MicroRNA-191 Regulates Intrahepatic Cholangiocarcinoma Via Epigenetic Mechanism

Background and Aims: Intrahepatic cholangiocarcinoma(ICC)remains difficult to cure because its molecular mechanisms are complex and not comprehensively understood.Mounting evidence suggests that microRNAs play a crucial role in ICC,but the roles of specific miRNAs involvement in the pathogenesis of this disease remain elusive.We performed a high-throughput microarray assay to detect ICC-specific miRNAs,followed by functional validation of the results.Methods: ICC miRNA profiles were generated using a high-throughput microarray assay in three pairs of ICC and normal duct bile.The significantly altered microRNAs were then validated using q RT-PCR in the remaining eighteen pairs of ICC and normal duct bile.Receiver-operating characteristic analysis was used to assess the prognosis accuracy of ICC patients.Kaplan-Meier and log-rank regression was used to analyze the relationship between miR-191 and patients survival of ICC.In vitro experiments,we study the function of miR-191 in cholangiocarcinoma cell proliferation,apoptosis,invasion and meastasis.Using bioinformatics software to predict the downstream target genes of miR-191.We used luciferase reporter gene,western blot and immunohistochemistry to identify the target gene,and its signaling pathway,clarifying miR-191 regulating downstream signaling pathways in the cholangiocarcinoma.In vivo tumor formation in nude mice and in situ intrahepatic cholangiocarcinoma model to explore the role of miR-191 promote tumor formation and invasion and metastasis.Results: We idenfified 72 significantly difference miRNAs included 23 upregulation and 49 downregulation from microarray analysis using p<0.05 and fold change?2 as cutoff value.Using Taqman q RCR in 18 pairs of ICC and normal dile duct tissues to verify the significantly different expression of microRNAs,miR-191 was found most meaningful.Kaplan-Meier revealed that miR-191 is closely related to the survival of patients with ICC.ROC analysis found that miR-191 is high sensitivity and specificity to predict the prognosis of postoperative cholangiocarcinoma;combination with miR-191 and TNM stage can better predict prognosis of ICC patients.miR-191 expression was significantly increased in ICC samples versus adjacent non-tumor tissues(P<0.001).miR-191 was an independent prognostic factor for overall survival(HR 3.742,95% CI 2.080 to 6.733;p<0.001)and disease-free survival(HR 2.331,95%CI 1.346 to 4.037;p= 0.003)of ICC patients.Ectopic expression of miR-191 mimics in ICC cell lines promoted proliferation,invasion and migration.Subcutaneous tumor formation in vivo and in situ model of ICC to reveal miR-191 overexpression promote tumor formation and ICC invasion and metastasis.Bioinformatics software prediction TET1 is a target gene of miR-191,and luciferase reporter prove miR-191 can be combined with TET1 3' UTR region.Western blot and IHC revealed that miR-191 directly interacted with its new target gene TET1 and negatively regulated the expression of TET1.Restoration of TET1 attenuated the miR-191-induced promotion of cholangiocarcinoma cell proliferation,invasion and migration.Furthere use of western blot,Co IP,and Ch IP identified that TET1 binding to the transcription start site(TSS)of p53 positively promoted p53 expression due to the demethylation of the p53 promotor region and the sites in proximity to the TSS region.Conclusions: First,our study demonstrated that,as an important oncogenic factor,miR-191 is up-regulated in a subset of human ICC tumors and cholangiocarcinoma cell lines and promoted cholangiocarcinoma proliferation,invasion and metastasis in vitro and in vivo.Second,we found that hypomethylation of miR-191 in ICC tissues and cell lines cause an increase in the expression of miR-191 in ICC tissues and cholangiocarcinoma cells.Third,we found that miR-191 can serve as a biomarker that can be useful for predicting the prognosis of ICC patients.Fourth,we demonstrated that TET1 is a miR-191 target gene.Finally,we demonstrated that TET1 as a tumor suppressor gene in ICC can bind to the TSS of the p53 to positively regulate p53 expression due to its demethylation.