RNA干扰HPV E6下调eIF4E转录表达抑制宫颈癌Hela细胞增殖并影响细胞周期进程研究

Study of RNA interference of HPV E6 down regulates the transcripiton of eIF4E and inhibits the cell proliferation and cell cycle of Hela cells

目的探讨宫颈癌Hella细胞中干扰HPV E6对eIF4E表达的调控,探寻HPV E6促癌的新途径,为宫颈癌诊断和治疗提供新靶点。方法构建高效的靶向HPV18 E6的shRNA质粒(shE6)并测序,而后转染人宫颈癌Hela细胞,荧光显微镜及流式细胞术检测转染效率。而后以Real-time PCR检测E6及eIF4E的mRNA水平,采用CCK-8法检测Hela细胞增殖能力的改变,流式细胞术检测细胞周期。结果测序结果显示成功构建shE6质粒。shE6-3转染人宫颈癌Hela细胞后48 h,E6及eIF4E mRNA表达的抑制率分别约为80%,76%。shE6-3对Hela细胞的增殖活性在48 h的抑制效果最明显,增殖抑制率约为21%;相对于NC组,shE6组G0/G1期细胞比例显著增高,而S期比例减少,G2/M期未见明显变化。结论 RNA干扰HPV E6能够下调eIF4E转录表达,抑制宫颈癌Hela细胞增殖,并阻滞细胞周期于G0/G1期。eIF4E有望成为宫颈癌防治的潜在靶点。

Objective To investigate RNA interference of E6 could regulate eIF4E in cervical cancer cells and to find new target gene for the diagnosis and therapy of cervical cancer.Methods ShRNAs of E6 were constructed by company and transfected into Hela cells.The transfcction rate was detected by fluorescence microscope and flowcytometry （FCM）.Real time PCR was used to detected mRNA of E6 and eIF4E.CCK-8 was employed for cell proliferation and FCM was used to detected the cell cycle.Results ShRNA plasmids of E6 were constructed successfully and transfected into Hela cells.48 h later,the mRNA of E6 and eIF4E were 80%,76%,respectively.ShE6-3 leaded to the inhibition of cell proliferation （21%） at 48 h.In the cell cyle detection,the cells of G0/G1 phase increased highly while the cells of S phase decreased.The cells of G2/M phase was the same to that of the control group.Conclusion ShE6 can inhibit the cell proliferatin of cervical cancer and can down regulate the transcription of eIF4E,block the cell cycle on G0/G1,which might be a potenitial target for cervical cancer.