Molecular Mechanism Of MiR-33b To Inhibit The Development Of Hepatocellular Carcinoma Through Targeting Fli-1

Background and ObjectiveHepatocellular carcinoma(HCC)is the most common digestive cancer and the third most common cancer-caused death in the worldwild.Despite significant therapeutic improvement in clinical surgical resection,chemoradiotherapy and targeted therapy,long-term therapeutic efficacy of HCC patients remains poor,mainly because of regional invasion and frequent metastasis.However,potential mechanisms of HCC development are not clearly understood,thus it is urgent to identify key biochemical pathways in metastasis to provide new therapeutic targets for HCC.The occurrence and development of hepatocellular carcinoma is a complicated process which is regulated by multiple genes and multiple cell signaling pathways.miRNA has been reported to play important roles in HCC pathogenesis.Many research reports confirmed miR-33b was closely related to many diseases,especially the development of tumor,however,whether miR-33b was involved in the pathogenesis of HCC is still unclear.So we plan to measure the expression level of miR-33b and study the biological effects of miR-33b in HCC tumorigenesis,in order to provide new therapeutic targets for HCC.Part Ⅰ Expression of miR-33b in HCC tumor tissues and cellsObjectiveTo investigate the expression level of miR-33b in HCC tissues and cell lines.MethodsWe utilized quantitative real-time PCR(qRT-PCR)to analyse the expression level of miR-33b in 50 HCC tumor tissues and BEL7402,PLC/PRF/5,SMMC7721 and MHCC97H cells.ResultsCompared with normal tissues,the expression of miR-33b in HCC tumor tissues was significantly down,regulated.The expression of miR-3 3b in BEL7402,PLC/PRF/5,SMMC7721and MHCC97H cell lines was also reduced.ConclusionThe expression of miR-3 3b in HCC tumor tissues and cell lines was significantly down-regulated.Part Ⅱ Validation of targets of miR-33b and identification of the biological characteristics of Fli-1 in HCCObjectiveTo predict and validate the targets of miR-33b and to identify the biological effects of miR-3 3b in HCC tumorigenesis.MethodsWe used bioinformatics software to predict the targets of miR-33b.PCR was used to amplify the 3 ’UTR sequence of the targets,which containing the binding site.We conducted pmirGLO-Fli-1 3’UTR carrier wild type(wt).Then we validated it by dual luciferase report gene experiment,qRT-PCR and western blot.We measured the expression level of Fli-1 in HCC tissues and cell lines by qRT-PCR and western blot;We next examined the biological role of Fli-1 by colony formation,wound healing assay and transwell assay.ResultsFli-1 was predicted as a potential target of miR-33b;dual luciferase report gene experimental results showed that co-transfection of pmirGLO-Fli-1 ’UTR(wt)and miR-33b effectively inhibited the luciferase activity,while after the mutation of the binding sites,the luciferase activity showed no obvious difference compared with negative control(NC);qRT-PCR results showed that,compared with NC group,Fli-1 mRNA expression had no obvious change;Subsequent western blot experiments found that Fli-1 expression significantly reduced after transfection with miR-33b mimic.Fli-1 was up-regulated in HCC tumor tissues and cell lines,it was closely correlated with distant metastasis.MiR-33b promoted the proliferation and metastatic ability of HCC tumor cells.ConclusionFli-1 was a direct target of miR-33b,it acted as an oncogene in HCC tumorigenesis.Part Ⅲ Biological Function and molecular mechanism of miR-33b in HCC tumorigenesisObjectiveTo ascertain the role of miR-33b in HCC tumorigenesis;To explore the molecular mechanisms of miR-33b in HCC.MethodsWe use transfection of miR-33b mimic/and pcDNA-3.1-Fli-1 to increase miR-33b and Fli-1 level respectively.We measured the tumor cell proliferation,metastasis and invasion by CCK-8 assay,wound healing assay and transwell assay.Finally,we observed MMP2 expression change by western blot.ResultsmiR-33b inhibited the proliferation and metastasis of tumor cells,We subsequently found that this inhibiting function of miR-33b could be counteracted by over-expression of Fli-1.MMP2 protein level was dramatically reduced after transfection with miR-33b mimic,while over-expression of Fli-1 increased MMP2 protein level.ConclusionmiR-33b plays a critical role in HCC tumorigenesis partly by targeting Fli-1,thus reducing MMP2 expression.