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SOX9, as a transcript factor, has been confirmed to boost proliferation and epithelial-mesenchymal transition (EMT) of hepatocellular carcinoma (HCC), but the underlying mechanism remains incompletely elucidated. A bioinformatics analysis web, Jaspar, manifested that SOX9 can transcriptionally regulate an lncRNA, MKLN1-AS. To determine the role of MKLN1-AS in HCC, this study measured MKLN1-AS expression in HCC and the paracancerous tissues and conducted a series of assays, including MTT, colony formation, and transwell assays, in vitro. EMT of HCC was evaluated by E-cadherin and vimentin protein levels. The regulatory effect of SOX9 on MKLN1-AS was determined using dual luciferase reporter and ChIP assays. Both MKLN1-AS and SOX9 were up-regulated in HCC tissues compared to paracancerous tissues. SOX9 promoted cell viability, proliferation, invasion, and EMT of HCCs, but these promoting effects of SOX9 were attenuated after the knockdown of MKLN1-AS. Overexpression of SOX9 increased MKLN1-AS in HCCs, whereas silencing SOX9 decreased MKLN1-AS expression. According to dual luciferase reporter and ChIP assays, SOX9 can bind to the promoter of MKLN1-AS gene to stimulate the expression. MKLN1-AS is transcriptionally regulated by SOX9 and mediates the effects of SOX9 on the proliferation and EMT of HCCs., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

To investigate the effect of long noncoding RNA (LINC01419)/miR-485-5p/LSM4 on the malignant behavior of hepatocellular carcinoma (HCC) cells. The expressions of LINC01419, miR-485-5p, and LSM4 were determined in HCC at the cellular and clinical levels, and cell biological behavior was evaluated. The relationships between LINC01419, miR-485-5p, and LSM4 were predicted and verified. Additionally, the subcellular localization of LINC01419 in HCC cells was analyzed. Finally, an animal experiment was conducted to confirm the effect of LINC01419 silencing on tumor growth. in HCC tissues and cells, LINC01419 and LSM4 were increasingly expressed, but miR-485-5p was decreasingly expressed. LINC01419 negatively regulated miR-485-5p- and miR-485-5p-targeted LSM4. LINC01419 was localized in the cytoplasm of HCC cells. Downregulation of miR-485-5p or upregulation of LSM4 reversed the inhibition of HCC cell malignant behavior by LINC01419 interference. LINC01419 sponges miR-485-5p to upregulate LSM4 expression, thereby facilitating the biological behavior of HCC cells., (© 2022 The Authors. The Kaohsiung Journal of Medical Sciences published by John Wiley & Sons Australia, Ltd on behalf of Kaohsiung Medical University.)

Background: Long noncoding RNA (lncRNA) small nucleolar RNA host gene 3 (SNHG3) is an oncogenic lncRNA that has been reported in many cancers, but the role of SNHG3 in cholangiocarcinoma (CCA) remains largely unknown. Bioinformatic analysis revealed a regulatory relationship among SNHG3, miR-3173-5p, and ERG. miR-3173-5p is a tumour suppressive miRNA, while ERG is an oncogene. In the present study, we focused on the regulatory effects and molecular mechanisms of SNHG3 in CCA., Method: The expression of SNHG3 and miR-3173-5p was evaluated using qRT-PCR analysis. Knockdown of SNHG3 was achieved by shRNA. Cell viability was assessed by MTT assay. Migration and invasion were determined by Transwell assay. Flow cytometry was used to assess cell apoptosis. Western blots were applied to quantify protein levels. Furthermore, using RNA pulldown and dual luciferase assays, the interactions between SNHG3 and miR-3173-5p and between miR-3173-5p and ERG in CCA cells were validated., Results: SNHG3 was significantly upregulated in CCA cells compared with normal human intrahepatic biliary epithelial cells. Knockdown of SNHG3 inhibited the proliferation and migration of CCA cells. Mechanistically, SNHG3-sponged miR-3173-5p, thus releasing the repression of ERG by miR-3173-5p. Rescue experiments showed that the miR-3173-5p/ERG axis mediated the oncogenic effect of SNHG3., Conclusion: Taken together, our data suggest that SNHG3 is a pleiotropic oncogenic lncRNA in CCA. Knockdown of SNHG3 expression suppressed malignant phenotypes in CCA cells via the miR-3173-5p/ERG axis., (© 2021. The Society for Surgery of the Alimentary Tract.)

