MiR-127-3p靶向MAPK4对葡萄膜黑色素瘤细胞增殖、凋亡、迁移和侵袭的影响  

Effect of MiR-127-3p on Proliferation,Apoptosis,Migration and Invasion of Uveal Melanoma Cells via Targeting MAPK4

作　　者：魏丽[1] 连红梅[2] 刘鹏[1] 刘兴华[1] 吴书一[3] 刘萌萌[1] WEI Li;LIAN Hong-mei;LIU Peng;LIU Xinghua;WU Shu-yi;LIU Meng-meng(Department of Pathology,Zhengzhou Third People′s Hospital,Zhengzhou 450000,China;Department of Anesthesia,The Third people′s Hospital Affiliated to Zhengzhou University,Zhengzhou 450000,China;Central Laboratory,Zhengzhou Third People′s Hospital,Zhengzhou 450000,China)

机构地区：[1]郑州市第三人民医院病理科 [2]郑州大学第三附属医院麻醉科室 [3]郑州市第三人民医院中心实验室,河南郑州450000

出　　处：《中山大学学报（医学版）》2020年第1期76-85,共10页Journal of Sun Yat-Sen University:Medical Sciences

基　　金：河南省科技攻关计划资助项目(2018020548)

摘　　要：[目的]探讨miR-127-3p对葡萄膜黑色素瘤细胞增殖、凋亡、迁移和侵袭的作用。[方法]通过RT-qPCR检测人葡萄膜黑色素瘤组织及细胞、正常组织及细胞中miR-127-3p与MAPK4mRNA的表达;通过Lipofectamine2000说明书将mimic-NC、miR-127-3pmimic、pc-MAPK4质粒分别或联合转染进入SP6.5或OM431细胞;通过双荧光素酶报告检测miR-127-3p与MAPK4复染靶向关系;通过CCK-8法检测细胞增殖,流式细胞术检测细胞凋亡,划痕实验检测细胞迁移能力,Transwell法检测细胞侵袭能力,蛋白印迹法检测AKT/mTOR通路蛋白相对表达水平。[结果]在葡萄膜黑色素瘤组织和细胞系中,miR-127-3p表达明显下调(P<0.01),而MAPK4表达明显上调(P<0.01);miR-127-3p与MAPK43′UTR区存在结合位点,miR-127-3p高表达明显抑制了含有野生型MAPK4质粒的荧光素酶活性(P<0.01),但对突变型MAPK4质粒的荧光素酶活性无影响;与Control组相比,miR-127-3pmimic组SP6.5细胞和OM431细胞增殖均明显下降(P<0.01),凋亡率均明显增加(P<0.01),划痕闭合率均明显降低(P<0.01),每视野侵袭细胞数目均明显减少(P<0.01),p-AKT(T308)/AKT、p-mTORr(S473)/mTOR蛋白表达均明显下调(P<0.01),共转染pc-MAPK4逆转上述变化。[结论]M iR-127-3p通过靶向下调MAPK4来抑制葡萄膜黑色素瘤细胞增殖、迁移和侵袭,诱导细胞凋亡,这可能与抑制AKT/mTOR通路激活有关。【Objective】To investigate the effect of miR-127-3p on proliferation,apoptosis,migration and invasion of uveal melanoma cells.【Methods】The expression of miR-127-3p and MAPK4 mRNA in human uveal melanoma tis⁃sues and cells,normal tissues and cells were detected by RT-qPCR.The mimic-NC,miR-127-3p mimic,pc-MAPK4 plasmids were transfected into SP6.5 or OM431 cells,respectively,by Lipofectamine 2000.The relationship between miR-127-3p and MAPK4 counterstaining was detected by dual luciferase assay.Cell proliferation was detected by CCK-8 method,apoptosis was detected by flow cytometry,cell migration ability was detected by scratch test,cell invasion ability was detected by Transwell method,and relative expression level of AKT/mTOR pathway protein was detected by Western blot.【Results】In uveal melanoma tissues and cell lines,the expression of miR-127-3p was down-regulated(P<0.01)while that of MAPK4 expression was significantly up-regulated(P<0.01).The binding site of miR-127-3p and MAPK43′UTR region,the high expression of miR-127-3p significantly inhibited the luciferase activity of wild-type MAPK4 plas⁃mid(P<0.01),but the mutant MAPK4 plasmid Luciferase activity has no effect.Compared with the Control group,the proliferation of SP6.5 cells and OM431 cells in miR-127-3p mimic group were significantly decreased(P<0.01),and the apoptotic rate was significantly increased(P<0.01).The scratch closure rate was obvious.The decrease(P<0.01),the number of invading cells per field was significantly decreased(P<0.01),and the expression of p-AKT(T308)/AKT and p-mTORr(S473)/mTOR protein were significantly down-regulated(P<0.01).Transfection of pc-MAPK4 reversed the above changes.【Conclusion】MiR-127-3p inhibits proliferation,migration and invasion of uveal melanoma cells and induces apoptosis by down-regulating MAPK4,which may be involved in the inhibition of AKT/mTOR pathway activation.

关 键 词：葡萄膜黑色素瘤 miR-127-3p MAPK4 增殖 迁移 侵袭 

分 类 号：R773.9[医药卫生—眼科]