Up-regulation Of MiR-499a By Hepatitis B Virus Regulates Hepatocellular Carcinogenesis Via Targeting MAPK6

ObjectiveTo reveal the molecular mechanisms of HBV affect hepatocellularcarcinogenesis by regulating miRNA and its target genes.Methods1. Real-time PCR were used to compare the expression of miR-499ain HepG2, HepG2.2.15cells and HBV adenovirus infected HepG2cells.Also the expressions of miR-499a were analyzed in SMMC7721cellstransfected with plasmids of four major protein of HBV. Luciferase reportconstruct of miR-499a promoter were generated. SMMC7721cells werecotransfected with pRL-TK plasmid, miR-499a promoter report construct,and HBV expression plasmid. Relative activity of miR-499a promoter wasdetected by dual-luciferase report assay system.2. The expression plasmid and the stable cell line of miR-499a wereconstructed. The MTS assay and clone formation assay were used toanalyze cell proliferation. Wound healing assay and transwell assay wereperformed to text cell migration. Analysis of tumorigenicity in nude mice used to explore cell proliferation by using miR-499a stable expression cellline.3. The MAPK63’UTR luciferase report plasmid and its mutantplasmid were constructed and co-transfected with miR-499a expressionplasmid into SMMC7721cells. Relative activity of luciferase was detectedby dual-luciferase report assay system. Real-time PCR and Western blotwere performed to detect the differential expression of MAPK6inSMMC7721cells transfected with pTarget-miR-499a or miR-499ainhibitor.4. After the MAPK6expression plasmid, si-MAPK6, pCH9/3091andpTarget-miR-499a transfected into SMMC7721cells, seperatetly, MTS andclone formation assay were carried out to analyze cell proliferation.Results1. HBV, especially HBx and HBs could increase miR-499a expression.HBx and HBs can increase the miR-499a promoter activity in SMMC7721cells.2. Both miR-499a and HBV could promote the proliferation andmigration of HCC cells.3. MAPK6was the target gene of miR-499a. its expression could beinhibited by miR-499a.4. MAPK6could inhibit the proliferation of liver cancer cell and thecell proliferation promoted by miR-499a may be regulated by MAPK6. ConclusionHBV could affect the hepatocellular carcinogenesis by up-regulatingthe expression of miR-499a via targeting MAPK6.