微小RNA-497负调控Wnt3a基因靶点抑制胶质瘤细胞增殖

MicroRNA-497 negatively regulates Wnt3a gene target and inhibits glioma cell proliferation

目的探讨微小RNA(miR)-497在胶质瘤细胞中表达及调控Wnt3a基因靶点抑制胶质瘤细胞增殖的影响。方法病毒载体质粒pLVTHM、元件质粒PsPAXZ和pMDZ.G 3个质粒共同组成慢病毒包装系统。重组质粒pGL3-WNT3a-wt 3’端非编码区(3’UTR)/pGL3-WNT3a-mut 3’UTR与miR-497共转染胶质瘤细胞株U251,检测处理组与对照组荧光素酶活性的变化,验证Wnt3a是miR-497作用靶点。反转录-聚合酶链反应(RT-PCR)和蛋白质印迹法(Western blot)检测胶质瘤细胞株U251转染miR-497后Wnt3a基因在mRNA和蛋白表达。荧光定量PCR检测胶质瘤细胞株U251感染病毒LV-miR-497后表达变化。噻唑蓝(MTT)检测胶质瘤细胞株U251感染病毒LV-miR-497和/或瞬时转染Wnt3a质粒后细胞生长曲线。结果荧光素酶实验验证miR-497与Wnt3a的3’UTR野生型结合位点(WT),与突变型Wnt3a质粒转染的U251细胞比较,野生型质粒转染细胞的荧光素酶活性显著降低。miR-497在mRNA和蛋白水平抑制Wnt3a的表达,miR-497与Wnt3a呈负相关,两组有差异统计学意义(0.95±0.23、0.31±0.09,P<0.05)。慢病毒载体转染miR-497建立稳定细胞株,与对照组比较,LV-miR-497转染后miR-497高表达,差异有统计学意义(0.86±0.14、8.51±1.32,P<0.05)。MTT检测胶质瘤细胞转染miR-497后增殖,结果提示过表达miR-497能够抑制胶质瘤细胞的生长,3组差异有统计学意义(0.78±0.41、0.54±0.22、0.60±0.09,P<0.05)。LV-miR-497处理的U251细胞比用LV-ctrl处理的细胞形成更小和更少的集落,3组差异有统计学意义(0.67±0.35、0.61±0.14、0.59±0.11,P<0.05)。结论 miR-497直接作用Wnt3a基因靶点,高表达miR-497抑制肿瘤细胞增殖。

Objective To investigate the expression of microRNA(miR)-497 in glioma cells and the effect of Wnt3a gene target on the proliferation of glioma cells.Methods Lentivirus packaging system was composed of three plasmids:virus vector plasmid pLVTHM,element plasmid PsPAXZ and pMDZ.G.The recombinant plasmid pGL3-WNT3a-wt 3′untranslated regions(3′UTR)/pGL3-WNT3a-mut 3′UTR and miR-497 were co transfected into glioma cell line U251.The changes of luciferase activity in the treatment group and the control group were detected to verify that Wnt3a is the target of miR-497.Reverse transcriptase-polymerase chain reaction(RT-PCR)and Western blotting were used to detect the mRNA and protein expression of Wnt3a gene in glioma cell line U251 transfected with miR-497.The expression changes of glioma cell line U251 infected with virus lv-miR-497 were detected by fluorescence quantitative PCR.Methyl thiazolyl tetrazolium(MTT)assay was used to detect the cell growth curve of glioma cell line U251 infected with virus lv-miR-497 and/or transiently transfected with Wnt3a plasmid.Results Luciferase experiment verified the 3′UTR wild-type binding site(WT)of miR-497 and Wnt3a.Compared with U251 cells transfected with mutant Wnt3a plasmid,the luciferase activity of cells transfected with wild-type plasmid decreased significantly.miR-497 inhibited the expression of Wnt3a at mRNA and protein levels.miR-497 was negatively correlated with Wnt3a.There was significant difference between the two groups(0.95±0.23,0.31±0.09,P<0.05).miR-497 was transfected with lentivirus vector to establish a stable cell line.Compared with the control group,miR-497 was highly expressed after lv-miR-497 transfection,and the difference was statistically significant(0.86±0.14,8.51±1.32,P<0.05).MTT assay showed that overexpression of miR-497 could inhibit the growth of glioma cells(0.78±0.41,0.54±0.22,0.60±0.09,P<0.05).U251 cells treated with lv-miR-497 formed smaller and fewer colonies than those treated with LV Ctrl.The difference between the three groups was statistically significant(0.67±0.35,0.61±0.14,0.59±0.11,P<0.05).Conclusion MiR-497 directly acts on Wnt3a gene target,and overexpression of miR-497 inhibits the proliferation of tumor cells.