siRNA抑制X连锁凋亡抑制蛋白基因对人HeLa细胞凋亡及周期的影响

摘要

目的：探讨特异性 siRNA 对宫颈癌 Hela 细胞 X 连锁凋亡抑制蛋白（XIAP）基因的抑制作用及其抑制后细胞凋亡和细胞周期的变化。方法设计合成 XIAP 特异性抑制 siRNA，并将 Hela 细胞分成4组，即观察组（pG-PU6／GFP／Neo ／XIAP -siRNA 转染细胞）、空白组（未加 siRNA 及转染试剂）、NC 组（阴性对照，为所选目的序列被无序打乱的 siRNA 转染）、Mock 组（转染试剂对照，加转染试剂未加 siRNA）。RT-PCR 法检测各组 XIAP mRNA 表达情况，流式细胞术检测各组细胞凋亡情况及细胞周期变化。结果转染48、72 h，观察组 XIAP mRNA 表达分别为20．20±0．10、19．99±0．14，空白组分别为21．43±0．04、20．95±0．15，两组比较均有统计学差异（P 均＜0．05）。转染48 h，观察组、空白组细胞凋亡率分别为（28．91±0．12）％、（9．73±0．02）％；转染72h，观察组、空白组细胞凋亡率分别为（37．29±0．10）％、（10．62±0．04）％；观察组细胞凋亡率均高于空白组（P 均＜0．05）。空白组、Mock组、NC 组间 XIAP mRNA 表达、细胞凋亡率比较均无统计学差异（P 均＞0．05）。与空白组相比，观察组转染48、72 h 后 G0～G1期细胞增多而 S 期细胞减少（P 均＜0．05）。结论特异性 XIAP-siRNA 可有效沉默宫颈癌 Hela 细胞XIAP 基因，抑制细胞增殖并促进细胞凋亡。%Objective To explore the inhibitory effect of small interfering RNA(siRNA)silencing human X-linked inhibitor of apoptosis protein (XIAP)gene on cell cycle and apoptosis of cervical HeLa cells.Methods DNA template coding XIAP specific siRNA was designed and synthesized.The Hela cells were divided into 4 groups:the observation group (pGPU6 /GFP/Neo /XIAP-siRNA-transfected cells),blank group (not added with siRNA and transfection reagent), negative control group (NC group,transfected by disrupted siRNA)and transfection reagent control group (Mock group, added with transfection reagents and without siRNA).The XIAP-siRNA was transfected into HeLa cells.The expression level of XIAP mRNA was assayed by RT-PCR.The apoptosis rate and cell cycle distribution were analyzed by flow cytome-try.Results Significant difference was found in the expression of XIAP mRNA at 48 and 72 h after transfection between the observation group and blank group (all P <0.05),but no significant difference was found among the blank group, Mock group and NC group (all P >0.05).In the observation group,the interference rates of HeLa cells transfected with XIAP-siRNA at 48 and 72 h were 27% and 59%.The apoptotic rate of the observation group was higher than that of the blank group (all P <0.05).Significant difference was found in the apoptosis rate of the observation group between the transfection of 48 h and 72 h (P <0.05).Compared with the blank group,the cells in the G0-G1 phrase increased and then decreased in the S phrase at 48 and 72 h after transfection (all P <0.05).Condnsion Specific XIAP-siRNA can ef-fectively silence the XIAP gene of cervical cancer Hela cells,inhibit the proliferation and promote the apoptosis.