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Yujie Xie,1 Fanglin Shao,1 Yuxiu Ji,1 Dechao Feng,1,2 Ling Wang,3 Zonghai Huang,3 Shengjian Wu,1 Fuhua Sun,1 Hong Jiang,4 Akira Miyamoto,5 Haiming Wang,6 Chi Zhang1 1Rehabilitation Medicine Department, The Affiliated Hospital of Southwest Medical University, Luzhou, Sichuan, People’s Republic of China; 2Department of Urology, Institute of Urology, West China Hospital, Sichuan University, Chengdu, Sichuan, People’s Republic of China; 3School of Information and Communication Engineering, University of Electronic Science and Technology, Chengdu, Sichuan, People’s Republic of China; 4Rehabilitation Medicine Department, Xichong County People’s hospital, Nanchong, Sichuan, People’s Republic of China; 5Faculty of Rehabilitation, Nishikyushu University, Kansaitama, Japan; 6Department of Rehabilitation Medicine, the First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan, People’s Republic of ChinaCorrespondence: Haiming Wang, Department of Rehabilitation Medicine, the First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan, People’s Republic of China, Tel +868950771, Fax +86 830 8950771, Email wanghaiming866@163.com Chi Zhang, Rehabilitation Medicine Department, The Affiliated Hospital of Southwest Medical University, Luzhou, Sichuan, People’s Republic of China, Email chizhang_swmu@126.comPurpose: To provide a comprehensive analysis of associated genes with osteoarthritis (OA). Here, we reported a network analysis of OA progression by using a Steiner minimal tree algorithm.Methods: We collected the OA-related genes through screening the publications in MEDLINE. We performed functional analysis to analyze the associated biochemical pathways of the OA-related genes. Pathway crosstalk analysis was constructed to explore interactions of the enriched pathways. Steiner minimal tree algorithm was used to analyze molecular pathway networks. The average clustering coefficient was compared with the corresponding values of the Osteoarthritis-specific network. The new finding RNA was compared with former single-cell RNA-seq analysis results.Results: A gene set with 177 members reported to be significantly associated with Osteoarthritis was collected from 187 studies. Functional enrichment analysis revealed a specific related-OA gene including skeletal system development, cytokine-mediated signaling pathway, inflammatory response, cartilage development, and extracellular matrix organization. We performed a pathway crosstalk analysis among the 72 significantly enriched pathways. A total of 151 of the 177 genes in the Osteoarthritis gene set were included in the human interactome network. There were 31 genes in the former single-cell RNA-seq analysis results. The CLU, ENO1, SRRM1, UBC, HMGB1, NR3C1, NOTCH2NL, and CBX5 have significantly increased expression in seven molecularly defined populations of OA cartilage.Conclusion: The Steiner tree-based approach finds new biological molecules associated with OA genes.Keywords: network analysis, Steiner minimal tree, osteoarthritis, bioinformatics, osteoarthritis genes

