下调miR-200a表达对结直肠癌细胞HCT116影响研究  

Down-regulation of miR-200a expression promotes the proliferation and migration and inhibits apoptosis of HCT116 cells in vitro

作　　者：吴共发[1] 李海刚[1] 赵海燕[2] 何楠[3] 韩慧霞[4] WU Gong-fa;LI Hai-gang;ZHAO Hai-yan;HE Nan;HAN Hui-xia(Department of Pathology,Zengcheng District People＇s Hospital of Guangzhou City,Guangzhou 511300,P.R.China;Department of Pathology,Longgang Central Hospital,Shenzhen 518116,P.R.China;Department of Pathology,Children Hospital of Zhengzhou,Zhengzhou 450053,P.R.China;Department of Pathology,School of Basic Medical Sciences,Southern Medical University,Guangzhou 510515,P.R.China)

机构地区：[1]广州市增城区人民医院.中山大学孙逸仙纪念医院增城院区病理科,广东广州511300 [2]深圳市龙岗中心医院病理科,广东深圳518116 [3]郑州市儿童医院病理科,河南郑州450053 [4]南方医科大学基础医学院病理学系,广东广州510515

出　　处：《中华肿瘤防治杂志》2018年第15期1065-1070,共6页Chinese Journal of Cancer Prevention and Treatment

基　　金：广州市科技计划(201804010043)

摘　　要：目的 miR-200a在多种肿瘤中被证实能调控上皮-间质转化(epithelial-mesenchymal transition,EMT),然而miR-200a在结直肠癌中调控EMT的机制仍未完全阐明。本研究探讨下调miR-200a表达对结直肠癌细胞系HCT116增殖、迁移和凋亡的影响及可能机制。方法实验分blank组(仅加LipofectamineTM2000)、NC组(转染miR-200anegative control)和inhibitor组(转染miR-200ainhibitor)3组,各组细胞处理48h后,CCK-8、Transwell迁移实验和TUNEL法分别检测细胞增殖、迁移和凋亡变化,免疫细胞化学及蛋白质印迹法检测相关蛋白变化。结果 miR-200a的抑制效率为50.9%,下调miR-200a后inhibitor组细胞增殖能力显著增强(A值为1.429±0.419),F=4.009,P=0.031;Transwell迁移结果显示,inhibitor组的穿膜数量显著增加至45.67±6.779,F=102.452,P<0.001;TUNEL结果显示,inhibitor组细胞的凋亡指数显著减低至(0.145±0.4585)%,F=19.932,P<0.001;下调miR-200a的表达后,免疫细胞化学检测显示,E-cadherin蛋白表达减弱,Vimentin表达增强;蛋白质印迹法显示,p-AKT和p-ERK1/2的表达增强,而AKT和ERK2表达无变化。结论在结直肠癌细胞中下调miR-200a能增强AKT和ERK信号通路活性,后两者可能诱导细胞发生EMT,最终导致促进细胞增殖和迁移,抑制细胞凋亡。OBJECTIVE MiR-200a has been reported to regulate epithelial-mesenchymal transition（EMT） in several tumours ; however, the mechanism of miR-200a regulate EMT in colorectal carcinoma （CRC） remains poorly understood. The present study was to explore the effect and mechanism of miR-200a down-regulation on the proliferation,migration, and apoptosis of HCTll6 cells in vitro. METHODS The expression was divided into blank group,NC group and inhibitor group,each group of cells were treaed at 48 h. Cell proliferation was studied by Cell Counting Kit-8（CCK 8） assay. Tran- swell migration assay and TUNEL assay were utilized to analyze the migration and apoptosis of HCTll6 cells. Proteins expression was investigated by immunocytochemistry and western blot. RESULTS The expression of miR-200a was de- clined 50.9% after transient transfection. After decreasing the expression of miR-200a, the proliferation of HCT116 cell was obviously increased （A value was 1. 429±0. 419, F= 4. 009, P = 0.031 ）, transwell migration assay indicated that the cells crossing the membrane were obviously increased to 45.67±6. 779 （F= 102. 452, P〈0. 001）, and TUNEL assay indicated that the percent of apoptotic cells was obviously decreased to （0. 145 ± 0.4585）% （F= 19. 932, P〈0. 001）. Im- muocytochemistry showed that the expressions of E-cadherin were obviously decreased, while Vimentin were apparently enhanced. Western blot showed that inhibition of miR-200a up-regulated p-AKT,p-ERK1/2 expression, but had no effect on total AKT and ERK2. CONCLUSION Down-regulation of miR-200a causes a distinct promotion of cells proliferation and migration, but reduction of apoptosis in CRC,its molecular mechanism may involve in the regulation of EMT through activation of AKT and ERK signaling pathway by miR-200a.

关 键 词：结直肠癌 miR-200a 上皮-间质转化 AKT ERK 

分 类 号：R735.3[医药卫生—肿瘤] R735.4[医药卫生—临床医学]