Cholangiocarcinoma (CCA) is a highly aggressive and malignant tumor. In this study, the effect and molecular mechanism of nuclear enriched abundant transcript 1 (NEAT1) in CCA were elucidated. The expressions of NEAT1, microRNA-186-5p (miR-186-5p), and PTP4A1 were measured by quantitative real-time PCR. The protein levels were measured by Western blotting. Kaplan-Meier analysis was performed to create survival curves. The interactions between NEAT1, miR-186-5p, and PTP4A1 were assessed through the dual luciferase reporter assay. Additionally, the cell proliferation, apoptosis, migration, and invasion were measured by colony formation, flow cytometry, the Transwell assay, and the wound healing assay, respectively. NEAT1 and PTP4A1 were significantly upregulated in CCA tissues and cells, but miR-186-5p was downregulated. NEAT1 expression was negatively correlated with the survival of CCA patients and has remarkable correlation with serum CA199 levels and lymph node metastasis. Besides, NEAT1 could act as a molecular sponge for miR-186-5p to upregulate PTP4A1 expression. More importantly, the knockdown of NEAT1 or overexpression of miR-186-5p inhibited the proliferation, migration and invasion of CCA cells, and the inhibition of miR-186-5p reversed the effects of the knockdown of NEAT1. In addition, NEAT1 could also activate the PI3K/AKT signaling pathway and regulate the epithelial-mesenchymal transition (EMT) through the miR-186-5p/PTP4A1 axis. In conclusion, NEAT1 was involved in cell proliferation, migration and invasion in CCA, and the NEAT1/miR-186-5p/PTP4A1/PI3K/AKT axis indicated novel regulatory mechanisms and therapeutics for the treatment of CCA., (© 2021 The Authors. The Kaohsiung Journal of Medical Sciences published by John Wiley & Sons Australia on behalf of Kaohsiung Medical University.)

Intrahepatic cholangiocarcinoma is a malignant tumor originating from intrahepatic bile ducts. Surgical therapy, radiotherapy, and chemotherapy are taken to treat this disease, but it is prone to recurrence and metastasis, with poor prognosis. Therefore, it is of great significance to explore new targets and molecular mechanisms for the development of cholangiocarcinoma cells. Clinical cholangiocarcinoma tissues from patients and four human cholangiocarcinoma cell lines were analyzed for microRNA-373 (miR-373) expression. For investigating whether miR-373 directly modulated unc-51 like autophagy activating kinase 1 (ULK1), dual-luciferase reporter assay was performed. In addition, CCK-8 assay, flow cytometry, western blot, and immunofluorescence were applied to evaluate the proliferation, apoptosis, and autophagy of cholangiocytic hepatocellular carcinoma cells. miR-373 downregulation was observed in clinical tissues and cell lines of cholangiocarcinoma. Overexpression of miR-373 reduced proliferation, enhanced apoptosis, and raised expression levels of pro-apoptosis proteins including BCL2 associated X (Bax), Caspase-3, and Caspase-9. Moreover, overexpression of miR-373 downregulated expression levels of microtubule-associated protein 1A/1B-light chain 3 (LC3)-II, Beclin-1, and promoted P62 expression on mRNA and protein levels. After miR-373 knockdown, all indexes of apoptosis and autophagy mentioned above were reversed. Luciferase activity was decreased after cotransfection of miR-373 mimic and wild-type ULK1 vector. Also, miR-373 overexpression inhibited ULK1 expression. Importantly, overexpression of miR-373 weakened expressions of ULK1, LC3, Beclin-1, and Bcl-2, and enhanced expressions of P62, Bax, Caspase-3, and Caspase-9. miR-373 mimic treatment and subsequent ULK1 overexpression, induced reverse regulation in expressions of these proteins, compared with overexpression of miR-373 only. miR-373 targeted ULK1 to initiate inhibition of autophagy and subsequent promotion of apoptosis in cholangiocarcinoma cells., (© 2020 The Authors. The Kaohsiung Journal of Medical Sciences published by John Wiley & Sons Australia on behalf of Kaohsiung Medical University.)

Leptin is an adipokine minimally known for its activities or underlying mechanisms in cholangiocarcinoma. The present study explored the effects of leptin on the epithelial‑mesenchymal transition (EMT) and pro‑angiogenic capability of cholangiocarcinoma cells, and investigated the underlying mechanisms. Cholangiocarcinoma cells were treated with leptin, and their migration and invasion rates were investigated using Transwell assays. Furthermore, conditioned medium was collected from cholangiocarcinoma cells following leptin treatment and applied to human umbilical vein endothelial cells to assess tube formation. The expression of EMT and pro‑angiogenic factors was examined by reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) and western blot analyses. Mechanistically, the function of pyruvate kinase muscle isozyme M2 (PKM2) was assessed in leptin‑induced phenotypes using siRNA targeting PKM2 (si‑PKM2). Bioinformatics screening and luciferase reporter assays were used to reveal microRNA (miR)‑122 as the potential mediator between leptin and PKM2. Finally, the associations between leptin and miR‑122 or PKM2 levels in patients with cholangiocarcinoma were assessed by ELISA and RT‑qPCR. Leptin significantly increased the EMT and pro‑angiogenic capability of cholangiocarcinoma cells, visibly inhibited endogenous miR‑122 expression, and upregulated PKM2. Furthermore, si‑PKM2 inhibited leptin‑induced migration, invasion, EMT‑associated marker expression levels and the pro‑angiogenic capability in cholangiocarcinoma cells. In addition, miR‑122 negatively regulated the expression of PKM2. When applied together with leptin, miR‑122 was sufficient to reverse the multiple malignancy‑promoting effects of leptin. Consistently, the serum leptin level positively correlated with that of PKM2, but negatively with that of miR‑122 in patients with cholangiocarcinoma. Leptin, by downregulating miR‑122 and elevating PKM2 expression, acts as a pleiotropic pro‑malignancy cytokine for cholangiocarcinoma. Therefore, increasing miR‑122 expression and inhibiting PKM2 may be future approaches for cholangiocarcinoma treatment.

