and its correlation with survival rate. The association between IL‑6 and epithelial‑mesenchymal transition (EMT)‑associated markers was also examined. Using immunohistochemistry, reverse transcription quantitative polymerase chain reaction (RT‑qPCR) and western blot analysis, the protein and mRNA expression levels of IL‑6, Twist, E‑cadherin and Vimentin in 20 GBC tissues were analyzed. The IL‑6, Twist and Vimentin proteins were overexpressed in 40, 20 and 70% of the human GBC samples, respectively. The protein expression of E‑cadherin was higher in only 5% of the GBC samples. These differences were significant (P

Background and Aim The biological function of tumor suppressor deleted in liver cancer 1 (DLC1) has been investigated in several types of human cancer, but its role in gallbladder cancer (GBC) is yet to be determined. In this research, we conducted in vitro and in vivo analysis to evaluate the inhibitory activities of DLC1 gene against GBC growth. Methods DLC1 expression in GBC tissues and cell lines was examined by immunohistochemical staining, reverse transcription polymerase chain reaction, and Western blot assay. The in vitro and in vivo effects of ectopic DLC1 expression on cell growth were evaluated. In addition, the effects of ectopic DLC1 expression on cell cycle, apoptosis, and migration were also evaluated. The expressions of cell cycle-related and apoptosis-related proteins were examined. Results The downregulation of DLC1 expression was a common event in GBC tissues and cell lines. Restoration of DLC1 expression in GBC-SD and NOZ cells significantly reduced cell proliferation, migration in vitro, and the ability of these cells to form tumors in vivo. Restoration of DLC1 expression arrested GBC-SD and NOZ cells in G0/G1 phase through inducing p21 in a p53-independent manner. In addition, restoration of DLC1 expression induced extrinsic and intrinsic apoptotic pathway through promoting the expressions of Fas L/FADD, Bax, cytochrome c, cleaved caspase-8, -9, -3, and cleaved poly-(ADP-ribose) polymerase and suppressing bcl-2 expression in GBC-SD and NOZ cells. Conclusions Our findings suggested that dysregulated expression of DLC1 is involved in proliferation and invasion of GBC cells and may serve as a potential therapeutic target.

Papillary thyroid cancer (PTC) is one of the endocrine cancers with high clinical and genetic heterogeneity. NOTCH signaling and its downstream NOTCH-Regulated Ankyrin Repeat Protein (NRARP) have been implicated in oncogenesis of many cancers, but the roles in PTCs are less studied. In this study, we show that NRARP is frequently over-expressed in thyroid carcinoma. The over-activation of NRARP is highly and positively correlated with NOTCH genes. Moreover, we find that the expression of NRARP is highly associated with several epithelial mesenchymal transition (EMT) markers and contributes to poor survival outcomes. Therefore, these results indicate that NRARP is an important clinical biomarker in thyroid carcinoma and it promotes EMT induction as well as the progression of PTCs via NOTCH signaling activation.

Background: The Notch-regulated ankyrin repeat protein (NRARP) is recently found to promote proliferation of breast cancer cells. The role of NRARP in carcinogenesis deserves extensive investigations. This study attempted to investigate the expression of NRARP in thyroid cancer tissues and assess the influence of NRARP on cell proliferation, apoptosis, cell cycle, and invasion in thyroid cancer. Methods: Thirty-four cases with thyroid cancer were collected from the Department of General Surgery, Xinhua Hospital, Shanghai Jiao Tong University School of Medicine between 2011 and 2012. Immunohistochemistry was used to detect the level of NRARP in cancer tissues. Lentivirus carrying NRARP-shRNA (Lenti-NRARP-shRNA) was applied to down-regulate NRARP expression. Cell viability was tested after treatment with Lenti-NRARP-shRNA using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Apoptosis and cell cycle distribution were determined by flow cytometry. Cell invasion was tested using Transwell invasion assay. In addition, expressions of several cell cycle-associated and apoptosis-associated proteins were examined using Western blotting after transfection. Student's t-test, one-way analysis of variance (ANOVA), or Kaplan–Meier were used to analyze the differences between two group or three groups. Results: NRARP was highly expressed in thyroid cancer tissues. Lenti-NRARP-shRNA showed significantly inhibitory activities against cell growth at a multiplicity of infection of 10 or higher (P < 0.05). Lenti-NRARP-shRNA-induced G1 arrest (BHT101: 72.57% ± 5.32%; 8305C: 75.45% ± 5.26%) by promoting p21 expression, induced apoptosis by promoting bax expression and suppressing bcl-2 expression, and inhibited cell invasion by suppressing matrix metalloproteinase-9 expression. Conclusion: Downregulation of NRARP expression exerts significant antitumor activities against cell growth and invasion of thyroid cancer, that suggests a potential role of NRARP in thyroid cancer targeted therapy.

