siRNA介导的hPOT1基因表达抑制对HeLa细胞凋亡的影响

Effects of RNAi-mediated gene silencing of hPOT1 expression on apoptosis of HeLa cell

目的 应用siRNA表达载体介导的RNAi技术,特异地抑制端粒保护蛋白hPOT1基因在HeLa细胞中的表达,并观察其对细胞凋亡的影响。方法利用先前构建的含hPOT1基因特异性序列(hsRNA)的3种重组siRNA表达质粒,由脂质体介导转染HeLa细胞,以RT PCR和电泳迁移率变化分析法检测转染细胞中的hPOT1基因的表达抑制效果,并通过流式细胞术、Hoechst荧光染色和DNA琼脂糖凝胶电泳检测细胞凋亡。结果 不同hPOT1特异性序列的3种的重组质粒转染HeLa细胞4 8h后,细胞中hPOT1基因mRNA和蛋白质表达水平降低,细胞凋亡水平明显增加。结论 siRNA表达载体介导的RNAi能有效地抑制HeLa细胞中hPOT1基因表达。

AIM To silence the expression of human telomere protection gene hPOT1 in HeLa cells using vector based on RNA interference (RNAi) technique and to investigate its effects on the apoptosis of HeLa cell. METHODS(Three recombinant) plasmids containing different hPOT1 target sequences were transfected into HeLa cells by liposome. The expression inhibition of hPOT1 was detected by RT-PCR and (EMSA). Cellular apoptosis was inspected by flow cytometry, Hoechst fluorescent staining and DNA agar electrophoresis. RESULTS After 48 h of transfecting three recombinant plasmids containing different hPOT1 target sequences, hPOT1 mRNA and protein level in HeLa cells reduced and the apoptosis increased evidently. CONCLUSION(hPOT1 expression) in HeLa cells can be inhibited significantly using plasmid-based RNAi and the down-regulation of hPOT1 expression can cause apoptosis in HeLa cell.