HPV16 E7 siRNA表达载体对宫颈癌CaSki细胞增殖及成瘤性的影响

The effect of HPV16 E7 specific siRNA expression vector on perliferation and tumorigenicity of cervical cancer CaSki cells

目的:通过载体介导的RNA干扰技术干扰E7癌基因的表达,探讨HPV16E7小干扰RNA(siRNA)表达载体对宫颈癌CaSki细胞增殖及成瘤性的影响,以及成为宫颈癌基因治疗新策略的可行性。方法:利用脂质体将HPV16E7特异性siRNA表达载体、空载体转染CaSki细胞,分别命名为CaSki-S、CaSki-P。通过荧光定量RT-PCR及流式细胞仪检测CaSki、CaSki-S、CaSki-P3种细胞E7mRNA和蛋白的变化,通过流式细胞仪及MTT法检测3种细胞的细胞周期及生长曲线变化,并检测3种细胞在裸鼠体内成瘤性及生长活性的差异。结果:荧光定量RT-PCR及流式细胞仪检测显示,CaSki-S细胞E7基因mRNA和蛋白的表达明显低于CaSki细胞,抑制率分别为92.86%、84.21%。MTT法检测显示,CaSki-S细胞生长明显慢于CaSki细胞。流式细胞仪检测细胞周期显示,CaSki-S细胞与未转染组CaSki细胞比较,G1期细胞比例明显增加,S期细胞比例则明显减少。裸鼠体内成瘤性及生长活性显示CaSki-S细胞显著低于CaSki细胞。而CaSki-P细胞无论是在E7mRNA和蛋白的表达,还是在肿瘤细胞生长、细胞周期的改变及成瘤性方面都与CaSki细胞无明显差异。结论:HPV16E7siRNA表达载体能有效地抑制宫颈癌CaSki细胞E7基因的表达,部分逆转其恶性表型。因此它有望成为宫颈癌基因治疗的新策略之一。

Objective：To interfere the HPV16 E7 in CaSki cells by vector-based RNA interference technique and investigate the effect of HPV16 E7 specific expression vector on perliferation and tumorigenicity of cervical cancer CaSki cells, and explore the feasibility of siRNA expression vector in treatment of cervical carcinoma. Methods： The HPV16 E7 siRNA expression vector and empty expression vector were transfected into CaSki cells by liposome, which named as CaSki-S, CaSki-P, respectively. CaSki group was the control group. The expression levels of HPV16 E7 mRNA and protein of the cells of the three group were detected by realtime RT-PCR and FCM. The cell cycle and cell proliferation were measured with flow cytometry and MTT method. The in vivo carcinogenic and growth activities of cancer cell in nude mice were observed. Results ：The expressions of HPV16 E7 mRNA and protein were significantly decreased in the CaSki-S cell compared with the CaSki cell, the inhibition rates were 92.86% and 84.21% ,respectively. The cell growth rate of CaSki-S cell was significantly inhibited. The ratio of G1 phase of CaSki-S cell was increased and the ratio of S phase was decreased. The nude mice tumorigenicity and growth activity of cancer cell were reduced in the CaSki-S cell.cell and CaSki ceil. Conclusion： inhibit the expression of E7 gene may be one of the ideal strategies for gene therapy of cervical cancer.