Niu Xiao-Yu, Peng Zhi-Lan, Wang He
Obstetrics and Gynecology Department, West China Second Hospital, Sichuan University, Chengdu, Sichuan 610 041, P.R. China.
Ai Zheng. 2004 Nov;23(11):1257-62.
BACKGROUND & OBJECTIVE: Tumorigenesis and progression of cervical cancer closely relate with human papilloma virus (HPV) E6 and E7 oncogenes. Ribozyme and antisense oligonucleotides had been used to inhibit the expression of HPV E6 or E7 oncogenes to treat cervical cancer, but problems, including low efficiency, short-period maintenance, hard work, and high costs, still exist. This study was to evaluate the specific inhibitory effect and time-efficiency of RNA interference (RNAi) on HPV16 E6 gene in cervical cancer cell line CaSki.
The specific small interfering RNA (siRNA) of HPV16 E6 modified by fluorescein was synthesized, and transfected into CaSki cells. The transfection efficiency of siRNA was evaluated by calculating the ratio of fluorescent cells to total cells. Cell apoptosis was evaluated by flow cytometry (FCM). The mRNA level of HPV16 E6 before and after siRNA transfection was measured by RT-PCR, and protein level of HPV16 E6 was measured by Western blot and FCM.
The transfection efficiency of siRNA was 81%. Apoptosis rates of CaSki cells at 1, 2, 5, and 9 d after transfection were 7.7%, 11.8%, 37.4%, and 12.6%, respectively. The mRNA level of HPV16 E6 at 1, 2, 5, and 9 d after transfection reduced by 77%, 83%, 59%, and 41%, respectively, but the mRNA level of beta-actin, as internal control, had no change. The inhibition rates of HPV16 E6 protein at 1, 2, 5, and 9 d after transfection were 79.7%, 80.4%, 71.3%, and 57.4%, respectively, but the protein level of Lamin A/C, as internal control, had no change at each time point.
RNAi exists in CaSki cells, and has specific high efficiency on HPV16 E6 gene.
宫颈癌的发生发展与人乳头瘤病毒（HPV）E6和E7癌基因密切相关。核酶和反义寡核苷酸曾被用于抑制HPV E6或E7癌基因的表达来治疗宫颈癌，但仍存在诸如效率低、维持时间短、操作困难和成本高等问题。本研究旨在评估RNA干扰（RNAi）对宫颈癌CaSki细胞系中HPV16 E6基因的特异性抑制作用和时效关系。
合成荧光素标记的HPV16 E6特异性小干扰RNA（siRNA），并转染至CaSki细胞。通过计算荧光细胞与总细胞的比例评估siRNA的转染效率。采用流式细胞术（FCM）评估细胞凋亡。用RT-PCR检测siRNA转染前后HPV16 E6的mRNA水平，用Western blot和FCM检测HPV16 E6的蛋白水平。
siRNA的转染效率为81%。转染后1、2、5和9天CaSki细胞的凋亡率分别为7.7%、11.8%、37.4%和12.6%。转染后1、2、5和9天HPV16 E6的mRNA水平分别降低了77%、83%、59%和41%，但作为内参的β-肌动蛋白的mRNA水平无变化。转染后1、2、5和9天HPV16 E6蛋白的抑制率分别为79.7%、80.4%、71.3%和57.4%，但作为内参的核纤层蛋白A/C的蛋白水平在各时间点均无变化。
RNAi存在于CaSki细胞中，对HPV16 E6基因具有特异性高效抑制作用。
