The Effect Of MiR-376a In The Pathogenesis Of Hepatocellular Carcinoma

MicroRNAs are endogenous non-coding RNAs of19-25nucleotides in length that act asposttranscriptional regulators by binding to the3′UTR of target mRNAs. Gradually, it be-came clear that dysregulation of miRNAs is involved in cancer development. Hepatocellularcarcinoma (HCC) is one of the most malignant tumor with high incidence and mortality, thesurvival rate within5years is less than40%. Therefore, elucidation the molecular mecha-nism of HCC development will improve the treatment and prognosis of HCC. In the recentyears, several studies revealed some deregulated miRNAin HCC and found many miRNAsplay an important role in the development of HCC of microRNA, such as miR-221, miR-222,miR-21, miR-122. Furthermore, many microRNA are related to tumor differentiation, tumormetastasis, prognosis and so on.Liver have extremely strong ability of adjusting their growth and size, the liver after resec-tion is in rapid proliferation state and cause a series of liver regeneration factors increased,such as: liver regeneration factor (HGF hepatocyte growth factor). On one hand, thesefactors make liver regenerate accurately; on the other hand, they have potential impacts onthe environment of tumor cells, leading to a recurrence of HCC. So many of the liver rege-neration factors play a similar role in the development of hepatocellular carcinoma.Some studies have strongly indicated that many factors involved in liver regeneration arealso implicated in the growth of occult tumors, and HCC pre-stage shares some resemblancewith liver regeneration in gene expression pattern. In a preliminary study on microRNAprofiling during early liver regeneration in mice, we found that mmu-miR-376b was down-regulated in regenerating livers. In view of the above analysis, we tried to explore the levelof miR-376b in HCC and its potential biological function in HCC. Hsa-miR-376b and hsa-miR-376a have strong homology in sequence, so we detected the level of miR-376a andmiR-376b in the HCC samples. The result indicated that tendency of miR-376a level de-creasing in tumor samples is more obvious, so we select miR-376a as the object to study themechanism of the HCC.Despite multiple microarray analysis show that miR-376a is downregulated in varioushuman cancers, including human intrahepatic cholangiocarcinoma and human epithelialovarian cancer, the biological roles of miR-376a in HCC still remain to be elucidated. Thus,this study sought to elucidate if and how miR-376a was involved in the development ofhepatocellular carcinoma, and this will provide basis to better illustrate the molecular mecha-nism of the development of hepatocellular carcinoma. The inquiry will also provide the basis for exploring therapeutic targets for HCC.Section I: Investigations on the miR-376a level in HCC tissues and hepatoma cell linesand its bioligocal functions.To determine the status of miR-376b and miR-376a expression in HCC, we conductedqRT-PCR analysis of41paired HCC and adjacent non-cancerous liver samples. Theseresults suggest that downregulation of miR-376a, not miR-376b, is a common event inhuman HCC tissues, which implicated that miR-376a may be involved in the development ofHCC. Therefore, we select miR-376a as the object to study the function and the clinicalsignificance. More interestingly, we found that the relative level of miR-376a in tumor/adjacent nontumor tissues was associated with the serum AFP level of patient. In otherwords, patients with higher relative level of miR-376a in tumor/adjacent nontumor tissuestended to have elevated serum AFP level.To determine the possible biological significance of miR-376a in tumorigenesis, we trans-fected HCC-derived cells (Huh7and HepG2) with the miR-376a mimics or the negative con-trol, then, we performed the MTT to detect its effect on the cell proliferation, and FACSanalysis to detect its effect on the cell apoptosis. Furthermore, nude mice bearing Huh7xenografts were used to assess the role of miR-376a mimics on tumor cell growth in vivo.These findings demonstrate that the growth inhibitory effect of miR-376a can also besustained in vitro and in vivo.Section II: Study on molecular mechanism that miR-376a exert its function.To explore the underlying molecular mechanism that miR-376a inhibited proliferation andinduced apoptosis in HCC cells, putative target genes were predicted using TargetScan (http://www.targetscan.org). More than200candidate target genes were predicted. Next, we ana-lyzed the pathway network of these candidate target genes using Molecule Annotation Sys-tem (MAS, http://bioinfo. capitalbio.com/mas) and we found PIK3R1was in the node of thecandidate gene network, namely hub gene. Hub genes efficiently disturbs the whole cellularfunction by destabilizing the network and are central to the underlying biological processes.In addition, the target genes of individual miRNAs tend to be hubs and bottlenecks in thenetworks. Moreover, the characterization of hub genes leads to a better understanding of therole of microRNAs. Based on the above reasons, the gene of PIK3R1was assayed as thetarget. To determine whether PIK3R1is regulated by miR-376a directly, we examined themRNA and protein levels of PIK3R1gene in miR-376a mimics treated Huh7and HepG2cell. QRT-PCR and western blotting analysis revealed that transfection of miR-376a caused a reduction of PIK3R1in mRNA level and protein level. Lastly, we confirmed thatmiR-376a target PIK3R1directly by luciferase activity assays. Moreover, we measured thelevels of p85α in18HCC samples. The p85α levels were obviously upregulated in HCCtissues relative to the corresponding adjacent tissues. Furthermore, these upregulation werecorrelated with the downregulation of mature miR-376a.To study the biological function of PIK3R1in HCC cells, Huh7cells were transfectedwith PIK3R1siRNA. QRT-PCR and western blotting results revealed that PIK3R1siRNAefficiently knocked down the expression of PIK3R1. Furthermore, after PIK3R1siRNAtreatment, MTT and apoptosis assays were performed. PIK3R1siRNA strongly inhibitedHuh7cell proliferation and induced apoptosis. These results confirmed that PIK3R1waspotentially involved in the miR-376a-regulated tumorgenesis and apoptosis. A previousstudy have demonstrated that miR-29activated p53by targeting p85α. Therefore, we wond-ered whether miR-376a could activate p53by targeting p85α. We detected the protein levelof p53in the miR-376a mimics, PIK3R1-siRNA or the negative control treated Huh7cells,the data showed that both the miR-376a mimics and the PIK3R1siRNA increased the levelof p53in Huh7. Theses finding indicated that miR-376a could upregulate p53expressionpossibly by targeting p85α.SectionIII: Regulation of miR-376a expressionPrevious reported that genomic and epigenetic alterations deregulate microRNA expres-sion in human epithelial ovarian cancer. In order to explore whether miR-376a expression isregulated by epigenetic alteration. DNA methylation enzyme inhibitors5-Aza-CdR andhistone acetylation enzyme inhibitors PBA were used to treat rats normal liver cells BRL-3Aand Huh7cells, the results showed that the expressioin of miR-376a increased after5-Aza-CdR and PBA were treated. The result indicated that epigenetic changes activate theexpression of miR-376a. It is reported that miRNA and epigenetics interacted each other andwere involved in cancer development. HDAC9, an epigenetics related gene, were found theputative target gene of miR-376a, then we confirmed that HDAC9was inhibited by miR-376a. Knowing that HDAC9repress MEF2activity through recruitment of multicomponentcomplexes and Mef2mediate the transcription of the miR379–410cluster. So we detectedthe expression of miR-376a after knockdown of HDAC9and MEF2by RNAi. As anticipa-ted, the result showed that the level of miR-376a increase after knockdown HDAC9whiledecrease after knockdown MEF2. These observations indicate that miR-376a is regulated byepigenetic changes, and the target gene, HDAC9, is involved in the regulation of miR-376a.