摘要
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 discuss the effect of Human Papilloma Virus 16 early protein 5 （HPV16-E5）-targeted siRNA on Caski cells apoptosis induced by TNF-related apoptosis-inducing ligand （TRAIL）. Methods HPV16-E5-targeted siRNA was transfected into Caski cells by lipofectamine. Forty-eight hours after the transfection, the expression of E5 mRNA was detected by RT- PCR; Cell apoptosis and caspase-8 activities in the blank control group, siRNA-E5-treated group, TRAIL-treated group, siRNA-E5 ＋ TRAIL-treated group and negative RNA ＋ TRAIL-treated group were deter- mined by flow cytometry and caspase-8 Activity Assay Kit. The expressions of receptors DR4 and DR5 on cell surface after RNA interference were tested by Real-Time PCR and Western blotting. Results RT-PCR revealed that the ex- pression of HPV16-E5 mRNA in the siRNA-E5 group was significantly lower than that in the blank and the negative control groups （ P 〈 0.05 ） ; The cell apoptosis rate in siRNA-E5 ＋ TRAIL group was significantly higher than that in the TRAIL group （P 〈 0.05 ）. There was no significant difference in cell apoptosis rate between the negative RNA ＋ TRAIL group and the TRAIL group（ P 〉 0.05 ） ; There was no difference in the expressions of receptors DR4 and DR5 after RNA interference （ P 〉 0.05 ） ; The activity of caspase-8 in siRNA-E5 ＋ TRAIL group was higher than that in TRAIL group, and the difference was statistical significant（ P 〈 0.05 ）. Conclusion HPV16-E5-Targeted siRNA could enhance the apoptosis mediated by TRAIL against Caski cancer cells in vitro, and the mechanism may be related to the increase of caspase-8 activity.
出处
《山东大学学报（医学版）》
CAS
北大核心
2013年第10期24-28,共5页
Journal of Shandong University:Health Sciences
基金
山东省自然科学基金(Y2006C44)