Jianjun Huang,1 Fangping Shao,2 Bin Chen,1 Guanrong Zheng,1 Jia Shen,1 Shenzhong Qiu1 1Department of Neurosurgery, The First People’s Hospital of Fuyang District of Hangzhou City, Hangzhou, People’s Republic of China; 2Emergency Department, The First People’s Hospital of Fuyang District of Hangzhou City, Hangzhou, People’s Republic of ChinaCorrespondence: Shenzhong Qiu, Department of Neurosurgery, The First People’s Hospital of Fuyang District of Hangzhou City, Hangzhou, People’s Republic of China, Email chengiftboy@163.comBackground: Secreted protein acidic and rich in cysteine-like 1 (SPARCL1) regulates synaptic stability and is up-regulated during axonal regeneration. Here, serum SPARCL1 was determined for estimating severity and prognosticating early neurological deterioration (END) and functional outcomes of acute intracerebral hemorrhage (ICH).Methods: In this prospective observational cohort study of 156 patients with supratentorial ICH, blood samples of 53 were acquired not only at admission but also ad days 1, 3, 5, 7 and 10. Another group of 53 healthy controls were recruited. The modified Rankin Scale (mRS) scores of 3– 6 at poststroke six months were regarded as poor prognosis.Results: As opposed to controls, serum SPARCL1 levels were markedly elevated during the early ten days after ICH, with the highest levels at days 1 and 3. Admission serum SPARCL1 levels were independently correlated with National Institutes of Health Stroke Scale scores and hematoma volume, were significantly increased in the order of six-month mRS scores from 0 to 6 and were independently correlated with six-month mRS scores. Serum SPARCL1 levels were linearly related to risks of poor six-month prognosis and END under restricted cubic spline, had significant efficiency under receiver operating characteristic (ROC) curve and were independently associated with END and poor prognosis. Subgroup analysis confirmed that no interactions existed for associations of serum SPARCL1 levels with other variables, such as age, gender and some specific vascular risk factors. END and poor prognosis prediction models integrating serum SPARCL1 were displayed using the two nomograms. The poor prognosis prediction model, but END prediction model not, performed well under calibration curve, decision curve and ROC curve.Conclusion: A substantial elevation of serum SPARCL1 levels during the early period after ICH is independently related to illness severity and poor neurological outcomes, thus signifying that serum SPARCL1 may appear as a prognostic biomarker of ICH.Keywords: Secreted protein acidic and rich in cysteine-like 1, intracerebral hemorrhage, early neurologic deterioration, prognosis, severity, biomarkers

Huang J, Shao F, Chen B, Zheng G, Shen J, Qiu S. Neuropsychiatr Dis Treat. 2023;19:2709–2728. The authors have advised that the funding statement on page 2727 is incorrect. The text “(No. 202107071918000249496)” should read “(No. 20201203B59)”. The authors apologize for this error.

Fengjin Shao,1– 3 Rui Liu,1 Xun Tan,1– 4 Qiaoyan Zhang,1– 3 Lujie Ye,1– 3 Bingxuan Yan,1– 3 Ying Zhuang,1,2,4 Jiaxue Xu1,2,4 1Department of Veterinary Medicine, Zhejiang University, Hangzhou, Zhejiang Province, People’s Republic of China; 2Veterinary Medical Center, Zhejiang University, Hangzhou, Zhejiang Province, People’s Republic of China; 3Institute of Preventive Veterinary Sciences, Zhejiang University, Hangzhou, Zhejiang Province, People’s Republic of China; 4Hainan Institute of Zhejiang University, Sanya, Hainan Province, People’s Republic of ChinaCorrespondence: Xun Tan, Department of Veterinary Medicine, Zhejiang University, Yuhangtang Road 866, Hangzhou, 310058, People’s Republic of China, Tel +86 571 8898 2393, Fax +86 571 8898 2310, Email tanxun@zju.edu.cnPurpose: Pulmonary arterial hypertension (PAH) is a progressive and fatal pulmonary vascular disease initiated by endothelial dysfunction. Mesenchymal stromal cells (MSCs) have been shown to ameliorate PAH in various rodent models; however, these models do not recapitulate all the histopathological alterations observed in human PAH. Broiler chickens (Gallus gallus) can develop PAH spontaneously with neointimal and plexogenic arteriopathy strikingly similar to that in human patients. Herein, we examined the protective effects of MSC transplantation on the development of PAH in this avian model.Methods: Mixed-sex broilers at 15 d of age were received 2× 106 MSCs or PBS intravenously. One day later, birds were exposed to cool temperature with excessive salt in their drinking water to induce PAH. Cumulative morbidity from PAH and right-to-left ventricle ratio were recorded. Lung histologic features were evaluated for the presence of endothelial damage, endothelial proliferation and plexiform lesions. Expression of proinflammatory mediators and angiogenic factors in the lung was detected. Matrigel tube formation assay was performed to determine the angiogenic potential of endogenous MSCs.Results: MSC administration reduced cumulative PAH morbidity and attenuated endothelial damage, plexiform lesions and production of inflammatory mediators in the lungs. No significant difference in the expression of paracrine angiogenic factors including VEGF-A and TGF-β was determined between groups, suggesting that they are not essential for the beneficial effect of MSC transplantation. Interestingly, the endogenous MSCs from birds receiving MSC transplantation demonstrated endothelial differentiatial capacity in vitro whereas those from the mock birds did not.Conclusion: Our results support the therapeutic use of MSC transplantation for PAH treatment and suggest that exogenous MSCs produce beneficial effects through modulating inflammation and endogenous MSC-mediated vascular repair.Graphical Abstract: Keywords: plexiform lesion, right ventricular hypertrophy, VEGF-A, TGF-β, angiogenesis, bone marrow

