In this study, we examined the effects of RhoC expression on the growth and apoptosis of human hepatocellular carcinoma cells (HCCs) in vitro in order to gain more understanding of its potential as a therapeutic target gene. We knocked down the endogenous expression levels of RhoC in human HCCs, BEL-7402, using siRNA and ectopically expressed RhoC in untransformed hepatocytes, HL7702. Stable cell lines were established, and cell growth was examined by MTT and colony formation assays, cell proliferation examined by silver nitrate staining of AgNORs, and cell cycle distribution examined by flow cytometry. RT-PCR analysis was performed to determine the mRNA expression levels of RhoC and cell cycle-related genes. Finally, the effect of RhoC expression on apoptosis was also examined by flow cytometry, agarose gel electrophoresis of fragmented DNA, Wright staining, and RT-PCR analysis for genes regulating apoptosis. Compared to the parental and control siRNA (siCtrl)-transfected BEL-7402 cells, the siRhoC-transfected cells exhibited significantly reduced cell growth, cell proliferation, percentage of cells in the S-G2/M phase, and expression of Cyclin D1, CDK4, and Bcl2. Knockdown of RhoC expression in BEL-7402 cells also significantly increased the percentage of cells in the G0/G1 phase, cellular apoptosis, and expression of p21, p16, and Bax. Furthermore, ectopic expression of RhoC in HL7702 cells led to a significant increase in cell growth compared to parental or siCtrl-transfected cells. These data suggest that RhoC is a key regulator of cell growth and apoptosis in HCC cells, making it a potential target for gene therapy.
[1]Jilin Univ, Hosp 1, Dept Gen Surg, Changchun 130021, Jilin Province, Peoples R China.
[2]Jilin Univ, Sch Basic Med Sci, Dept Immunol, Changchun 130021, Jilin Province, Peoples R China.
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