miR-182靶向调控FOXO3a表达对胶质瘤细胞增殖和侵袭能力的影响

Effects of FOXO3a expression by miR-182 targeted regulation on proliferation and invasion of glioma cells

目的探讨小分子核苷酸序列182(miR-182)靶向调控"叉头蛋白"的O亚家族(FOXO3a)蛋白表达对胶质瘤细胞U251增殖与侵袭能力的影响。方法将无义序列与miR-182 micmics脂质体转染入U251细胞内,MTT法测定细胞的增殖数量,Transwell小室测定细胞的转移。荧光素酶报告实验检测miR-182对FOXO3a的靶向作用,应用Western blot与qRT-PCR测定FOXO3a蛋白与mRNA的表达。将FOXO3a野生型与突变型质粒导入U251细胞,应用MTT与Transwell实验测定U251细胞的增殖与转移能力变化。结果与对照组及无义序列组比较,在第1～3天,转染miR-182 micmics脂质体组的细胞数显著增加(P<0.05);第4～5天,miR-182 micmics脂质体组的细胞数呈减小趋势,较其他两组细胞数仍显著增加(P<0.05)。荧光素酶报告实验检测显示,共转染突变型pc DNA3/EGFP(增强绿色荧光蛋白)-FOXO3a 3′-UTR(非翻译区)质粒和miR-182 mimics组细胞荧光素酶活性最低(P<0.01)。与对照组及无义序列组比较,miR-182 mimics组FOXO3a的蛋白及mRNA含量显著减少(P<0.01)。转染突变型FOXO3a脂质体的U251细胞,与野生型组比较,其细胞增殖数量与转移数量均显著减少(P<0.05)。结论 miR-182可促进胶质瘤U251细胞的增殖与转移,FOXO3a蛋白表达量的增加会造成胶质瘤U251细胞的增殖与转移能力降低,miR-182对FOXO3a起负性调控的作用。

Objective To explore the effects of FOXO3 a protein expression by miR-182 targeted regulation on the proliferation and invasion of glioma cell U251.Methods The nonsense sequence and miR-182 micmics liposome were transfected into U251 cells.The proliferation of cells was determined by MTT assay and the cell metastasis was measured by transwell chamber.The Luciferase Reporter Assay was used to detect the targeting effect of miR-182 on FOXO3 a,and the expression levels of FOXO3 a protein and mRNA were determined by Western blot and qRT-PCR.The FOXO3 a wild-type and mutant-type plasmids were introduced into U251 cells,and the proliferation and metastasis ability of U251 cells were measured by MTT and transwell.Results Compared with control group and nonsense sequence group,the number of cells in miR-182 micmics liposome group was significantly increased on the 1 st to 3 rd d(P<0.05).On the 4 th to 5 th d,the number of cells in miR-182 micmics liposome group showed a decreasing trend,but the number of cells was significantly increased in other two groups(P<0.05).In the Luciferase Reporter Assay,the mutant pc DNA3/EGFP-FOXO3 a 3′-UTR plasmid and miR-182 mimics were transfected,and the luciferase activity in miR-182 mimics group was the lowest(P<0.01).Compared with control group and nonsense sequence group,the levels of FOXO3 a protein and mRNA in miR-182 mimics group were significantly decreased(P<0.01).The quantities of cell proliferation and metastasis in U251 cells which were transfected with mutant FOXO3 a liposome were significantly decreased compared with those in wild-type group(P<0.05).Conclusion miR-182 can promote the proliferation and metastasis of glioma cell U251.The increase of FOXO3 a protein expression will decrease the proliferation and metastasis ability of glioma cell U251,and miR-182 may negatively regulate FOXO3 a.