Upregulated microRNA-194 impairs stemness of cholangiocarcinoma cells through the Rho pathway via inhibition of ECT2.

Cholangiocarcinoma (CCA) is devastating for its delayed presence, difficulty in diagnosis, and high mortality. Other studies have supported the important role of microRNAs (miRNAs) in the pathogenesis of CCA, and the role of miR-194 was investigated in several human cancers, though, the molecular mechanism of miR-194 in CCA stem cells remains largely unknown. We aimed to identify the functional significance of miR-194 in CCA. The microarray-based analysis was applied to detect the epithelial cell transforming sequence 2 (ECT2) expression and predict the miRNA-regulated ECT2, followed by the identification of relationship between ECT2 and obtained miRNA by dual-luciferase reporter gene assay. The effects of depletion or ectopic expression of miR-194 on Rho pathway and the biological characteristics of CCA were assessed by reverse transcription quantitative polymerase chain reaction, immunoblotting, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide, scratch test, Transwell, and flow cytometry. Lastly, tumor growth was assessed by xenograft tumor in nude mice. ECT2 was highly expressed while miR-194 was poorly expressed in CCA stem cells, and the targeting relation between ECT2 and miR-194 was proved. More important, the elevated expression of miR-194 or ECT2 silencing inhibited the Rho pathway, and further promoted the apoptosis and suppressed the stem cell proliferation, migration, and invasion of CCA in vitro. miR-194 inhibited the tumor growth in vivo. In a word, miR-194 inhibits ECT2 and blocks the activation of Rho signaling pathway, thus promoting apoptosis, inhibiting proliferation and migration of CCA stem cells, and suppressing tumor growth. The mechanism can be regarded as a target for treating CCA.

Gao J ，Dai C ，Yu X ，Yin XB ，Zhou F ... -  《-》

MicroRNA-494-dependent WDHDI inhibition suppresses epithelial-mesenchymal transition, tumor growth and metastasis in cholangiocarcinoma.

Cholangiocarcinoma (CCA) represents a devastating malignancy characterized by high mortality, and notoriously problematic to diagnose. Recently, microRNAs (miRs) have been intensively investigated due to their potential usefulness from a tumor treatment perspective. The current study was aimed to investigate whether miR-494 influences epithelial-mesenchymal transition (EMT), tumor growth and metastasis of CCA. The regulatory miRNAs of WDHD1 in CCA expression chip were predicted, followed by determination of the miR-494 and WDHD1 expression in normal cholangiocyte tissues and CCA tissues. The related protein levels were determined. CCA cell migration, invasion, viability, and cell cycle distribution and the dosage-dependent effect of miR-494 on CCA cell growth were subsequently detected. Finally, tumorigenicity and lymph node metastasis (LNM) were measured. Initially, miR-194 affected the CCA development via negatively regulating WDHD1 and miR-494 which were downregulated while WDHD1 was upregulated in CCA. In addition, miR-494 overexpression elevated E-cadherin expression while decreased expressions of WDHD1, N-cadherin, Vimentin, Snail, Twist and MMP-9. Finally, overexpressed miR-494 was observed to suppress EMT, cell viability, migration, invasion, arrest cell cycle progression, tumor formation, and LNM while accelerating cell apoptosis in vivo. This study indicated that miR-494 overexpression suppresses EMT, tumor formation and LNM while promoting CCA cell apoptosis through inhibiting WDHD1 in CCA.

Liu B ，Hu Y ，Qin L ，Peng XB ，Huang YX ... -  《-》

MicroRNA-21 regulates biological behavior by inducing EMT in human cholangiocarcinoma.

Liu Z ，Jin ZY ，Liu CH ，Xie F ，Lin XS ，Huang Q ... -  《International Journal of Clinical and Experimental Pathology》

LncRNA HOTAIR suppresses cell apoptosis, autophagy and induces cell proliferation in cholangiocarcinoma by modulating the miR-204-5p/HMGB1 axis.

Cholangiocarcinoma (CCA) is a malignant tumor in the world. LncRNA HOX transcript antisense intergenic RNA (HOTAIR) was identified as a crucial regulator in various cancers including CCA. This study aimed to unravel the functions of HOTAIR and its biological mechanism in CCA, hinting for the new therapeutic targets in CCA. The levels of HOTAIR, miR-204-5p and HMGB1 in CCA tissues and cell lines (HuB28 and HuCCT1) were measured by quantitative real-time polymerase chain reaction (qRT-PCR). Western blot was conducted to detect the protein levels of LC3-I, LC3-II, Beclin-1 and HMGB1. The relationships among HOTAIR, miR-204-5p and HMGB1 were examined by dual-luciferase reporter assay, RNA immunoprecipitation (RIP) assay and RNA pull down assay. Cell proliferation ability and apoptosis rate were assessed by CCK8 assay and flow cytometry, respectively. in vivo experiment was conducted to examine the bio-functions of HOTAIR in nude mice. HOTAIR and HMGB1 were over-expressed, while miR-204-5p was lowly expressed in CCA tissues and cells. The dual-luciferase reporter assay indicated that miR-204-5p was a target of HOTAIR, and HMGB1 was a target of miR-204-5p. The restoration experiments showed that HOTAIR repressed cell apoptosis, autophagy and promoted cell proliferation via miR-204-5p/HMGB1 axis. Additionally, HOTAIR silencing retarded the xenograft tumor growth by up-regulation of miR-204-5p and down-regulation of HMGB1. These data unraveled that lncRNA HOTAIR regulated HMGB1 to suppress cell apoptosis, autophagy and induce cell proliferation by sponging miR-204-5p in CCA. Thus, this new regulatory pathway may provide new therapeutic targets for CCA.

Lu M ，Qin X ，Zhou Y ，Li G ，Liu Z ，Yue H ，Geng X ... -  《-》

MiR-124 induces autophagy-related cell death in cholangiocarcinoma cells through direct targeting of the EZH2-STAT3 signaling axis.

Cholangiocarcinoma (CCA) is a lethal cancer associated with chronic inflammation that has increased in prevalence in recent decades. The dysregulated expression of microRNAs (miRNAs) has been detected in various types of malignancies, and depending on the target genes this can result in miRNAs functioning as tumor suppressors or oncogenes. In this study, we investigated the role of miR-124 in cholangiocarcinoma (CCA) and found that its expression was significantly downregulated in the tumor tissue of patients and in CCA cell lines. Our results provided evidence that miR-124 induces apoptotic cell death and triggers the autophagic flux in CCA cells. EZH2 and STAT3 were identified as direct targets of miR-124. The effect of miR-124 on EZH2 expression in CCA cells was evaluated using cell transfection, xenotransplantation into nude mice and a luciferase reporter assay. Silencing of EZH2 restored the effects of miR-124, whereas overexpression of EZH2 abrogated the effects of miR-124. Silencing of Beclin1 or ATG5 abrogated the effects of miR-124 or siEZH2. In vivo, overexpression of miR-124 dramatically induced autophagy-related cell death and suppressed tumorigenicity. Taken together, our findings indicated that downregulation of miR-124 expression was associated with disease progression in human CCA and we revealed that miR-124 exerts a tumor suppressive function in CCA by inducing autophagy-related cell death via direct targeting of the EZH2-STAT3 signaling axis.

Ma J ，Weng L ，Wang Z ，Jia Y ，Liu B ，Wu S ，Cao Y ，Sun X ，Yin X ，Shang M ，Mao A ... -  《-》