Anaplastic thyroid cancer (ATC) represents the most aggressive subtype of thyroid cancer and has a poor prognosis. In addition to surgery, chemotherapy is an important treatment for ATC; however, the therapeutic effects of current chemotherapies for ATC are not particularly promising. There is a high proportion of side population (SP) cells in ATC, which may be a reason for its drug resistance. In the present study, the antitumor activities of combined octreotide (OCT) and cisplatin (DDP) on the proliferation and apoptosis of ATC SP cells were evaluated. First, SP cells from 8305C and BHT101 cell lines were detected and sorted. Following in vitro culture for 1 week, cluster of differentiation (CD)44, CD133, ATP-binding cassette (ABC) subfamily B member 1 (ABCB1), ABC subfamily G member 2 (ABCG2) and somatostatin receptor expression was detected to characterize the SP cells. An MTT assay was performed to investigate the combined effects on 8305C-SP cell proliferation in vitro, and a mouse model was used to investigate the combined effects on 8305C-SP cell proliferation in vivo. Annexin V/propidium iodide staining was used to investigate the combined effects on 8305C-SP cell apoptosis. Chemotherapeutic drug resistance-associated protein expression and apoptosis-associated protein expression were also detected following combined treatment. As a result, SP cells were identified in 8305C and BHT101 cells, and the proportion of 8305C-SP cells was increased compared with that of BTH101-SP cells. SP cells have enhanced proliferation, tumorigenicity and drug resistance compared with main population cells. The combined treatment of OCT with DDP suppressed the proliferation of 8305C-SP cells in vitro and in vivo, and induced 8305C-SP cell apoptosis. Combined treatment decreased the ABCB1 and ABCG2 expression by SP cells and activated mitochondrial apoptotic signaling, resulting in cell apoptosis. In conclusion, these data support the hypothesis that combined treatment with OCT and DDP induces ATC cell apoptosis and suppresses cell proliferation. These data provide a theoretical basis for further combined chemotherapy clinical applications.

Primary gallbladder cancer (GBC) is the most common malignancy of the digestive system. Due to its resistance to standard chemotherapy, no effective treatments are available at present. Artemisinin, a plant-derived anti‑malarial drug, has recently been shown to have anti-proliferative effects on a range of human cancer cell types. However, the efficacy of artemisinin against gallbladder cancer has not been reported. The present study investigated the effects of artemisinin on the proliferation, cell cycle and apoptosis of gallbladder cancer cell lines. A cell viability assay and an in vivo xenograft study demonstrated that artemisinin significantly inhibited the growth of gallbladder cancer. Western blot analysis indicated that artemisinin induced the expression of p16, while down‑regulating phosphorylated extracellular signal‑regulated kinase (ERK)1/2, CDK4 and cyclin D1 expression, leading to inhibition of the ERK1/2 pathway. Furthermore, flow cytometry and western blot analysis showed that artemisinin caused G1-phase arrest of the cell cycle, promoted the generation of reactive oxygen species (ROS), led to a collapse of the mitochondrial membrane potential and to triggered cytochrome c release from the mitochondria into the cytoplasm, which finally activated caspase‑3‑mediated apoptosis. In conclusion, the present study demonstrated that artemisinin inhibits the proliferation of gallbladder cancer cells in vitro as well as in vivo and induces apoptosis via induction of ROS and cell cycle arrest. These results suggested that artemisinin may be suitable for the treatment of gallbladder cancer.

Gallbladder carcinoma (GBC) has a high mortality rate, requiring synergistic anti-tumor management for effective treatment. The myxoma virus (MYXV) exhibits a modest clinical value through its oncolytic potential and narrow host tropism.We performed viral replication assays, cell viability assays, migration assays, and xenograft tumor models to demonstrate that bone marrow-derived stem cells (BMSCs) may enhance efficiency of intravenous MYXV delivery.We examined the permissiveness of various GBC cell lines towards MYXV infection and found two supported single and multiple rounds of MYXV replication, leading to an oncolytic effect. Furthermore, we found that BMSCs exhibited tropism for GBC cells within a Matrigel migration system. BMSCs failed to affect the growth of GBC cells, in terms of tumor volume and survival time. Finally, we demonstrated in vivo that intravenous injection of MYXV-infected BMSCs significantly improves the oncolytic effect of MYXV alone, almost to the same extent as intratumoral injection of MYXV.This study indicates that BMSCs are a promising novel vehicle for MYXV to clinically address gallbladder tumors.

