Up-regulated MicroRNA-181a Induces Carcinogenesis In Hepatitis B Virus-related Hepatocellular Carcinoma By Targeting E2F5

ObjectiveTo further understand the molecular mechanisms of hepatitis B virus(HBV) regulated miR-181a expression in Hepatoma cells and the effect ofmiR-181a up-regulation on hepatocellular carcinoma develpment.Methods1. qRT-PCR was performed to compare miR-181a expression in HepG2and HepG2.2.15cells, HepG2cells infected with adenovirus expressionHBV (Ad-HBV-HepG2) and HepG2cells infected with adenovirusexpression GFP (Ad-GFP-HepG2).2. The miR-181a promoter luciferase reporter plasmid was transfectedinto HepG2, HepG2.2.15cells, Ad-HBV-HepG2and Ad-GFP-HepG2cells.Then luciferase reporter assay was used to measure the miR-181a promoteractivity.3. miR-181a expression plasmid and miR-181a inhibitor weretransfected into SMMC7721cells. And then MTS was used to test cellproliferation of high-expression and low-expression cells. Meanwhile, MTS was also used to test cell growth of miR-181a stable expression cells(p-miR-181a) and its control (p-control).4. TargetScan and MiRanda were used to predict potential target genesof miR-181a. Luciferase reporter assay was used to screen out the realtarget genes of miR-181a.5. qRT-PCR and Western Blot were used to exam E2F5expression inmiR-181a high-expression and low-expression SMMC7721cells.6. MTS and Colony formation assay were used to analyze the cellproliferation of SMMC7721cells transfected with E2F5siRNA.7. qRT-PCR and Western Blot were used to exam the effect of HBV onE2F5expression. MTS was performed to exam the influence of HBV oncell proliferation. MTS and colony formation assay were used to analyzethe cell proliferation of HepG2.2.15cells transfected with miR-181ainhibitor.8. Tumor growth assay was used to investigate the effect of miR-181aand E2F5on the tumor growth in nude mouse.Results1. HBV up-regulated the expression of miR-181a by enhancing itspromoter activity.2. MiR-181a overexpression induced cell proliferation in HCC cells.3. E2F5was a target gene of miR-181a.4. E2F5was involved in miR-181a inducing promotion of HCC cells proliferation.5. HBV down-regulated the expression of E2F5.6. MiR-181a promoted tumor growth of SMMC7721cells in nude mice.ConclusionThese results suggested that up-regulation of miR-181a by HBV inhepatoma cells may contribute to the progression of HCC possibly bytargeting E2F5, suggesting miR-181a plays important role in HCCdevelopment.