Expression Of MicroRNA-29a And Suppression Cell Proliferation And Migration In Hepatocellular Carcinoma

Hepatocellular carcinoma(HCC)is one kind of cancer sourced in liver and the fourth most common cause of cancer-related death worldwide.Chronic hepatitis B and C virus infections,exposure to aflatoxin and other contribute to development of HCC.HCC has the characteristics of onset hidden,difficulty of effectively diagnosing,high malignant degree,poor prognosis and rapid progression.Only about 10% to 20% of patients with HCC are currently eligible for surgical treatment.Therefore,it is urgent to develop novel and effective markers for diagnosis,prognosis,and treatment for patients with this disease.miRNAs are a class of short,evolutionarily conserved,endogenous,singlestranded,non-coding RNA molecules that are approximately 21-24 nucleotides in length which regulate gene expression at the post-transcriptional level.miRNAs play a major role in many fundamentally important biological processes,such as cell cycle,differentiation,development,and metabolism.miRNAs can work as tumor suppressors and oncogenes.Such dual function prompt many researchers focus on miRNAs and cancers.Emerging evidence indicates that some tumor-specific miRNAs are widely deregulated or upregulated in HCC and closely associated with the occurrence and development of HCC.Recent studies have shown that there are 17 miRNAs with high expression in HCC(including miR-181,miR-182,miR-183,miR-224,mi R-21,mi R-222,mi R-96,miR-9,mi R-216,miR-324-5p,miR-186,miR-155,mi R-301,mi R-221,miR-151,miR-374 and mi R-106b).Additionally,there are 9 mi RNAs with low expression in HCC(miR-139,mi R-214,miR-199a-3p,miR-199a-5p,mi R-125 a,miR-195,miR-34 a,miR-200 a and miR-122a).microRNA-29a(miR-29a),currently one of the most interesting miRNAs families in humans,has been shown to be silenced or down-regulated in many different types of cancer,such as prostate cancer,renal cell carcinoma,gastric cancer and pancreatic cancer;which has also been shown to mediate either tumor suppressive or oncogenic functions in distinct malignancies.However,the roles of miR-29 a in HCC remain to be clarified.In the present work,we found that mi R-29 a was downregulated in HCC cell lines and primary tumor samples,and miR-29 a was further identified to be a tumor suppressor,as restoration of mi R-29 a expression in HCC cell lines could reduce cell proliferation and suppressed cell migration and tumor formation.Part One The expression level of miR-29 a in HCC cell and HCC samples and prognosis of HCC paientFirstly,we used real-time PCR to detect the expression of miR-29 a in HCC cell and 62 cases of HCC patients in clinical specimens respectively.At the same time,the expression level of miR-29 a in patients with HCC and survival curve were analyzed.The results demonstrate that the down-regulation of miR-29 a expression in hepatocellular carcinoma cell HepG2,Huh7,MHCC7721,and Hep3 B compared with normal hepatic cells Chang liver,LO2.Down-regulation of miR-29 a expression was highly significant in 62 HCC patients tissues compared with adjacent non-tumorous liver tissues,miR-29 a expression level is closely related with the overall survival of HCC patients.It shows that miR-29 a is involved in the occurrence and development of HCC.Part Two The biologic function of miR-29 a in HepG2 cellsThe exploration of biologic functions of mi RNAs provided new means to study the interaction of main tumors in HCC.Therefore,it is necessary to elucidate the biologic function of miR-29 a in hepatocellular carcinoma.firstly,we transfected miR-29 a mimics into HepG2 cells.Then we observed effect of miR-29 a on Hep G2 cells by wound healing assay,soft agar colony formation assay,CCK8 assay,apoptosis assay,transwell migration assay,and nude mouse engrafts,respectively.The results suggested that overexpression of miR-29 a significantly inhibited cell proliferation and migration in HepG2 cells.miR-29 a had no effect on cell apoptosis in HepG2 cells,but it caused the G0/G1 phase cell cycle arrest by Flow Cytometry.mi R-29 a inhibited the migration of HepG2 cells in wound healing assay and transwell migration assay.Moreover,overexpression of miR-29 a obviously suppressed tumor growth in a nude mouse model.Part Three The prediction and preliminary identification of target gene of miR-29aThe results showed that two parts above miR-29 a was involved in the occurrence and development of HCC.In order to study the molecule mechanism of miR-29 a in HCC further,preliminary identification were predicted for its target gene.We found the target genes of miR-29 a with the application of bioinformatics tools and selected out the potential target genes of miR-29 a.Western blot assessed potential target genes expression,shown that HepG2 cells transfected with miR-29 a mimics reduced expression of target genes Insulin-like growth factor 1 receptor(IGF1R).In addition,we also proved that inhibiting the expression of IGF1 R also inhibited invasion of HepG2 cells with the help of siRNA technique.Interrelated analyses conclude that IGF1 R was the target gene of miR-29 a,and it played part in inhibiting the invasion of HepG2 cells by regulating the expression level of IGF1 R.In summary,our research results suggest that the involvement of miR-29 a in the proliferation and migration of HCC partly by down-regulating IGF1 R expression.Meanwhile it may be useful as a novel biomarker for diagnosis,prognosis,and treatment for patients with HCC.