摘要
目的探讨microRNA-205(miR-205)表达对人类黑素瘤细胞株(A375和A2058)迁移和侵袭的作用及其分子机制。方法转染miR-205过表达慢病毒(Lenti-miR-205)构建miR-205过表达的黑素瘤细胞A375和A2058细胞系(miR-205组),转染空白对照慢病毒作为对照细胞系(NC组)。通过划痕实验和Transwell实验分别检测两组细胞迁移和侵袭能力,激光共聚焦扫描显微镜观察细胞形态和表达钙黏蛋白E(E-cadherin)的细胞数量与荧光强度,蛋白质印迹法检测E-cadherin、Twist、整合素(intergrin)、波形蛋白(vimentin)、Zeb1、基质金属蛋白酶(MMP)-2、MMP-9蛋白表达。结果培养8 h和24 h时miR-205组A375与A2058细胞的迁移能力均大于NC组(P<0.01);miR-205组两种细胞的侵袭能力均低于NC组(P<0.01)。MiR-205过表达后A375和A2058细胞均由梭形、基质型向圆形、表皮型转化。与NC组相比,miR-205组细胞E-cadherin蛋白表达增加(P<0.01),且表达E-cadherin的细胞数量和荧光强度均增加;Twist、intergrin、vimentin、Zeb1、MMP-2和MMP-9蛋白表达水平降低(P<0.01)。结论 MiR-205通过诱导E-cadherin的表达逆转上皮间质转化,从而抑制黑素瘤细胞的迁移和侵袭。
Objective To explore the effect of microRNA-205 （miR-205） expression on migration and invasion of human melanoma cell lines （A375 and A2058） and its related molecular mechanism. Methods The miR-205 over-expressed melanoma cells （miR-205 group） were constructed via transfecting A375 and A2058 cell lines with Lenti-miR-205, and the cells transfected with blank vector virus were taken as control group （NC group）. The migration and invasion ability of the cells in two groups were detected by scratch assay and Transwell assay, respectively. The confocal laser scanning microscopy was employed to observe the cell morphology and the cell number and fluorescence intensity of the cells expressing E-cadherin protein. Western blotting was performed to detect the protein expressions of E-cadherin, Twist, intergrin, vimentin, Zeb1, matrix metalloproteinase （MMP）-2 and MMP-9. Results At 8 h and 24 h of cells culture, the migration ability of A375 and A2058 cells in miR-205 group was significantly higher than that in NC group （P〈0.01）; the invasion ability of two kinds of cells in miR-205 group was lower than that in NC group （P〈0.01）. A375 and A2058 cells over-expressing miR-205 were transformed from shuttle, stroma type to round and epidermal type. Compared with the NC group, the E-cadherin expression of the cells in miR-205 group was significantly increased （P〈0.01） and the cell number and fluorescence intensity of the cells expressing E-cadherin was increased, while the protein expressions of Twist, intergrin, vimentin, Zeb1, MMP-2 and MMP-9 were significantly decreased （P〈0.01）. Conclusion MiR-205 can inhibit the migration and invasion abilities of melanoma cells by inducing E-cadherin expression and reversing epithelial-mesenchymal transition.
出处
《第二军医大学学报》
CAS
CSCD
北大核心
2017年第5期576-582,共7页
Academic Journal of Second Military Medical University
基金
上海市卫生局科研课题(20134363)~~