Background/aims: Acute pancreatitis contributes to high mortality in pancreatitis patients, and miRNAs play a vital role in the development of acute pancreatitis (AP), however, its precise biological role remains largely elusive., Methods: To clarify the potential mechanisms of miRNAs in AP, we built mouse models of mild acute pancreatitis (MAP) and moderate/ severe acute pancreatitis (SAP). MiRNA microarray analysis and Real-time quantitative PCR (qRT-PCR) were used to analyze the expression of miRNA in MAP/SAP. TargetScan software, dual-luciferase gene reporter assays and Western blotting were used to assess the target genes of miR-155-5p in AP., Results: miR-155-5p was significantly decreased in MAP/SAP mice compared to controls. In pancreatic acinar AR42J cells transfected with miR-155-5p mimic, the expression of Rela and Traf3 notably decreased in both the caerulein- and TLC-S-induced groups compared with the negative control (NC); however, the expression of Rela and Traf3 notably increased after transfection with miR-155-5p inhibitor. Combined analysis using the TargetScan software and dual-luciferase gene reporter assays indicated that Rela and Traf3 were both targeted by miR-155-5p. Meanwhile, the expression of Ptgs2 also decreased after transfection of the AR42J cells with miR-155-5p mimic. The opposite results were found when miR-155-5p inhibitor was transfected into the AR42J cells. In addition, we treated caerulein- and TLC-S-induced AR42J cells with the Rela inhibitor helenalin and found that the expression of Rela, Traf3 and Ptgs2 decreased compared with the NC, while the expression of miR-155-5p did not show any significant difference. Furthermore, we found that miR-155-5p was significantly down-regulated in pancreatitis patients., Conclusion: miR-155-5p inversely regulated AP development through the Rela/Traf3/Ptgs2 signaling pathway., (© 2018 The Author(s). Published by S. Karger AG, Basel.)

It has been reported that hsa‑microRNA (miRNA/miR)‑372 functions as a tumor suppressor or oncogene in various digestive system tumors, however, its roles in gallbladder cancer (GBC) are yet to be established. The present study aimed to determine the expression and clinical relevance of hsa‑miR‑372 in GBC. The expression of hsa‑miR‑372 in 80 pairs of human GBC tissues and adjacent normal gallbladder tissues was measured by reverse transcription‑quantitative polymerase chain reaction. Subsequently, the associations between hsa‑miR‑372 expression levels and the clinicopathological characteristics of patients with GBC were determined using χ2 test. Furthermore, Kaplan‑Meier method and Cox regression analysis were performed to evaluate the association between hsa‑miR‑372 expression and the prognosis of patients with GBC. Furthermore, a dual‑luciferase reporter assay and western blot analysis were performed to predict and verify the target gene of hsa‑miR‑372. The results demonstrated that markedly lower hsa‑miR‑372 expression was observed in GBC tissues, which was associated with poor prognosis in patients with GBC. Downregulated expression of hsa‑miR‑372 was negatively associated with tumor histological grade, tumor‑node‑metastasis stage, lymph node metastasis and distant metastasis, however, no association was observed between reduced hsa‑miR‑372 expression and patient gender, age, tumor size and gallbladder stones. Multivariate Cox regression analysis revealed that hsa‑miR‑372 expression, histological grade and lymph node metastasis were independent prognostic factors for overall survival in patients with GBC. Chloride intracellular channel 1 (CLIC1) was previously reported to be an effective biomarker for predicting the prognosis of GBC. Notably, the results of the present study indicated that CLIC1 may be a direct target gene of hsa‑miR‑372. In conclusion, the current study provides the first statistically convincing evidence that downregulation of hsa‑miR‑372 may occur in GBC tissues, which may be associated with aggressive and progressive tumor behavior by affecting CLIC1 expression.