摘要
背景与目的:研究表明宫颈癌的发生发展与人乳头瘤病毒(humanpapillomavirus,HPV)E6、E7癌基因密切相关,于是人们采用核酶或HPVE6、E7反义寡核苷酸抑制其表达来治疗宫颈癌,虽然取得了一定的效果,但仍面临基因抑制效率低、维持时间短、工作量大、耗费多等问题。本研究采用最新的RNA干扰技术干扰宫颈癌CaSki细胞中HPV16E6转录,以了解其能否特异性抑制HPV16E6基因及其时效性如何。方法:设计合成针对HPV16E6的荧光标记siRNA,借脂质体转染宫颈癌CaSki细胞,通过荧光照片计数荧光细胞占所有细胞的比例计算转染效率;测定转染后不同时间点的细胞凋亡率;RT-PCR测定HPV16E6mRNA变化,Westernblot和流式细胞仪检测转染前后蛋白表达情况。结果:相差显微镜荧光照片显示细胞转染的效率为81%。HPV16E6siRNA转染细胞后24h、48h、5天凋亡率分别为7.7%、11.8%和37.4%,转染9天时凋亡率下降至12.6%。RT-PCR扩增结果显示,细胞转染前HPV16E6mRNA的量与siRNA阴性对照比较没有显著性差异,但转染后24h、48h、5天和9天分别减少了77%、83%、59%和41%;而作为内对照的β-actinmRNA在转染前后无变化。流式细胞仪定量测定HPV16E6蛋白,结果显示转染后24h、48h和5天,蛋白表达抑制率分别为79.7%、80.4%和71.3%;9天时抑制率有下降,但仍可达57.
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. METHODS: 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. RESULTS: 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 β 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. CONCLUSION: RNAi exists in CaSki cells, and has specific high efficiency on HPV16 E6 gene.
出处
《癌症》
SCIE
CAS
CSCD
北大核心
2004年第11期1257-1262,共6页
Chinese Journal of Cancer
基金
国家自然科学基金(No.30371483)~~