Ubiquitin-like containing PHD and RING finger domains 1 (UHRF1), overexpressed in various human malignancies, functions as an important regulator in cell proliferation and epigenetic regulation. Depletion of UHRF1 has shown potential antitumor activities in several types of cancer. However, the role of UHRF1 in gallbladder cancer (GBC) has not been investigated. RT-PCR, western blotting and immunohistochemistry were performed to examine UHRF1 expression at mRNA and protein levels in GBC tissues and cell lines. UHRF1 siRNA and UHRF1 shRNA were used to deplete the expression of UHRF1. The results showed that UHRF1 was overexpressed in GBC and its expression correlated with advanced TNM stage and presence of lymph node metastasis. UHRF1 depletion in GBC-SD and NOZ cells markedly inhibited proliferation, migration in vitro and the ability of these cells to form tumors in vivo. UHRF1 depletion upregulated the expression of PML and triggered extrinsic and intrinsic apoptotic pathways by promoting the expression of FasL/FADD, bax, cytosolic cytochrome c, cleaved caspase-8, -9 and -3 and cleaved PRAP and by suppressing bcl-2 expression in GBC-SD and NOZ cells. In addition, UHRF1 depletion induced cell cycle arrest at G1/S transition by inducing p21 in a p53-independent manner in GBC-SD and NOZ cells. Our findings suggest that UHRF1 is involved in the proliferation and migration of GBC cells and may serve as a biomarker or even a therapeutic target for GBC.

The biological function of tumor suppressor deleted in liver cancer 1 (DLC1) has been investigated in several types of human cancer, but its role in gallbladder cancer (GBC) is yet to be determined. In this research, we conducted in vitro and in vivo analysis to evaluate the inhibitory activities of DLC1 gene against GBC growth.DLC1 expression in GBC tissues and cell lines was examined by immunohistochemical staining, reverse transcription polymerase chain reaction, and Western blot assay. The in vitro and in vivo effects of ectopic DLC1 expression on cell growth were evaluated. In addition, the effects of ectopic DLC1 expression on cell cycle, apoptosis, and migration were also evaluated. The expressions of cell cycle-related and apoptosis-related proteins were examined.The downregulation of DLC1 expression was a common event in GBC tissues and cell lines. Restoration of DLC1 expression in GBC-SD and NOZ cells significantly reduced cell proliferation, migration in vitro, and the ability of these cells to form tumors in vivo. Restoration of DLC1 expression arrested GBC-SD and NOZ cells in G0/G1 phase through inducing p21 in a p53-independent manner. In addition, restoration of DLC1 expression induced extrinsic and intrinsic apoptotic pathway through promoting the expressions of Fas L/FADD, Bax, cytochrome c, cleaved caspase-8, -9, -3, and cleaved poly-(ADP-ribose) polymerase and suppressing bcl-2 expression in GBC-SD and NOZ cells.Our findings suggested that dysregulated expression of DLC1 is involved in proliferation and invasion of GBC cells and may serve as a potential therapeutic target.

Tissue factor pathway inhibitor-2 (TFPI-2) has been identified as a tumor suppressor gene in several types of cancers, but its role in gallbladder carcinoma (GBC) is yet to be determined. In the present study, TFPI-2 expression in GBC tissues was examined, and its inhibitory activities against GBC growth were evaluated in vitro and in vivo after adenovirus-mediated gene transfer of TFPI-2 (Ad5-TFPI-2) was constructed to restore the expression of TFPI-2 in GBC cell lines (GBC-SD, SGC-996, NOZ) and xenograft tumors. Immunohistochemical staining showed that TFPI-2 was significantly downregulated in GBC tissue specimens. Ad5-TFPI-2 could significantly inhibit GBC growth both in vitro and in vivo. Apoptosis analysis and western blotting assay demonstrated that Ad5-TFPI-2 could induce the apoptosis of both GBC cell lines and tissues by promoting the activities of cytochrome c, Bax, caspase-3 and -9 and suppressing Bcl-2 activity. These data indicated that TFPI-2 acts as a tumor suppressor in GBC, and may have a potential role in gene therapy for GBC. (Cancer Sci 2012; 103: 723–730)

Protein-coding genes account for only ~2% of the human genome, whereas the vast majority of transcripts are non-coding RNAs (ncRNAs) including long ncRNAs (lncRNAs). A growing volume of literature has proposed that lncRNAs are important factors in cancer. Colon cancer-associated transcript-1 (CCAT1), an lncRNA, which was first identified in colon cancer, was previously shown to promote tumor development and be a negative prognostic factor in gastric cancer. However, the mechanism through which CCAT1 exerts its oncogenic activity remains largely unknown. Recently, a novel regulatory mechanism has been proposed in which RNAs can cross-talk with each other via competing shared for microRNAs (miRNAs). The proposed competitive endogenous RNAs could mediate the bioavailability of miRNAs on their targets, thus imposing another level of posttranscriptional regulation. In this study, we demonstrated that CCAT1 was upregulated in gallbladder cancer (GBC) tissues. CCAT1 silencing downregulated, whereas CCAT1 overexpression enhanced the expression of miRNA-218-5p target gene Bmi1 through competitively ‘spongeing’ miRNA-218-5p. Our data revealed that CCAT1 knockdown impaired the proliferation and invasiveness of GBC cells, at least in part through affecting miRNA-218-5p-mediated regulation of Bmi1. Moreover, CCAT1 transcript level was correlated with Bmi1 mRNA level in GBC tissues. Together, these results suggest that CCAT1 is a driver of malignancy, which acts in part through ‘spongeing’ miRNA-218-5p.