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目的探讨靶向人乳头瘤病毒16早期蛋白5(HPV16-E5)的siRNA对TRAIL蛋白诱导子宫颈癌Caski细胞株凋亡的调节作用。方法 HPV16-E5特异性siRNA转染48 h后,RT-PCR检测HPV16-E5 mRNA的表达;流式细胞术及caspase-8活性检测试剂盒分别检测空白对照组、siRNA-E5单独作用组、TRAIL单独作用组、siRNA-E5+TRAIL作用组、无关序列RNA+TRAIL作用组细胞的凋亡率及caspase-8的活性水平;RT-PCR及Western blotting检测siRNA干扰后Caski细胞表面TRAIL死亡受体DR4、DR5的表达。结果 RT-PCR检测结果显示,转染48 h后,siRNA-E5组HPV16-E5 mRNA的表达较空白对照组和无关序列对照组下调(P<0.05);siRNA-E5+TRAIL作用组较TRAIL单独作用组相比细胞凋亡率明显增加(P<0.05),而无关序列RNA+TRAIL作用组较TRAIL单独作用组细胞凋亡率无明显变化(P>0.05);siRNA-E5组TRAIL受体DR4、DR5的表达较无关序列对照组、空白对照组无明显差异(P>0.05);caspase-8活性水平在siRNA-E5+TRAIL作用组明显较TRAIL单独作用组高,差异有统计学意义(P<0.05)。结论靶向HPV16-E5的siRNA可增强TRAIL诱导宫颈癌Caski细胞的凋亡作用,其机制与增强caspase-8的活化有关。
Objective To investigate the effect of siRNA targeting HPV16-E5 on the apoptosis of Caski cells induced by TRAIL. Methods The expression of HPV16-E5 mRNA was detected by RT-PCR 48 h after transfected with HPV16-E5 specific siRNA. The expression of HPV16-E5 mRNA was detected by flow cytometry and caspase-8 activity assay kit respectively. The apoptosis rate and the activity of caspase-8 in the cells treated with siRNA-E5 + TRAIL alone or in combination with RNAi without TRAIL were detected by RT-PCR and Western blotting. The expressions of TRAIL death receptor DR4 , DR5 expression. Results The results of RT-PCR showed that the expression of HPV16-E5 mRNA was down-regulated in siRNA-E5 group 48 h after transfection compared with blank control group and non-related sequence control group (P <0.05) (P <0.05). However, the apoptosis rate of TRAIL receptor DR4 in siRNA-E5 group was not significantly different from that of TRAIL alone group (P> 0.05) (P> 0.05). The activity of caspase-8 in the siRNA-E5 + TRAIL group was significantly higher than that in the TRAIL group (P <0.05), and the difference was statistically significant (P <0.05). Conclusion The siRNA targeting HPV16-E5 can enhance the apoptosis of Caski cells induced by TRAIL. The mechanism is related to the enhancement of caspase-8 activation.