The Expression Of MicroRNA-144and Its Function In Cholangiocacinoma

Objective:To screen microRNA expression by Microarray in cholangiocarcinoma tumor and adjacent normal biliary tissues for finding research subjects.Methods:The microRNA profiles were measured in three pairs of cholangiocarcinoma tumor specimens and adjacent normal biliary tissues using Agilent microRNA microarrays. The Gene Ontology (GO) function and pathway analysis was completed according to the results of the Microarray, the microRNAs signal pathways and targets related to cholangiocarcinoma progression were predicted. The expression of selected miRNAs was analyzed in CCA tissues and CCA cell lines by qRT-PCR.Results:There were a total of28differentially expressing microRNAs, in which19were upregulated, and9downregulated; The results of GO and Pathyway analysis showed that miR-144might play an important role in cholangiocarcinoma occurrence and development. The expression of miR-144was low in both cholangiocarcinoma tissue and cholangiocarcinoma cells with statistical significance, which was consistent with the results of Microarray.Conclusions:The expression of miRNAs in cholangiocarcinoma tissues and normal bile duct tissues adjacent to carcinoma is different obviously. MiR-144is low-expressing in both cholangiocarcinoma tissues and cholangiocarcinoma cells, which may serve as an object for further study. Objective:To explore the effect of miR-144on proliferation, migration and invasion of cholangiocarcinoma cells in vitro, and investigate the potential target proteins of miR-144.Methods:MiR-144overexpressing cholangiocarcinoma cells HCCC-9810and CCLP1were first constructed by transfecting lenti-virus vector PCDH-miR-144. Proliferation of cholangiocarcinoma cells was evaluated by CCK-8assay, and the migration and invasion ability was evaluated by the scratch experiment and transwell assay. Western blot test was used to observe level change of proteins related to proliferation and invasion.Results:MiR-144was overexpressed in constructed cholangiocarcinoma cells successfully; the proliferation, migration and invasion ability of cholangiocarcinoma HCCC-9810and CCLP1cells was decreased by miR-144significantly. miR-144could also inhibit the expression of p-AKT and MMP-2in protein level.Conclusions:MiR-144inhibit the proliferation, migration and invasion ability of cholangiocarcinoma cells HCCC-9810and CCLP1, which might by decreasing the expression of p-AKT and MMP-2. Objective:To screen and validate the potential targets of miR-144, and then further clarify the biological function of miR-144.Methods:Predicted and verified the potential target genes of miR-144by miRanda, PicTar and Target Scan software; established the dual luciferase report system, then validated the direct combination of miR-144to target genes, and clarified their binding sites; detected the effect of LIS1on proliferation, migration and invasion ability of cholangiocarcinoma cells by using specific siRNA targeted LIS1; Western blot test was used to detect the influence of LIS1on signaling pathways related to proliferation, migration and invasion; validated the functional consistency of miR-144and LIS1.Results:LIS1was one of the potential target genes of miR-144; miR-144could combine with LIS1at two sites of3’UTR to regulate LIS1expression; knockdown of LIS1could reduce the protein level of p-AKT and MMP2, which affects the proliferation and invasion of cholangiocarcinoma cells.Conclusions:MiR-144decreased the expression of p-AKT and MMP2after binding to LIS1directly, which influenced the proliferation and invasion ability of cholangiocarcinoma cells eventually. Objective:To validate the inhibition of miR-144on cholangiocarcinoma proliferation and metastasis in vivo; Analyze the correlation of miR-144and LIS1in clinical samples; Analyze the relationship between miR-144and clinical pathological features of cholangiocarcinoma patients.Methods:Tumorigenicity assays in nude mice and cholangiocarcinoma liver metastasis model were used to analyze the effect of miR-144on cholangiocarcinoma proliferation and metastasis in vivo; The pathological information of53cholangiocarcinoma patients in Tongji hospital was collected from March2008to September2012, the expression of miR-144and LIS1was detected, then analyzed the correlation; immunohistochemistryl was used to detect the expression of LIS1, AKT, p-AKT, and MMP2in clinical specimens; the relationship between miR-144and the clinical pathology was analyzed.Results:MiR-144could inhibit tumor growth and metastasis in mice model; there was no correlation between miR-144and LIS1in clinical samples; miR-144was negatively related with LIS1, p-AKT and MMP2expression; there existed a correlation in miR-144expression, lymph node metastasis and pathologic differentiation degree of the tumor.Conclusions:MiR-144regulate the expression of p-AKT and MMP2by targeting LIS1, thus inhibit cholangiocarcinoma growth and metastasis in vivo; miR-144is negatively related to the lymph node metastasis and pathologic differentiation degree, which is likely to be a new target for diagnosis and treatment of cholangiocarcinoma.