HPV16 E7通过调控miR-29a-CDK6信号通路促进肿瘤细胞增殖  

HPV16 E7 Promotes Tumor Cell Proliferation by Regulating the miRNA-29a-CDK6 Signaling Pathway

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 Gynaecology and Obstetrics,Affiliated Ren-he 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）》2021年第6期744-749,共6页Acta Medicinae Universitatis Scientiae et Technologiae Huazhong

基　　金：湖北省卫生健康委员会联合基金青年人才项目(No.WJ2019H556);宜昌市医疗卫生研究项目(No.A20-2-042)。

摘　　要：目的探究人乳头瘤病毒16 E7(HPV16 E7)通过调控细胞内miR-29a表达促进细胞增殖的分子机制,从miRNA的视角探讨高危型HPV的致癌机制。方法将HPV16 E7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;通过半定量RT-PCR检测细胞周期相关基因及miR-29a潜在靶基因CDK6、HBP1的表达水平,通过荧光素酶报告实验确定miR-29a的靶基因;将miR-29a mimic及inhibitor分别瞬时转染PC3-E7和PC3-3.1细胞,Western blot检测CDK6蛋白表达,流式细胞术检测细胞周期分布。结果与对照组相比,克隆细胞株PC3-E7中CDK4、CDK6、Cyclin E2、E2F1等细胞周期相关调节因子及miR-29a的潜在靶基因HBP1、CDK6等表达均有不同程度的上调(均P<0.01);而PC3-E7细胞中CDK6蛋白表达水平亦明显高于对照组(P<0.01);miR-29a通过靶向3′-UTR区域调控CDK6基因转录和蛋白表达,E7高表达后下调了miR-29a的表达水平,使得CDK6表达升高,而加速细胞周期G;期向S期进展。上调miR-29a后可以抑制CDK6的表达,并调控下游细胞周期,部分逆转E7的促增殖效应。结论miR-29a与CDK6 mRNA 3′UTR结合并下调其表达,从而抑制细胞的增殖,E7-miR-29a-CDK6信号通路可能是高危型HPV致细胞发生恶性增殖的重要途径。Objective To explore the molecular mechanism of HPV16 E7 promoting cell proliferation by regulating intracellular miR-29 a expression,and to investigate the carcinogenic mechanism of high risk type HPV at miRNA level.Methods We constructed recombinant plasmid pcDNA3.1(-)-E7.Recombinant plasmid and empty-loading plasmid were transfected into prostate cancer cells PC3,respectively.Cell-cycle-related genes,miR-29 a potential target genes CDK6,HBP1 were detected by semi-quantitative RT-PCR.miR-29 a target genes were determined by Luciferase reporter assay.PC3-E7 and PC3-3.1 cells were transiently transfected with miR-29 a mimics/inhibitors,respectively.CDK6 protein was detected by Western blotting.Cell Cycle distribution was measured by flow cytometry.Results Compared with the control group,the expression of CDK4,CDK6,cyclin E2,E2 F1 and the potential target genes of miR-29 a,such as HBP1 and CDK6,were up-regulated in PC3-E7 cell line(P<0.05).The expression level of CDK6 protein in PC3-E7 cells was significantly higher than that in the control group(P<0.01).miR-29 a regulated CDK6 gene transcription and protein expression by targeting the 3′-UTR region,and the expression level of miR-29 a was down-regulated after E7 was highly expressed,which increased the expression of CDK6 and accelerated the progression of G1 to S phase of the cell cycle.However,upregulation of miR-29 a could inhibit CDK6 expression,regulate downstream cell cycle,and partially reverse the proliferative effect of E7.Conclusion miR-29 a binds to CDK6 mRNA 3′UTR and down-regulates its expression,thus inhibiting cell proliferation.The E7-miR-29 a-CDK6 signaling pathway may be a new and important pathway for malignant cell proliferation caused by high-risk HPV.

关 键 词：人乳头瘤病毒16 E7 miR-29a CDK6 细胞增殖 

分 类 号：R737.2[医药卫生—肿瘤]