Protein Tyrosine Phosphatase-PEST Enhance Proliferation Of HepG2 Cell

Objective:A protein tyrosine phosphatase-PEST (PTP-PEST) stable over-expressed HepG2 cell line was established by gene transfection. We detected the expression of PTP-PEST related gene by RT-PCR,real-time quantitative PCR and western blot. Then cell proliferation was measured by MTT. Base on the experimental data, to investigate the function of PTP-PEST in hepatocellular carcinoma and the signal transduction PTP-PEST might be involved in.Methods:1. PCR was done to amplify the cDNA sequences of PTP-PEST from prepared cDNA and the product was subcloned into pcDNA3.1 mammalian expression vector, named pcDNA3.1/PTP-PEST.2. PTP-PEST mRNA and PTP-PEST protein were analyzed by RT-PCR, real-time quantitative PCR and western blot. The stable over-expression PTP-PEST HepG2 cell line was obtained.3. Using real-time PCR, western blot and cell proliferation-MTT, then the effect of PTP-PEST in HepG2 cells proliferation, and changes of related gene were investigated in vitro.Results:1. HepG2 cells were stably transfected with recombinant plasmids and empty vectors respectively. A over-expression cell line was screen by RT-PCR, real-time quantitative PCR and western blot.2. The effects of PTP-PEST over-expression in HepG2 cell:â‘ PTP-PEST increase the cell proliferation;â‘¡PTP-PEST promote Ras, Grb2 and PSTPIP expression in HepG2 at gene level, then Grb2 protein was confirmed increase by western blot.Conclusion:1. A PTP-PEST stable over-expressed HepG2 cell line was successfully constructed. 2. PTP-PEST over-expression increase growth of HepG2 cell in vitro, up-regulated Grb2, Ras and PSTPIP at gene level, and promoted Grb2 protein expression.3. Base on the function of Grb2 which is on the upstream in Ras signal construction, we speculated that the regulation of HepG2 growth by PTP-PEST was via to Ras signal construction.