Yan Zheng,1,* Yibo Wen,2,* Huixia Cao,1 Yue Gu,1 Lei Yan,1 Yanliang Wang,1 Limeng Wang,1 Lina Zhang,1 Fengmin Shao1 1Henan Provincial Key Laboratory of Kidney Disease and Immunology, Henan Provincial People’s Hospital, Zhengzhou, 450052, Henan, People’s Republic of China; 2Clinical Systems Biology Laboratories, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, 450052, Henan, People’s Republic of China*These authors contributed equally to this workCorrespondence: Fengmin ShaoHenan Provincial Key Laboratory of Kidney Disease and Immunology, Henan Provincial People’s Hospital, Henan, People’s Republic of ChinaEmail fengminshao01@163.comBackground: Immunotherapy has revolutionized the treatment of clear cell renal cell carcinoma (ccRCC). However, the therapy is constrained by drug resistance. Therefore, further characterization of immune infiltration in ccRCC is needed to improve its efficacy.Methods: Here, we adopted the CIBERSORT method to analyze the level of 22 immune cells, and analyzed the correlation of immune cells and clinical parameters in ccRCC in The Cancer Genome Atlas. We used consensus clustering to cluster ccRCC and identified differently expressed genes (DEGs) between hot and cold tumors using the “Limma” package, and then performed enrichment analysis of DEGs. Finally, we constructed and validated a Cox regression model using the “survival”, “glmnet”, and “survivalROC” packages, implemented in R.Results: Regulatory T cells upregulated in tumor tissue increased during tumor progression, and correlated with poor overall survival in ccRCC. Consensus clustering identified four clusters of ccRCC. To elucidate the underlying mechanisms of immune cell infiltration, we subdivided these four clusters into two major types, immune hot and cold, and identified DEGs between them. The results revealed different transcription profiles in the two tumor types, with hot tumors being enriched in immune-related signaling, whereas cold tumors were enriched in extracellular matrix remodeling and the phosphatidylinositol 3-kinase–AKT (PI3K/AKT) pathway. We further identified hub genes and prognostic-related genes from the DEGs, and constructed a Cox regression model for predicting the overall survival of patients with ccRCC. The areas under the receiver operating characteristics curve for the risk model for the training, testing, and external Zhengzhou validation cohorts were 0.834, 0.733, and 0.812, respectively. Notably, gene sets in the prediction model could also predict the overall survival of patients receiving immunotherapy.Conclusion: These findings provide a comprehensive characterization of immune infiltration in ccRCC, while the constructed model can be used effectively to predict the overall survival of ccRCC patients.Keywords: clear renal cell carcinoma, transcriptome profiling, immune infiltration, Cox risk model

