HPV16阳性宫颈癌细胞株中miR-497-5p靶向PD-L1的免疫调控作用  

Immune Regulation of miR-497-5p Targeting PD-L1 in HPV16-Positive Cervical Cancer Cell Line

作　　者：王亚菲 杨英捷 孙清华 田琴 任婕 Wang Yafei;Yang Yingjie;Sun Qinghua;Tian Qin;Ren Jie(Department of Obstetrics,the Second People’s Hospital of Guiyang,Guiyang 550081,Guizhou,China;Department of Gynaecology,Affiliated Tumor Hospital of Guizhou Medical University,Guiyang 550000,Guizhou,China;School of Clinical Medicine,Guizhou Medical University,Guiyang 550025,Guizhou,China)

机构地区：[1]贵阳市第二人民医院产科,贵阳550081 [2]贵州医科大学附属肿瘤医院妇科,贵阳550000 [3]贵州医科大学临床医学院,贵阳550025

出　　处：《肿瘤预防与治疗》2023年第3期200-207,共8页Journal of Cancer Control And Treatment

基　　金：贵州省科技计划项目(编号:黔科合基础-ZK[2021]一般469)。

摘　　要：目的:探讨miR-497-5p靶向PD-L1在HPV16阳性宫颈癌细胞株中的免疫调控作用。方法:通过免疫共沉淀(co-immunoprecipitation,Co-IP)验证宫颈癌细胞中HPV16 E6和E7蛋白对PD-L1蛋白是否有直接作用;对宫颈癌组织差异表达的miRNA进行生信分析筛查;以TCGA宫颈癌数据为基础,探讨miRNA与预后之间的关系;E6、E7 siRNA由上海吉玛制药技术有限公司设计合成,通过脂质体在HPV16阳性宫颈癌细胞株中转染E6、E7 siRNA,下调E6、E7的表达;构建E6、E7基因表达质粒,于HPV阴性子宫颈癌细胞株中表达E6、E7。利用qRT-PCR对不同组细胞表达miRNA进行检测;miR-497-5p mimics和inhibitor转染HPV16阳性宫颈癌细胞株,通过Western blot检测各组PD-L1蛋白的表达情况;通过预测软件,在PD-L1 mRNA 3’UTR上寻找miR-497-5p的作用靶点;利用双荧光素酶报告实验,验证miR-497-5p是否与PD-L1 mRNA 3’UTR靶向结合。结果:经Co-IP实验验证,HPV16 E6和E7对PD-L1无直接作用关系;在宫颈癌细胞中对E6和E7表达进行上调和下调后,发现E6、E7与hsa-miR-497-5p表达存在负调控关系;miR-497-5p mimics和inhibitor转染HPV16阳性宫颈癌细胞株后发现,hsa-miR-497-5p可抑制PD-L1蛋白的表达水平;hsa-miR-497-5p inbibitor可提高PD-L1蛋白的表达水平;双荧光素酶活性实验结果提示miR-497-5p可靶向结合PD-L1的3’-UTR序列。结论:HPV16 E6/E7可能通过miR-497-5P/PD-L1轴促进宫颈癌细胞免疫逃逸。Objective:To investigate the immune regulation of miR-497-5p targeting PD-L1 in HPV16-positive cervical cancer cell line(H16CC).Methods:The direct effect of HPV16 E6 and E7 proteins on PD-L1 protein in cervical cancer cells was determined by co-immunoprecipitation(Co-IP).Differential expression of miRNA in cervical cancer tissue was screened by bioassay.The relationship between miRNA and prognosis of cervical cancer was explored based on TCGA data.E6 and E7 siRNA were designed and synthesized by Shanghai GenePharma Co.,Ltd.The expressions of E6 and E7 siRNA were down-regulated by transfecting E6 and E7 siRNA in H16CC with liposomes.Construction of E6,E7,E6E7 expression plasmid were construced,and E6 and E7 were expressed in HPV-negative cancer cell line.qRT-PCR was used to detect miRNA expression in cell lines.H16CC was transfected with miR-497-5p mimics and inhibitor.The expression of PD-L1 protein was detected by Western blot.The target of miR-497-5p on PDL1 mRNA 3’UTR was searched by prediction software.Whether miR-497-5p acts directly with the target of PD-L1 mRNA 3’UTR was verified by the dual-luciferase reporter assay.Results:The CO-IP assay showed that HPV16 E6 and E7 had no direct effect on PD-L1.After up-and down-regulation of E6 and E7 expressions in cervical cancer cells,it was found that E6 and E7 were negatively regulated by the expression of hsa-miR-497-5p.After H16CC was transfected with miR-497-5p mimics and inhibitor,it was found that hsa-miR-497-5p inhibited the expression of the PD-L1 protein;however,the hsa-miR-497-5p inhibitor could increase the expression level of the PD-L1 protein.The results of the dual-luciferase reporter assay suggested that miR-497-5p could target the 3’-UTR sequence of PD-L1.Conclusion:HPV16 E6/E7 may promote cervical cancer cell immune escape through miR-497-5p/PD-L1 axis.

关 键 词：子宫颈鳞癌 微小RNA 程序性死亡受体配体-1 人乳头瘤病毒16 免疫调控 

分 类 号：R737.33[医药卫生—肿瘤]