MicroRNA-483-3p对人神经胶质瘤细胞的作用  

Effects of microRNA-483-3p on human glioma cells

作　　者：赵克 刘康栋 

机构地区：[1]郑州大学附属肿瘤医院神经外科,河南郑州450008

出　　处：《中国病理生理杂志》2017年第7期1163-1170,共8页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No.81372269)

摘　　要：目的:探讨microRNA(miRNA)-483-3p对人神经胶质瘤细胞A172生长和迁移能力的影响及潜在作用机制。方法:实时荧光定量聚合酶链式反应(RT-q PCR)检测人肾胚细胞系HEK-293和不同神经胶质瘤细胞株(A172、U251和SHG44)中miRNA-483-3p的表达水平。转染miRNA-483-3p抑制序列(miRNA-483-3p inhibitor)下调A172细胞中miRNA-483-3p的表达,采用CCK-8法和流式细胞术检测细胞活力和周期分布;Transwell实验检测细胞的迁移;Western blot检测周期相关调控因子及上皮-间充质转化相关蛋白的水平。双萤光素酶报告基因分析法预测及验证其可能的靶基因。结果:miRNA-483-3p在各型神经胶质瘤细胞中高表达。沉默miRNA-483-3p后,A172细胞的活力下降并呈现出明显的周期阻滞,且细胞迁移率也显著降低。同时细胞中cyclin D1、周期蛋白依赖性激酶4、磷酸化视网膜母细胞瘤蛋白、N-cadherin及vimentin的蛋白表达水平均显著降低,E-cadherin和β-catenin的蛋白表达水平显著升高。双萤光素酶报告基因分析显示Smad4是miRNA-483-3p的可能作用靶点,A172细胞共转染miRNA-483-3p inhibitor和Smad4 siRNA可部分逆转miRNA-483-3p介导的细胞增殖及迁移抑制。结论:沉默miRNA-483-3p可通过靶向Smad4抑制神经胶质瘤细胞株A172的生长及迁移。AIM： To investigate the effects of microRNA （miRNA） ^l83-3p on the growth and migration of hu-man glioma cell line A172 and its potential mechanisms. METHODS： The abundance of miRNA^l83-3p in human embry-onic kidney 293 cells and different human glioma cell lines （A172, U251 and SHG44） was measured by RT-qPCR. After down-regulation of miRNA^l83-3p by transfection of inhibitor in the A172 cells, the cell viability, cell cycle distribution and cell migration were detected by CCK-8 assay, flow cytometry and Transwell assay, respectively. Furthermore, the pro-tein levels of cell cycle-related molecules and epithelial-mesenchymal transition markers were measured by Western blot. Luciferase reporter assay was used to predict and verify the target gene of miRNA^l83-3p. RESULTS： miRNA^l83-3p was highly expressed in human glioma cells. Knockdown of miRNA-483-3p inhibited A172 cell viability, arrested cell cycle and decreased cell migration rate. Furthermore, the protein levels of cyclin D1, cyclin-dependent kinase 4, phoshorylated reti-noblastoma protein, N-cadherin and vimentin were significantly decreased after knockdown of miRNA-483-3p, accompanied with the up-regulation of E-cadherin and p-catenin protein expression. Luciferase reporter assay demonstrated that Smad4 was a potential target gene of miRNA^l83-3p. Down-regulation of Smad4 in the A172 cells transfected with miRNA^l83-3p inhibitor partially reversed the effect of miRNA^l83-3p on cell viability and migration. CONCLUSION ： Knockdown of miRNA-483-3p restrains the growth and migration of A172 cells by targeting Smad4.

关 键 词：MicroRNA-483-3p 神经胶质瘤 细胞活力 细胞迁移 SMAD4 

分 类 号：R739.41[医药卫生—肿瘤] R730.23[医药卫生—临床医学]