Effect Of GP73 Gene Silence Induced Autophagy On Viability And Apoptosis In Hep G2 Cell Line

Background: Our previous study showed there was a relatively high level of autophagy existed after GP73 gene silence in Hep G2 cell. And as a molecular biological phenomenon, autophay has already been demonstrated to play important roles to a series of actions in kinds of tumor cells.Objective: The aim of this study was to further compare the level of GP73 gene silence induced autophagy with control, and to explore its effect on the viability and apoptosis in Hep G2 cell line.Methods: Specific stealth RNA(siRNA) targeting human GP73 gene was designed and used to silence its expression in Hep G2 cell. The silence effect was demonstrated by reverse transcriptase polymerase chain reaction(RT-PCR) in the level of message RNA(mRNA) and enzyme-linked immuno sorbent assay(ELISA)in the level of protein. Monodansylcadaverine(MDC) and Beclin 1 were adopted to indicate the autophagy level in aspect of general morphology and qualitative analysis.Combined with autophagy inhibitor 3-methyladenine(3-MA) and inducer rapamycin,the effect of autophagy were studied by observing the changing tread of autophagy, as well as the effect on cell viability and apoptosis by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) assay and flow cytometry.Results: After GP 73 gene silence, the GP73 m RNA level and protein expression were both markedly reduced(P< 0.05). MDC staining showed that the fluorescent density was higher and the number of MDC-labeled particles was greater, and the level of autophagy specific gene of Beclin 1 was also increased in the silence group than that in the control group(P< 0.05). And MTT assay and flow cytometry confirmed that reduced GP73 protein expression inhibited cell proliferation and induced apoptosis. Both 3-MA and rapamycin significantly influenced the level of autoghagy. 3-MA inhibited the autophagy, and further affected the viability and apotosis(P< 0.05); while rapamycin did not lead to significantly changes in the mentioned aspects.Conclusions: GP73 gene silence would induce high level of autoghagy in Hep G2 cell line, and this autophagy might further influence the viability and apoptosis.