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Received:July 29, 2022 Published Online:September 20, 2022
Received:July 29, 2022 Published Online:September 20, 2022
中文摘要: 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。
Abstract: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 (H16CC) through LncRNA GABPB1-AS1/miR-497-5p. 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-497-5p and prognosis was analyzed based on The Cancer Genome Atlas（TCGA） cervical cancer data. After the H16CC were transfected with miR-497-5p mimics and inhibitor, the expression of PD-L1 was detected by western blot. E6, E7 siRNAs were constructed and transfected into H16CC by liposomes. The expression of LncRNA GABPB1-AS1 and miR-497-5p 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-497-5p by qRT-PCR. The target relationship between miR-497-5p and PD-L1, LncRNA GABPB1-AS1 and miR-497-5p 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-497-5p had a better prognosis. The expression of PD-L1 was negatively correlated with miR-497-5p (P<0.05). After inhibiting the expression of E6, E7 in H16CC cell lines, the expression of LncRNA GABPB1-AS1 was decreased, and the expression of miR-497-5p 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-497-5p was decreased (P<0.05). The targeting relationship between miR-497-5p and PD-L1, miR-497-5p and LncRNA GABPB1-AS1 was confirmed by dual luciferase reporter assay. Conclusion miR-497-5p 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-497-5p. LncRNA GABPB1-AS1/miR-497-5p may mediate the immunoregulatory effect of E6，E7 on PD-L1 in HPV infected cervical cancer.
keywords: Cervical cancer Human papillomavirus Human papillomavirus 16 positive primary cervical cancer cell line Long non-coding RNA GA-binding protein 1-AS1 MicroRNA-497-5p Programmed death ligand-1
文章编号： 中图分类号：R-33 R737.33 文献标志码：A
基金项目:贵州省科技计划项目（黔科合基础-ZK［2021］一般469）
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