MiR-133a And MiR-326Co-contribute To HepG2Drug Sensitivity By Targeting ABCC1Directly

Objective: To study the effects of miR-133a and miR-326on regulation of ABCC1gene in human hepatocellular carcinoma (HCC) HepG2cells, and the effects ofHepG2cells to5-FU、ADM and DDP.Methods: Computer programs were taken to predict the miRNA binding sites ofABCC1gene3′UTR. These binding sites were confirmed by dual luciferase reporterassay. HCC cell line (HepG2) was cultured in vitro. miRNA mimics and negativecontrol were transfected into HepG2cells. Transfection efficiency and ABCC1mRNA level were tested by qRT-PCR. ABCC1protein expression was detected bywestern blot analysis in HepG2cells after transfection with miR-133a or miR-326mimics. The effects of the two miRNAs on5-FU、ADM and DDP sensitivity toHepG2cells were determined by MTT assay.Results: Computational programs predict one binding site for miR-133a and twobinding sites for miR-326in3′UTR of human ABCC1.QRT-PCR showed that miRNA expression in HepG2cells transfected withmiR-133a or miR-326mimics was significantly up-regulated in comparison withcontrol cells and untreated cells. The datas also showed that both of miR-133a and miR-326decreased the ABCC1mRNA level compared with the NC.Western blot analysis showed that enhanced expression of miR-133a or miR-326significantly decreased the amount of ABCC1protein, compared with NC and anduntreated cells.MTT cell proliferation assay showed that the transfection of miR-133a ormiR-326mimics significantly sensitize HepG2cells to5-FU、ADM and DDP,compared with NC and and untreated cells. Luciferase reporter assays showed that the luciferase activity of cells transfectedwith miR-133a (or miR-326) mimics+wild type ABCC13′UTR was inhibited bytransfection of miR-133a (or miR-326) relative to cells transfecting with NC+wildtype ABCC13′UTR. Whie, compared with NC+mutant type ABCC13′UTR, theluciferase activity of cells transfected with miR-133a (or miR-326) mimics+mutanttype ABCC13′UTR had no significantly changed.Conclusion: MiR-133a and miR-326down-regulated the ABCC1expression ofHepG2cells; MiR-133a and miR-326increased the sensitivity of HepG2cells to5-FU、ADM and DDP; MiR-133a and miR-326mediate hepatocellular carcinoma cell lineHepG2drug sensitivity by targeting ABCC1directly.