miRNA-200b通过靶向VEGF抑制视网膜母细胞瘤细胞生长与侵袭  

miRNA-200b inhibits retinoblastoma cell proliferation and invasion by targeting VEGF

作　　者：刘越峰[1] 罗卫民[1] 张勇[1] 钟晓东[1] 

机构地区：[1]湖北医药学院附属十堰市太和医院眼科中心,十堰442000

出　　处：《新医学》2016年第8期521-526,共6页Journal of New Medicine

基　　金：十堰市科技课题(14Y40);湖北省教育厅科学研究计划指导性项目(B2015477)

摘　　要：目的明确miRNA-200b是否通过调控靶向血管内皮生长因子(VEGF)的表达而抑制视网膜母细胞瘤细胞的生长与侵袭,进一步揭示miRNA-200b抗肿瘤的分子机制。方法将视网膜母细胞瘤Y79细胞分成6组:miRNA-200b、miRNA-NC、sh-VEGF、shRNA-NC、miRNA-200b+shVEGF、miRNA-NC+shRNA-NC组。荧光素酶报告系统检测miR-200b对VEGF的3'-非翻译区荧光素酶活性的影响;蛋白免疫印迹法检测sh-VEGF转染的干扰效果;MTS实验与Transwell侵袭实验分别检测上述各组对视网膜母细胞瘤细胞增殖与侵袭的影响。结果荧光素酶报告检测结果显示,miR-200b能特异性地与VEGF的3'-非翻译区结合,并抑制其荧光素酶的活性;蛋白免疫印迹法显示,shVEGF转染组中VEGF的蛋白表达水平较对照组明显下调;MTS实验结果显示,转染miRNA-200b组与sh-VEGF组48 h后视网膜母细胞瘤细胞的生存率均低于各自对照组(P均<0.05);而共转染miRNA-200b+sh-VEGF组48 h后视网膜母细胞瘤细胞的生存率低于对照组、miRNA-200b组、sh-VEGF组(F=9.887,P均<0.05);Transwell侵袭实验显示,转染miRNA-200b组与sh-VEGF组48 h后视网膜母细胞瘤细胞的穿膜能力均低于各自对照组(P均<0.05);而共转染miRNA-200b+sh-VEGF组48 h后视网膜母细胞瘤细胞的穿膜能力低于对照组、miRNA-200b组、sh-VEGF组(P均<0.05)。结论 miRNA-200b通过靶向调控VEGF的表达而抑制视网膜母细胞瘤细胞的生长与侵袭。Objective To explore whether miRNA-200 b inhibits retinoblastoma cell proliferation and invasion by targeted-regulating the expression of vascular endothelial growth factor（ VEGF）,and further elucidate the molecular mechanism underlying the anti-tumor function of miRNA-200 b. Methods Retinoblastoma cell line Y79 was divided into miRNA-200 b,miRNA-NC,sh-VEGF,shRNA-NC,miRNA-200 b ＋ sh-VEGF and miRNA-NC ＋ shRNA-NC groups. The effect of miRNA-200 b upon the luciferase activity of VEGF 3＇-UTR was evaluated by luciferase assay. The intervention effect of sh-VEGF transfection was assessed by Western blot. The proliferation and invasion ability of Y79 cells in each group were evaluated by MTS and Transwell assays when transfected with miRNA-200 b and miRNA-NC,sh-VEGF and shRNA-NC,miRNA-200 b ＋ sh-VEGF and miRNA-NC ＋ shRNA-NC,respectively. Results Luciferase assay revealed that miRNA-200 b could specifically bind with the 3＇-UTR of VEGF and suppress the luciferase activity. Western blot detected that the expression of VEGF protein in the sh-VEGF transfection group was significantly down-regulated compared with that in the shRNA-NC group（ P〈0. 05）. MTS assay revealed that the survival rates of Y79 cells at 48 h after miRNA-200 b and sh-VEGF transfection were considerably lower compared with those in corresponding control groups（ both P〈0. 05）. At 48 h following co-transfection with miRNA-200 b and sh-VEGF,the survival rate of Y79 cells was significantly lower than those in corresponding control,miRNA-200 b and sh-VEGF groups（ all P〈0. 05）. Transwell assay showed that the invasion ability of Y79 cells through the basement membrane was significantly lower compared with those at 48 h after miRNA-200 b and sh-VEGF transfection（ both P〈0. 05）.At 48 h after co-transfection with miRNA-200 b and sh-VEGF,the invasion ability of Y79 cells through the basement membrane was considerably lower than those in the corresponding control,miRNA-200 b and shVEGF groups（ all P〈0. 05）. Conclusion mi

关 键 词：视网膜母细胞瘤 微小RNA-200b 血管内皮生长因子生长 侵袭 

分 类 号：R739.7[医药卫生—肿瘤]