HBV Inhibit Expression Of CFLAR By Regulating MIR-499A In Hepatoma Cells

ObjectiveTo investigate the association of HBV, miR-499a and CFLAR in hepatoma cells.Methods1. To ascertain whether HBV could influence expression of miR-499a, its expressions in HepG2 and HepG2.2.15 cells(expressing HBV) were measured with qRT-PCR.In addition, miR-499a expressions were detected HepG2 cells infected with recombinant adenoviruses expressing HBV (Ad-HB V) or GFP (Ad-GFP).2. CFLAR 3’UTR luciferase reporter vectors were constructed to determine whether miR-499a was capable of targeting CFLAR. Luciferase activity assay was performed 48h after cotransfection of miR-499a with wild-type 3’UTR CFLAR or mutated 3’UTR CFLAR luciferase reporter vector.2. MiR-499a expression plasmid was generated and the expression was verified by using qRT-PCR. Predicted target gene were selected through Targetscan and miRDB. QRT-PCR and Western blot analysis demonstrated that CFLAR expressions were inhibited after miR-499a was overexpressed in SMMC7721 cells.QRT-PCR and Western blot analysis revealed that CFLAR expression were up-regulated when miR-499a was inhibited in SMMC7721 cells.4. To determine whether HBV could regulate the expression of CFLAR by using qRT-PCR and Western blot.Results1. MiR-499a was up-regulated in HepG2.2.15 cells compared to HepG2.2.15 cells. MiR-499a was obviously higher in Ad-HBV-HepG2 cells than that in Ad-GFP-HepG2 cells.2. MiR-499a regulated CFLAR expression by targeting its 3’UTR.3. Mir-499a was capable of targeting CFLAR and down-regulate CFLAR expression in SMMC7721 cells and CFLAR had a higher expression when miR-499a was inhibited.4. HBV up-regulate CFLAR expression in mRNA and protein levels.ConclusionCFLAR was one target gene of miR-499a and HBV inhibited expression of CFLAR by up-regulating miR-499a.