Effect On Biological Characteristics Of Human Gastric Carcinoma Cell Line With Silencing Human Protection Of Telomeres 1 (hPOT1) By RNA Interference

Objective Telomeres are nucleoprotein structures that cap the ends of eukaryotic chromosomes. Senescence and tumor develop with disfunction of telomeres. Telomere–specific binding proteins play crucial roles in keeping integrity and normal function of telomeres. Protection of telomeres 1 (POT1),found in 2001, protects chromosome ends and regulates telomere length.To investigate the function of hPOT1 in human gastric cancer cell.The expression of POT1 was inhibited by siRNA, the change in biological characteristics,telomere length and the expression of TRF1,TRF2,Tankyrase,hTERT and mutation of p53 were observed ..Methods The siRNA eukaryotic expression vector targeting POT1 was constructed, and transfected into human gastric carcinoma cell line BGC823 through lipofectamine 2000 .The expression of POT1 in mRNA level were detected by semi-determinalion RT- PCR. Cell morphology ,cell growth curve, plating efficiency, senescence were observed, apoptotic population was analysed by FCM and telomere length was measured by real-time quantitive PCR. The expression of TRF1,TRF2,Tankyrase,hTERT and mutation of P53 in mRNA level were detected by semi-determinalion RT- PCR.Results 1,The mRNA level of POT 1 was inhibited significantly in the cells transfected with POT1 siRNA. 2,More larger and multinuclear cells , lower growth rate, a decrease in plating efficiency,premature senescene, apoptotic and shorter telomere length were observed in cell group of siRNA.3,A significant increase of TRF1,a marked decrease of TRF2 and Tnakyrase in mRNA level were detected. No change in hTERT expression and little decrase in mutations p53 expression were observed in cells group of siRNA.Conclusion 1,With the expression of hPOT1 in human gastric carcinoma cell Line BGC823 was inhibiated by siRNA, biological characteristics including cell growth , plating efficiency, senescence, apoptotic were effected ,may due to the destabilization of telomere and DNA damage. 2,The decrase in telomere length show that POT1 play a positive role in telomere length control of BGC 823 cells. 3,(1)The significant change of TRF1,TRF2,Tnakyrase mRNA indicate POT1 correlate with the other three telomere–specific binding proteins intimately. (2)No change in hTERT mRNA mean POT1 regulate telomere length without depending on telomerase. (2)Little decrase of mutation of p53 show POT1 correlate with it in certain degree.