HPV16 E7 siRNA表达载体对宫颈癌CaSki细胞生物学活性的影响

摘要

Objective To investigate a new potential therapy to achieve targeted inhibition of HPV16 E7 oncogene in tumor cells using small interfering RNAs (siRNA). Methods The HPV16 E7 siRNA and scramble control expression plasmids were structured and transfected into CaSki cells by liposome. The stable cell lines of HPV16 E7 siRNA were established by Hygromycin screening. The expression levels of HPV16 E7 mRNA and protein were detected by RT-PCR, immunofluorescence and flowcytometry. The cell cycle and cell proliferation were measured with flowcytometry and MTT method. Meanwhile the cisplatin chemotherapy sensitivity was observed in the cells. Results Plasmids HPV16 E7 siRNA and scramble control were constructed and confirmed by sequencing. HPV16 E7 mRNA and protein expressions were significantly decreased in the HPV16-E7Si cells compared with the CaSki-Si control cells. The cell growth rate of HPV16-E7Si cells was significantly inhibited. Flowcytometry analysis revealed a significant increase in apoptosis. In addition, after cis-platin( DDP) treatment for 48 hours the ratio of G0-1 phase of HPV16-E7Si cells was increased compared with control cells. Conclusion HPV16 E7 siRNA could successfully suppress CaSki cell growth and induce the cell apoptosis, and increases sensitivity of the cisplatin chemotherapy.%目的 探讨人乳头瘤病毒16型(HPV16) E7基因小干扰RNA(siRNA)表达载体对宫颈癌CaSki细胞生物学活性的影响.方法 利用脂质体HPV16 E7特异性siRNA表达载体(HPV16-E7Si)、空载体(CaSki-Si)转染CaSki细胞,荧光定量RT-PCR、流式细胞仪检测HPV16-E7Si、CaSki-Si细胞E7 mRNA和蛋白的变化；流式细胞仪及MTT法检测2种细胞的细胞周期及生长曲线变化,并检测2种细胞对顺铂的敏感性.结果 成功获得沉默E7基因的克隆细胞株CaSki-E7Si及阴性对照CaSki-Si；CaSki-E7Si细胞生长明显慢于CaSki-Si细胞(P＜0.05),且前者细胞的凋亡峰明显增多.2种细胞加用顺铂作用48 h后发现,CaSki-Si细胞周期阻滞在S～ G2期,HPV16-E7Si阻滞在G0～1期.结论 HPV16 E7 siRNA表达载体能有效地抑制宫颈癌CaSki细胞E7基因的表达,促进肿瘤细胞凋亡,并增加顺铂的敏感性.