摘要
目的探讨TSPAN8表达对抗肿瘤免疫应答的影响及其可能机制。方法通过CCLE(Cancer Cell Line Encyclopedia)数据库找到Tetraspanin 8(TSPAN8)高表达的人三阴乳腺癌细胞MDA-MB 231。构建siR-TSPAN8质粒并转染MDA-MB 231细胞,以空载体转染组作为对照组,首先使用qRT-PCR(Quantitative Real-Time Polymerase Chain Reaction)及Western blotting方法比较对照组及siR-TSPAN8组TSPAN8的表达水平。从健康志愿者外周血提取单个核细胞(PBMC)并分别与对照组及siR-TSPAN8组细胞进行共培养。使用CCK-8法比较24h,48h,72h在不同效靶比(E/T)的条件下PBMC对肿瘤细胞的杀伤水平。流式细胞仪检测PBMC中CD3^(+)T细胞、NK细胞及NKT细胞百分比;检测肿瘤细胞表面程序性死亡配体1(PD-L1)的表达水平。结果通过CCLE数据库发现多株人乳腺癌细胞高表达TSPAN8,选用人三阴乳腺癌细胞MDA-MB 231细胞进行后续转染实验。与对照组相比,转染siR-TSPAN8的细胞TSPAN8的mRNA及蛋白表达均下调。与PBMC共培养实验结果显示下调TSPAN8后免疫细胞杀伤水平显著增加。流式细胞仪检测结果显示,下调TSPAN8增加共培养PBMC细胞中CD8^(+)T细胞比例,同时抑制肿瘤细胞PD-L1表达水平。结论Tetraspanin 8通过调控三阴乳腺癌细胞PD-L1的表达,影响抗肿瘤免疫反应。
Objective To investigate the effect of tetraspanin 8(TSPAN8)expression on anti-tumor immune response and its possible mechanism.Methods High expression of TSPAN8 was found in MDA-MB 231cells by using Cancer Cell Line Encyclopedia(CCLE)database.Si-TSPAN8 was constructed and transfected into MDA-MB 231 cells.The expression levels of tspan8in the control group and si-TSPAN8 group were compared by Quantitative Real-time Polymerase Chain Reaction(qRT-PCR)and Western blotting.Peripheral blood mononuclear cell(PBMC)were isolated from the healthy volunteers and co-cultured with control or si-TSPAN8 cells.Cell cytotoxicity at different effector/target(E/T)ratios was measured by CCK-8 method at 24h,48h,and 72h.The percentage of CD3^(+)T cells,NK cells and NKT cells in PBMC,and the expression level of programmed death-ligand 1(PD-L1)on tumor cells were detected by the flow cytometry.Results Several human breast cancer cell lines were discovered with high expression of tspan8 through CCLE database.Human triple negative breast cancer cell MDA-MB 231 was selected for subsequent transfection experiment.Compared with the control group,the mRNA and protein expression of tspan8 were down regulated in si-TSPAN8 transfected cells.Co-culture results showed that down-regulation of TSPAN8 significantly increased cell cytotoxicity.Down-regulation of tspan8 promoted the proportion of CD8^(+)T cells and inhibited the expression of PD-L1 on the tumor cells.Conclusion TSPAN8 inhibits the cytotoxicity of immune cells by regulating the expression of PD-L1on breast cancer cells.
作者
郝毅
HAO Yi(College of Basic Medicine Sciences,Jilin University,Jilin Changchun 130021,China)
出处
《中国实验诊断学》
2021年第7期1061-1065,共5页
Chinese Journal of Laboratory Diagnosis