Sisi Wang,* Junying Du,* Fangbing Shao, Wen Wang, Haiju Sun, Xiaomei Shao, Yi Liang, Boyi Liu, Jianqiao Fang, Junfan Fang Department of Neurobiology and Acupuncture Research, The Third Clinical Medical College, Zhejiang Chinese Medical University, Key Laboratory of Acupuncture and Neurology of Zhejiang Province, Hangzhou 310053, People’s Republic of China*These authors contributed equally to this workCorrespondence: Jianqiao Fang; Junfan Fang Email fangjianqiao7532@163.com; fangjunfan0223@163.comBackground: Acute pain can transition to chronic pain, presenting a major clinical challenge. Electroacupuncture (EA) can partly prevent the transition from acute to chronic pain. However, little is known about the mechanisms underlying the effect of EA. This study investigated the effect of EA on pain transition and the activation of metabotropic glutamate receptor 5 (mGluR5)–protein kinase C epsilon (PKCϵ) signaling pathway in the dorsal root ganglia (DRG).Methods: The hyperalgesic priming model was established by the sequential intraplantar injection of carrageenan (1%, 100 μL) and prostaglandin E2 (PGE2) into the left hind paw of rats. EA treatment (2/100 Hz, 30 min, once/day) was applied at bilateral Zusanli (ST36) and Kunlun (BL60) acupoints in rats. Von Frey filaments were used to investigate the mechanical withdrawal threshold (MWT) at different time points. The protein expression levels of mGluR5 and PKCϵ in the ipsilateral L4-L6 DRGs of rats were detected by Western blot. Some pharmacological experiments were performed to evaluate the relationship between mGluR5, PKCϵ and the MWT. It was also used to test the effects of EA on the expression levels of mGluR5 and PKCϵ and changes in the MWT.Results: Sequential injection of carrageenan and PGE2 significantly decreased the MWT of rats and up-regulated the expression level of mGluR5 and PKCϵ in the ipsilateral L4-L6 DRGs. EA can reverse the hyperalgesic priming induced by sequential injection of carrageenan/PGE and down-regulate the protein expression of mGluR5 and PKCϵ. Glutamate injection instead of PGE2 can mimic the hyperalgesic priming model. Pharmacological blocking of mGluR5 with specific antagonist MTEP can prevent the hyperalgesic priming and inhibit the activation of PKCϵ in DRGs. Furthermore, EA also produced analgesic effect on the hyperalgesic priming rats induced by carrageenan/mGluR5 injection and inhibited the high expression of PKCϵ. Sham EA produced none analgesic and regulatory effect.Conclusion: EA can regulate pain transition and it may relate with its inhibitory effect on the activation of mGluR5-PKCϵ signaling pathway in the DRGs.Keywords: electroacupuncture, pain transition, hyperalgesic priming, mGluR5, PKCϵ, DRGs

Zhao Wang,* Qiang Zhang,* Yungang Sun, Feng Shao Department of Thoracic Surgery, The Nanjing Chest Hospital, Nanjing, Jiangsu, People’s Republic of China*These authors contributed equally to this workCorrespondence: Feng ShaoDepartment of Thoracic Surgery, The Nanjing Chest Hospital, No. 215, Guangzhou Road, Nanjing, Jiangsu 210029, People’s Republic of ChinaTel +86 25 5861 9746Email g74414010yung@163.comBackground: Non-small cell lung cancer (NSCLC) is a common malignancy over the world. Previous report indicated that the plasmacytoma variant translocation 1 (PVT1) has been documented to function as an oncogene in various types of human cancers. However, the biological mechanism of PVT1 was still rarely reported in NSCLC.Methods: The levels of PVT1, miR-17-5p, and bone morphogenetic protein and activin membrane-bound inhibitor (BAMBI) in NSCLC tissues (n=64) and cells (H1299 and A549) were detected by qRT-PCR and immunohistochemistry (IHC). The protein level of BAMBI was measured by Western blot assay. Cell viability and apoptotic rate were evaluated by MTT assay and flow cytometry, respectively. The migrated and invaded abilities were assessed by Transwell assay and Wound healing assay. The interactions between miR-17-5p and PVT1 or BAMBI were predicted by starBase v2.0 and TargetScan, respectively, and then dual-luciferase reporter assay and RNA pull-down assay were performed to verify these interactions. The mice model experiments were constructed to further validate the roles of PVT1 in vivo.Results: The levels of PVT1 and BAMBI were both apparently increased, and miR-17-5p was declined in NSCLC tissues and cells. The depletion of PVT1 or BAMBI blocked cell viability, migrated and invaded abilities but impelled apoptotic rate in A549 and H1299 cells. PVT1 was validated as a sponge to miR-17-5p and BAMBI was a direct target of miR-17-5p. PVT1 promoted cell viability, migrated and invaded abilities but repressed apoptotic rate by targeting BAMBI. MiR-17-5p regulated cell behaviors mediated by PVT1. PVT1 silencing decreased BAMBI expression by sponging miR-17-5p. In addition, PVT1 knockdown blocked the xenograft tumor growth in vivo.Conclusion: These results manifested that PVT1 modulated BAMBI to promote tumor progression in NSCLC by sponging miR-17-5p. Thus, the novel regulatory pathway may provide a new therapeutic target for NSCLC patients.Keywords: PVT1, miR-17-5p, BAMBI, tumor progression, non-small cell lung cancer

