HPV16E7下调miR-29a促进PC3细胞增殖  

HPV16E7 Promotes Cellular Proliferation by Down-regulating MicroRNA-29a

作　　者：熊焱强[1,2] 冯晶 刘朝奇[2] 李志英[1] Xiong Yanqiang;Feng Jing;Liu Chaoqi(Department of Gynecology and Obstetrics,Affiliate Renhe Hospital,China Three Gorges University,Yichang 443001,China;Hubei Key Laboratory of Tumor Microenvironment and Immunotherapy,China Three Gorges University,Yichang 443002,China)

机构地区：[1]三峡大学附属仁和医院妇产科,宜昌443001 [2]三峡大学肿瘤微环境与免疫治疗湖北省重点实验室,宜昌443002

出　　处：《华中科技大学学报（医学版）》2018年第2期145-149,共5页Acta Medicinae Universitatis Scientiae et Technologiae Huazhong

基　　金：肿瘤微环境与免疫治疗湖北省重点实验室(三峡大学)2015年度开放基金资助项目(No.2015KZL06)

摘　　要：目的探讨人乳头瘤病毒16E7(human papillomavirus V16E7,HPV16E7)能否通过调控细胞内miR-29a表达促进细胞增殖,从microRNA的角度探讨高危型HPV的致癌机制。方法将HPV16E7基因构建到真核表达载体pcDNA3.1(-),将重组质粒及空载质粒分别转染人前列腺癌细胞PC3,经G418筛选,通过RT-PCR、流式细胞术检测稳定转染效果;用MTT法、流式细胞术检测克隆细胞株的生物学活性,半定量RT-PCR检测E7对PC3细胞中miR-29a表达的影响。结果重组质粒经酶切及测序鉴定显示构建成功,并获得稳定表达E7的PC3克隆细胞株。与阴性对照组PC3-3.1(-)比较,克隆细胞株PC3-E7增殖能力显著增强(P<0.01),G0/G1期细胞明显减少,而S期和G2/M期细胞明显增多(均P<0.05);PC3-E7细胞中miR-29a表达量显著低于对照组(P<0.01)。结论 HPV16E7可以加速细胞周期G1向S期进展,其机制可能与其下调miR-29a有关。Objective To explore whether human papillomavirus type 16E7(HPV16E7) promotes cell proliferation by down-regulating miR-29a,and the carcinogenic mechanism of high-risk HPV from the perspective of microRNA.Methods The HPV16E7 gene was constructed into the eukaryotic expression vector pcDNA3.1(-).The recombinant plasmid and empty vector plasmid were transfected into human prostate cancer cell line PC3,respectively.After G418 selection,stable transfection was detected by RT-PCR and flow cytometry.The biological activity of the cloned cell line was detected by MTT assay and flow cytometry.Semi-quantitative RT-PCR was used to detect the effect of HPV16E7 on miR-29a expression in PC3 cells.Results Restriction enzyme digestion and sequencing showed that the recombinant plasmid was successfully constructed.A PC3 clone cell line stably expressing HPV16E7 was obtained.Compared with PC3-3.1(-)in negative control group,the proliferation ability of cloned cell line PC3-E7 was significantly increased(P<0.01),cells at G 0/G 1 phase were significantly decreased,and those at S phase and G 2/M phase significantly increased(P<0.05).The expression of miR-29a in PC3-E7 cells was significantly lower than that in the control group(P<0.01).Conclusion HPV16E7 can accelerate the progression from cell cycle G 1 to S phase,and its mechanism may be related to the down-regulation of miR-29a.

关 键 词：宫颈癌 人乳头瘤病毒16E7 miR-29a 细胞增殖 

分 类 号：R737.3[医药卫生—肿瘤]