MicroRNA-16 Mediates The Mechanism Of Yap1 In The Occurrence And Development Of Cholangiocarcinoma

Screening and validation of microRNA expression in cholangiocarcinomaObjective: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 20 pairs of cholangiocarcinoma tumor specimens and adjacent normal biliary tissues using high-throughput microRNA microarrays.The expression of selected miRNAs was analyzed in CCA tissues and CCA cell lines by qRT-PCR.Results:There were a total of 10 differentially expressing microRNAs,in which 6 were upregulated,and 4 downregulated;The expression of miR-16 was 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 obviously different.MiR-16 is low-expressing in both cholangiocarcinoma tissues and cholangiocarcinoma cells,which may serve as an object for further study.Part Two Effect of miR-16 on proliferation and invasion of cholangiocarcinoma CellsBackground:Cholangiocarcinomas(CCA)are aggressive cancers,with a high mortality and a poor survival rate.MicroRNAs are small non-coding RNAs that play important roles in tumor initiation and progression in a variety of cancers,including cholangiocarcinomas.In this study,we aimed to investigate the effect and mechanism of miR-16 in CCAs.Methods:miR-16 profiles were analyzed in 45 couples of primary CCAs and corresponding normal bile duct specimens using Real-Time PCR.Expressions of miR-16 was further confirmed in 4 kinds of human CCA cell lines.The effect and mechanism of miR-16 in CCA cell proliferation,migration and apoptosis were studied in vitro study by cell transfecting through Mimics and Inhibitors.Results:miR-16 was significantly downregulated in human CCA tissues compared with their corresponding adjacent normal bile duct tissues.In vitro functional study,overexpression of miR-16 dramatically suppressed CCA cells proliferation and migration and promoted cells apoptosis.Furthermore,knock-down of miR-16 promoted CCA cells proliferation and migration and inhibited cells apoptosis.Conclusion:miR-16 inhibited CCA cells growth and migration and promoted apoptosis.miR-16 could suppress tumor in CCA and may possess potential prognostic and therapeutic value for CCA patients.Part Three The screening and verification of miR-16 targetsObjective:To screen and validate the potential targets of miR-16,and then fur ther clarify the biological function of miR-16.Methods:Predicted and verified the potential target genes of miR-16 by Target Scan,PicTar and miRanda software;established the dual luciferase report syst em,then validated the direct combination of miR-16 to target genes,and clarif ied their binding sites;Detected the expression of Yapl in CCA cell lins what had been overexpression or kockdown by miR-16;detected the effect of CCA cells on proliferation,migration and invasion ability by regulating the expressi on of miR-16 and Yapl at the same time by rescue experiment.validated the f unctional consistency of miR-16 and Yapl.Results:Yapl was one of the potential target genes of miR-16;miR-16 could combine with Yapp 1 at two sites of 3’UTR to regulate Yap1 expression;regula tion of miR-16 could affect the protein level of Yapl,which affects the prolife ration and invasion of CCA cells.Conclusions:Part Four Functional validation of miR-16 in vivo and its clinical significanceObjective:To validate the inhibition of miR-16 on cholangiocarcinoma proliferation and metastasis in vivo;Analyze the correlation of miR-16 and Yapl in clinical samples;Analyze the relationship between miR-16 and clinical pathological features of cholangiocarcinoma patients.Evaluate the prognostic significance of miR-16 expression in cholangiocarcinoma patients.Methods:Tumorigenicity assays in nude mice and cholangiocarcinoma lung metastasis model were used to analyze the effect of miR-16 on cholangiocarcinoma proliferation and metastasis in vivo;The pathological information of 45 cholangiocarcinoma patients in the First Affiliated Hospital of Nanjing Medical University was collected from April 2008 to June 2012,the relationship between miR-16 and the clinical pathology was analyzed.In addition,to analyze the prognostic significance of miR-16 expression,Kaplan-Meier survival curve was carried out in a set of 70 CCA patients with integral follow-up data.Results:MiR-16 could inhibit tumor growth and metastasis in mice model;Statistical results showed that miR-16 expression was remarkably associated with tumor size,metastasis,and AJCC TNM stage in CCA patients.Patients with low expression of miR-16 exhibited a worse disease-free survival(DFS)than those with high expression of miR-16.Likewise,a statistically significant association between low miR-16 expression and short overall survival(OS)was also demonstrated in CCA patients.Conclusions:MiR-16 could inhibit cholangiocarcinoma growth and metastasis in vivo through directly targeting Yapl in vivo.MiR-16 might be a promising therapeutic target or prognosis biomarker for CCA.