Hongjin Shao,1 Dianbo Dong,1 Feng Shao2 1Department of Proctology, Liaocheng People’s Hospital, Liaocheng, Shandong 252000, People’s Republic of China; 2Department of Gastrointestinal Surgery, Liaocheng People’s Hospital, Liaocheng, Shandong 252000, People’s Republic of ChinaCorrespondence: Feng ShaoDepartment of Gastrointestinal Surgery, Liaocheng People’s Hospital, Liaocheng, Shandong 252000, People’s Republic of ChinaTel +86 1 396 956 7952Email Shaofeng7952@126.comIntroduction: Taurine up-regulated 1 (TUG1) was reported to be over-expressed and involved in various human malignancies. However, its expression status and mechanistic importance in colorectal cancer (CRC) were yet to be defined.Methods: Relative expressions of TUG1, miR-153-1 and Kruppel-like factor 4 (KLF4) were analyzed by real-time PCR. The potential influences of TUG1-proficiency and miR-153-1-deficiency on cell proliferation, migration and viability were determined by colony formation, wound healing and CCK-8 assays, respectively. Cell invasion was evaluated by transwell chamber assay. The regulatory effect of KLF4 on miR-153-1 was interrogated by luciferase reporter assay. Direct association between KLF4 and miR-153-1 promoter was measured by chromatin immunoprecipitation (ChIP) assay. Subcellular localization of TUG1 was determined by fractionization PCR. Enrichment of EZH2 on KLF4 promoter was analyzed by ChIP-PCR. The pro-tumoral activity of TUG1 was determined using xenograft tumor model.Results: We demonstrated the over-expression of TUG1 and down-regulation of miR-153-1 in CRC. Knockdown of TUG1 or ectopic over-expression of miR-153-1 in SW480 significantly suppressed cell proliferation, migration and viability. TUG1 negatively modulated miR-153-1 expression, and simultaneous expression of TUG1 completely abolished the anti-tumor effect of miR-153-1. We further identified KLF4 as a transcription factor of miR-153-1, which was negatively regulated by TUG1 along with EZH2.Conclusion: Our study unravels the critical involvement of TUG1/KLF4/miR-153-1 axis in CRC.Keywords: TUG1, miR-153-1, colorectal cancer, KLF4, EZH2

Fu-Tao Meng,1,2,* Mei Huang,1,2,* Fang-Fang Fan,3,* Feng Shao,1,2 Chao Wang,1,2 Qiang Huang1,2 1Department of General Surgery, Anhui Provincial Hospital Affiliated to Anhui Medical University, Hefei, Anhui Province, People’s Republic of China; 2Anhui Province Key Laboratory of Hepatopancreatobiliary Surgery, Anhui Provincial Hospital, Hefei, Anhui Province, People’s Republic of China; 3Perelman School of Medicine, University of Pennsylvania, Pennsylvania, PA, USA *These authors contributed equally to this work Background: This study explored a simple, high-yield method for isolating quiescent human pancreatic stellate cells (PSCs) to provide sufficient and reliable raw materials for PSC-related studies. Materials and methods: Single-cell suspensions were prepared from normal human pancreatic tissue specimens using the gentleMACS™ tissue processor, which enhanced the yield and viability of the suspensions. Percoll density gradient centrifugation was then performed to isolate quiescent normal PSCs (NPSCs). Cell viability was determined by trypan blue staining, and the states of the NPSCs were determined by autofluorescence and oil red O staining. The purity of human activated PSCs (APSCs) was determined by immunofluorescence assays. Results: The yield of NPSCs was ~(2.75±0.65)×106 cells/g. The maximum cell viability was 92%, whereas the maximum cell purity was 95%. Conclusion: The method employed in this study to isolate PSCs is a simple, high-yield and stable method that is worth popularizing. Keywords: pancreatic stellate cells, gentleMACS™ Dissociator, Percoll, primary cell culture

