Effects Of Rpb5-mediating Protein (RMP) On Cell Proliferation Of Hepatocellular Carcinoma Cells

Objective:The aim of the test is to investigate the effects of RMP on cell proliferation and apotosis of human hepatocellular carcinoma cells and to explore mechanisms both in vitro and in vivo.Methods:Establish the RMP silenced and RMP over-expression transient expression cell lines transfected with lipofectamine 2000. RT-PCR was used to detect the expression of RMP on cell lines. The cellular proliferation and adhesion capacity was detected by MTT assay. Wound healing test was used to observe the cell migration ability. Flow cytometry was applied to measure the effects of RMP on cell cycle and apoptosis. The expressions of related genes of cell apoptosis were detected by Western-Blot. And we also establish model of liver tumor in nude mice. Athymic nude mice bearing liver tumors were intratumorally injected with liposome-plasmid complexes. Tumor growth was recorded. All nude mice were killed at the end of the experiment to observe the growth of xenografts. Morphology of the transplation human hepatoma cancer tumors in nude mice was detected by HE staining. The expressions of RMP, APF, Bcl-2, Bax and P53 proteins in tumor tissues were checked by immunohistochemistry.Results:In vitro: Results of RT-PCR showed that RMP interference and RMP over-expression plasmid could expression in HCC/liver cells. Higher expression of RMP in tumor cells than normal cell. MTT assay showed that RMP promoted the growth of hepatoma and hepatic cells. The adhesion capability was decreased but cell migration was enhanced in RMP over-expression group. Flow cytometry analyses showed that RMP depletion induced G2/M arrest and resulted higher numbers of apoptotic in hepatoma cells but there was no significant changes in normal liver cells. RMP over-expression decreased apoptosis rate in hepatoma and hepatic cells. Western-blot showed that RMP over-expression group significantly up-regulated the expression of Bcl-2 and down-regulated the expression of Bax and Caspase3. In vivo:The model of xenograft tumor in nude mice were successfully constructed. Compared with controls the tumor volumes and carcinoma weights in RMP interference group were significantly reduced and induced apoptosis by up-regulating the level of Bax,P53 proteins expression and down- regulating AFP,Bcl-2 expression.Conclusion:The results of experiments indicated that RMP could express widely in human cell lines, and its expression was higher in tumor cells. Interference of RMP inhibited hepatoma cell proliferation and induced cell apoptosis in vitro and in vivo. The results indicated that RMP may be a promising target in gene therapy of the liver cancer. The study we found that RMP over-expression could promote HL-7702 cell proliferation, enhanced cell migration ability and decreased cell adhesion. So our supposition is RMP might cause normal cells to become cancerous tumoe cell. The mechanism of RMP inhibit cell apoptosis may implement through up-regulated Bcl-2 while down- regulated Bax and P53.