Effects Of GABA-GABRQ On Proliferation Of HepG-2 Cell Line

Objective: To study the effects ofγ-aminobutyric acid(GABA) on proliferation and malignant phenotypes of hepatocellular carcinoma cell line HepG-2, and to investigate the effects of RNA interference-induced gene silence of GABRQ on proliferation of HepG-2 cells.Methods: HepG-2 cells were cultured by routine method,and then treated with different concentrations of GABA.The proliferation of HepG-2 cells were measured by MTT,mean doubling time and cell cycles by flow cytometry. The malignant phenotypes were investigated by soft agar colony formation assay and tumor formation in Balb/c nude mice; Hepatoclullar carcinoma cell line HepG-2 was transfected by GABRQ small interfering RNA(siRNA), and the level of mRNA of GABRQ was determined by Semiquantitative PCR assay.The growth and the migration ability was examined using MTT, FCS, colony formation and wound healing assay.Results: Compared with control group, GABA efficiently stimulated the proliferation of HepG-2 cells in a dose-dependent manner and affected the distribution of cell cycles of HepG-2 cells. The mean doubling time of control group and different concentration of GABA-treated groups were 5.13,2.54,2.11,1.62,1.38 and 4.35 days respectively.The colony formation rates were 3.8%, 6.2%, 8.1%, 8.9%, 9.1%, 4.6% respectively. Tumorigenicity test showed that the average weights of tumors were 1.382g and 0.285g respectively.The difference between the control group and the GABA-treated was significant (P<0.01); The mRNA level of HepG-2 cells transfected with si-mock, si-lGABRQ,si-2GABRQ,si-3GABRQ,si-4GABRQ were detected by RT-PCR, the results indicated that GABRQ mRNA can't be detected In HepG-2/si-1GABRQ group; In vitro experiment showed that the growth and the migration ability of HepG-2 cells transfected with si-1GABRQ were reduced significantly. The growth ability of HepG-2 cells transfected with si-mock was up-regulated after treated with GABA, but such phenomenon did not occur in si-1-transfected group.Conclusion: GABA can enhance proliferation and malignant phenotypes of HepG-2 cells; GABRQ may play an important role in the growth and metastasis of human hepatoclullar carcinoma cells, and RNAi-induced GABRQ down-regulation can inhibit cell proliferation ability. The growth ability of HepG-2 cells transfected with si-mock was up-regulated after treated with GABA,but such phenomenon did not occur in si-1-transfected group.