Feng Shao,* Wei Qi,* Fu Tao Meng,* Lujun Qiu, Qiang Huang Department of Biliary-Pancreatic Surgery, Anhui Provincial Hospital Affiliated with Anhui Medical University, Hefei, Anhui, People’s Republic of China *These authors contributed equally to this work Background: This population-based study evaluated the overall (OS) and cancer-specific survival (CSS) benefit from palliative radiotherapy (RT) in patients with unresectable intrahepatic cholangiocarcinoma (ICC). Methods: We queried The Surveillance, Epidemiology, and End Results (SEER) database for the patients with unresectable ICC diagnosed from 1973 to 2013. Propensity score–matched analysis was performed to reduce the impact of the selection bias between the palliative RT group and the nonpalliative RT group. Kaplan–Meier survival curves were used to estimate the survival outcome before and after propensity score matching. OS and CSS were compared between patients with and without palliative RT using univariate and multivariate Cox proportional hazards regression analyses. Results: A total of 4,027 of 15,803 patients diagnosed with ICC were included in this study. Of those, 847 (21%) patients underwent palliative RT, whereas 3,180 (79%) did not. In the unmatched population, patients treated with palliative RT had improved OS and CSS relative to those treated without palliative RT (adjusted HR =0.9065, 95% CI =0.8360–0.982, P=0.01735) and CSS (adjusted HR =0.8874, 95% CI =0.8160–0.9652, P=0.00532). After propensity score matching, palliative RT was associated with a significantly improved OS (adjusted HR =0.8544, 95% CI =0.7722–0.9453, P=0.00228) and CSS (adjusted HR =0.8563, 95% CI =0.7711–0.9509, P=0.0037). Conclusion: Palliative RT seems to improve the prognosis of patients with unresectable ICC. Keywords: radiation therapy, SEER, unresectable intrahepatic cholangiocarcinoma

Yichen Wang, Feng Shao, Lu Chen Department of Gynecologic Oncology, Zhejiang Cancer Hospital, Hangzhou, China Abstract: Epithelial ovarian cancer is the deadliest gynecological malignancy worldwide. A better understanding of epithelial ovarian cancer pathogenesis and the molecular mechanism underlying its metastasis may increase overall survival rates. Previous studies have indicated that aldehyde dehydrogenase 1 family member A2 (ALDH1A2) is a candidate tumor suppressor in epithelial ovarian cancer. However, the potential role of ALDH1A2 in the molecular mechanisms of epithelial ovarian cancer remains largely unclear. In the present study, we found lower expression of ALDH1A2 in high-grade epithelial ovarian cancer tissues than in low-grade epithelial ovarian cancer tissues. Overexpression of ALDH1A2 decreased the proliferation and migration of epithelial ovarian cancer cell lines, whereas ALDH1A2 knockdown significantly increased cell growth and migration. Moreover, upregulation of ALDH1A2 also reduced the activation of signal transducer and activator of transcription 3 (STAT3). In conclusion, these findings suggest that ALDH1A2 suppresses epithelial ovarian cancer cell proliferation and migration by downregulating STAT3. Keywords: ALDH1A2, epithelial ovarian cancer cell, STAT3, migration, cell growth

Liya Liu, Hao Yu, Lihong Huang, Fang Shao, Jianling Bai, Donghua Lou, Feng Chen Department of Epidemiology and Biostatistics, School of Public Health, Nanjing Medical University, Nanjing, People’s Republic of China Background: The correlation between overall survival (OS) and progression-free survival (PFS) has been evaluated in patients with metastatic or advanced gastric cancer who have received first-line and/or second-line chemotherapy. However, no corresponding analysis has been done for patients who have undergone third-line or later-line chemotherapy.Methods: A total of 303 patients from the Phase II/III studies of apatinib were pooled (the Phase II study as a training data set, the Phase III study as a testing data set). Landmark analyses of PFS at 2 months from randomization were performed to minimize lead time bias. The Cox proportional hazard model was used to test for the significance effect of PFS rate at 2 months in predicting OS. Additionally, the PFS/OS correlations were evaluated by the normal induced copula (National Institute for Health and Care Excellence) estimation model.Results: The median OS was 3.37 months (95% confidence interval 2.63–3.80) in patients who experienced progression at 2 months and 5.67 months in patients who did not (95% confidence interval 4.83–6.67; P