LncRNA GABPB1-AS1靶向miR-497-5p在宫颈癌细胞中的免疫调控作用  

Immunomodulatory effect of LncRNA GABPB1-AS1 targeting miR-497-5p in cervical cancer cells

作　　者：孙清华 任婕 王亚菲 田琴 SUN Qing-hua;REN Jie;WANG Ya-fei;TIAN Qin(School of Clinical Medicine,Guizhou Medical University,Guiyang,Guizhou 550004,China;不详)

机构地区：[1]贵州医科大学临床医学院,贵州贵阳550004 [2]贵州医科大学附属医院妇科,贵州贵阳550004

出　　处：《中国临床研究》2022年第9期1193-1199,共7页Chinese Journal of Clinical Research

基　　金：贵州省科技计划项目(黔科合基础-ZK[2021]一般469)。

摘　　要：目的 探讨E6、E7可能通过长链非编码RNA GA结合蛋白1-AS1(LncRNA GABPB1-AS1)/miRNA-497-5p实现对程序性死亡配体1(PD-L1)在人乳头瘤病毒(HPV)16阳性原代宫颈癌细胞株(H16CC)中的免疫调控作用。方法 使用免疫共沉淀法(Co-IP)验证宫颈癌细胞中HPV E6、E7蛋白和PD-L1蛋白是否有直接作用。基于癌症基因组图谱(TCGA)的宫颈癌数据分析hsa-miR-497-5p与预后的关系。构建miR-497-5p mimics和inhibitor转染H16CC后,通过western blot方法检测PD-L1的表达情况;构建E6、E7 siRNA,经脂质体转染H16CC,通过qRT-PCR方法检测细胞中LncRNA GABPB1-AS1、miR-497-5p表达的情况,同时构建E6、E7基因表达质粒,在HPV阴性细胞株(HNCC)表达E6、E7,qRT-PCR法检测其细胞中LncRNA GABPB1-AS1、miR-497-5p的表达;通过软件及双荧光素酶活性实验确定miR-497-5p与PD-L1、LncRNA GABPB1-AS1与miR-497-5p有无直接靶向关系。结果 HPV E6、E7和PD-L1蛋白无直接作用;通过TCGA的数据发现hsa-miR-497-5p高表达的宫颈癌患者预后较好;PD-L1的表达与miR-497-5p呈负相关(P<0.05);抑制H16CC细胞株中E6、E7表达后,LncRNA GABPB1-AS1表达下调,miR-497-5p与之反向变化(P<0.05);而过表达HNCC细胞株中E6、E7后,LncRNA GABPB1-AS1表达上调,miR-497-5p表达下调,差异有统计学意义(P<0.05);双荧光素酶活性实验证实miR-497-5p与PD-L1、miR-497-5p与LncRNA GABPB1-AS1存在靶向结合关系。结论 miR-497-5p能抑制PD-L1蛋白的表达水平,E6、E7蛋白与LncRNA GABPB1-AS1表达呈正向调控关系,与miR-497-5p表达呈负向调控关系,LncRNA GABPB1-AS1/miR-497-5p可能介导HPV E6、E7对PD-L1在HPV感染宫颈癌中的免疫调控作用。Objective To explore the immunomodulatory effect of E6 and E7 on programmed death ligand-1(PD-L1)in human papillomavirus(HPV) 16 positive primary cervical cancer cell line(H16 CC) through LncRNA GABPB1-AS1/miR-497-5 p. Methods Co-immunoprecipitation(Co-IP) was used to verify whether HPV E6, E7 protein and PD-L1 protein in cervical cancer cells have a direct effect. The relationship between hsa-miR-497-5 p and prognosis was analyzed based on The Cancer Genome Atlas(TCGA) cervical cancer data. After the H16 CC were transfected with miR-497-5 p mimics and inhibitor, the expression of PD-L1 was detected by western blot. E6, E7 siRNAs were constructed and transfected into H16 CC by liposomes. The expression of LncRNA GABPB1-AS1 and miR-497-5 p in the cells was detected by qRT-PCR. Plasmids of E6, E7 gene were constructed and overexpressed in HPV negatiue cell line(HNCC) to detect the expression of LncRNA GABPB1-AS1 and miR-497-5 p by qRT-PCR. The target relationship between miR-497-5 p and PD-L1, LncRNA GABPB1-AS1 and miR-497-5 p were determined by dual luciferase reporter assay. Results Direct interaction between HPV E6, E7 and PD-L1 proteins was not found. TCGA data showed that cervical cancer patients with high expression of hsa-miR-497-5 p had a better prognosis. The expression of PD-L1 was negatively correlated with miR-497-5 p(P<0.05). After inhibiting the expression of E6, E7 in H16 CC cell lines, the expression of LncRNA GABPB1-AS1 was decreased, and the expression of miR-497-5 p was reversed(P<0.05). After the overexpression of E6, E7 in HNCC cell lines, the expression of LncRNA GABPB1-AS1 was increased and the expression of miR-497-5 p was decreased(P<0.05). The targeting relationship between miR-497-5 p and PD-L1, miR-497-5 p and LncRNA GABPB1-AS1 was confirmed by dual luciferase reporter assay. Conclusion miR-497-5 p can inhibit the expression level of PD-L1 protein. E6,E7 protein is positively regulated with the expression of LncRNA GABPB1-AS1, and negatively regulated with the expression of miR-497-5 p.

关 键 词：宫颈癌 人乳头瘤病毒 人乳头瘤病毒16阳性原代宫颈癌细胞株 长链非编码RNA GA结合蛋白1-AS1 微小RNA-497-5p 程序性死亡配体1 

分 类 号：R-33[医药卫生] R737.33