miR-21通过靶向TIMP3调控黑色素瘤细胞的免疫抑制  

miR-21 regulates immunosuppression of melanoma cells by targeting TIMP3

作　　者：陈敏[1] 郭爱元 付楚涵 窦建华 丁玉芳 黄进华(指导) CHEN Min;GUO AI-Yuan;FU Chu-Han;DOU Jian-Hua;DING Yu-Fang;HUANG Jin-Hua(Dermatology Department,the Third Xiangya Hospital of Central South University,Changsha 410013,China)

机构地区：[1]中南大学湘雅医学院附属三医院皮肤科,长沙410013

出　　处：《中国免疫学杂志》2020年第16期1973-1977,1982,共6页Chinese Journal of Immunology

摘　　要：目的:探讨微小RNA-21(miR-21)是否通过调节金属蛋白酶组织抑制因子3(TIMP3)的表达影响黑色素瘤细胞分泌免疫抑制因子。方法:实时荧光定量聚合酶链式反应(qRT-PCR)检测黑色素瘤组织和细胞中miR-21的表达水平。脂质体介导的细胞转染法上调或下调黑色素瘤A375细胞中miR-21的表达。ELISA检测细胞培养上清液中IL-10、转化生长因子-β(TGF-β)和血管内皮生长因子(VEGF)的含量。双荧光素酶报告基因实验检测miR-21与TIMP3靶向关系。Western blot检测细胞中TIMP3蛋白表达水平。结果:黑色素瘤组织和不同黑色素瘤细胞株中miR-21的表达量较癌旁组织和正常黑色素细胞明显降低(P<0.05)。在A375细胞中转染miR-21 mimic后,细胞中miR-21表达量显著上升,分泌的免疫抑制因子IL-10、TGF-β和VEGF的含量显著升高(P<0.05)。转染miR-21 inhibitor后,细胞中miR-21表达量显著降低,分泌的免疫抑制因子IL-10、TGF-β和VEGF的含量显著降低(P<0.05)。生物信息学软件预测miR-21和TIMP3存在靶向结合位点,双荧光素酶报告基因实验验证了miR-21和TIMP3靶向结合关系,miR-21能够负向调节TIMP3蛋白的表达。结论:miR-21能够靶向调控TIMP3的表达影响黑色素瘤细胞分泌的免疫抑制因子IL-10、TGF-β和VEGF的含量。Objective:To investigate whether miR-21 affects the secretion of immunosuppressive factors by regulating the TIMP3 gene in melanoma cells.Methods:qRT-PCR for detection of miR-21 expression in melanoma tissues and cells.Liposomal-mediated cell transfection up-regulated or down-regulated miR-21 expression in melanoma A375 cells.The levels of IL-10,TGF-βand VEGF in the cell culture supernatant were determined by ELISA.Dual luciferase reporter assay was wed to detect miR-21 and TIMP3 targeting.The expression level of TIMP3 protein in the cells was detected by Western blot.Results:The expression of miR-21 in melanoma tissues and different melanoma cell lines was significantly lower than that in adjacent tissues and normal melanocytes(P<0.05).Transfection of miR-21 mimic upregulated miR-21 expression in cells.The levels of secreted immunosuppressive factors IL-10,TGF-βand VEGF were significantly increased(P<0.05).Transfection of miR-21 inhibitor could inhibit the expression of miR-21 in cells.The levels of secreted immunosuppressive factors IL-10,TGF-βand VEGF were significantly decreased(P<0.05).Bioinformatics software predicted the presence of targeted binding sites or miR-21 and TIMP3.Dual luciferase reporter assays demonstrated a targeted binding relationship between miR-21 and TIMP3,and miR-21 negatively regulated TIMP3 protein expression.Conclusion:miR-21 can regulate the expression of TIMP3 and affect the secretion of immunosuppressive factors IL-10,TGF-βand VEGF by melanoma cells.

关 键 词：MIR-21 TIMP3 黑色素瘤细胞 免疫抑制因子 

分 类 号：R737.31[医药卫生—肿瘤]