siRNA介导hPOT1基因表达抑制对HeLa细胞Caspase-3的影响

目的应用siRNA表达载体介导的RNA i技术,特异地抑制端粒保护蛋白POT1基因在HeLa细胞中的表达,并观察其对细胞Caspase-3活化的影响。方法利用作者课题组先前构建的含hPOT1基因特异性序列(hsDNA)的3种重组siRNA表达质粒(pmU6-shRNA1、pmU6-shRNA2和pmU6-shRNA3),由脂质体介导转染HeLa细胞,以RT-PCR和EM-SA法检测转染细胞中的hPOT1基因的表达抑制效果,并通过RT-PCR、W estern b lot和Caspase-3检测试剂盒分析Caspase-3表达和活化。结果3种pmU6-shRNA重组质粒转染HeLa细胞48 h后,细胞中hPOT1基因mRNA和蛋白质表达水平均降低,Caspase-3 mRNA表达水平基本无变化,Caspase-3酶原水平降低,而Caspase-3酶活性增高。结论siRNA表达载体介导的RNA i能有效地抑制HeLa细胞中hPOT1基因表达,hPOT1基因表达的抑制导致细胞Caspase-3活化。

Effect of inhibition of hPOT1 expression mediated by siRNA on Caspase-3 in HeLa cells

Aim To inhibit the expression of human protection of telomere gene hPOT1 in HeLa cells using vector based on RNA interference(RNAi) technique and to investigate its effects on the activation of Caspase-3 in HeLa cell.Methods Three recombinant plasmids(our group constructed before) containing different hPOT1 target sequences(pmU6-shRNA1,pmU6-shRNA2 and pmU6-shRNA3)were transfected into HeLa cells by liposomal.The expression inhibition of hPOT1 was detected by RT-PCR and EMSA.The expression and activation of Caspase-3 were inspected by RT-PCR,Western blot analysis and Caspase-3 kit.Results After 48 hour′s transfecting,hPOT1 mRNA and protein level in HeLa cells reduced and the expression of Caspase-3 mRNA had no change.Caspase-3 zymogen decreased but its activity increased.Conclusions hPOT1 expression in HeLa cells can be significantly inhibited by using siRNAvectored RNAi and the down-regulation of hPOT1 expression can activate Caspase-3.