微小RNA-30a-5p/Ovo样锌指蛋白2轴通过Wnt/β-连环蛋白信号通路调控人恶性脑胶质瘤细胞增殖能力

MicroRNA-30a-5p/ovo like zinc finger 2 axis regulates the proliferation of human malignant glioma cells via Wnt/β-catenin signaling pathway

目的探讨微小RNA(miR)-30a-5p/OVOL2轴对人恶性脑胶质瘤(HMG)U-87 MG细胞增殖能力的调控作用及信号通路机制。方法利用定量聚合酶链反应(qPCR)和蛋白质印迹法(Western blot)分别检测miR-30a-5p和OVOL2在HA1800和U-87 MG细胞系中的mRNA水平和蛋白水平;通过细胞计数试剂盒(CCK-8)检测抑制miR-30a-5p表达后U-87 MG的细胞增殖能力和过表达OVOL2后U-87 MG细胞的增殖能力;用qPCR和Western blot实验检测抑制miR-30a-5p表达后U87-MG细胞中OVOL2的mRNA和蛋白表达水平;通过miRNA靶基因网站预测OVOL2 mRNA的3’端非编码区(3’UTR)与miR-30a-5p的结合序列,通过双荧光素酶报告基因实验验证靶向结合关系;利用CCK-8检测下调miR-30a-5p表达的同时下调OVOL2表达后U-87 MG的增殖水平,利用Western blot检测下调miR-30a-5p表达或上调OVOL2表达后U-87 MG细胞中总β-连环蛋白(t-β-catenin)和磷酸化β-catenin(p-β-catenin)的蛋白表达水平。两组间比较采用t检验。结果在U-87 MG中miR-30a-5p表达高于HA1800细胞,而OVOL2表达低于HA1800细胞;下调miR-30a-5p的表达可以抑制U-87 MG细胞的增殖能力;上调OVOL2的表达可以抑制U-87 MG细胞的增殖能力;miR-30a-5p通过靶向抑制OVOL2的表达调控U-87 MG细胞的增殖能力;下调OVOL2的表达逆转了下调miR-30a-5p表达所导致的U-87 MG细胞增殖能力的抑制,下调miR-30a-5p或上调OVOL2的表达抑制了该细胞中Wnt/β-catenin信号通路的水平。结论miR-30a-5p/OVOL2轴通过Wnt/β-catenin信号通路调控HMG细胞的增殖能力。

Objective To explore the regulatory effects of microRNA(miR)-30a-5p/OVOL2 axis on the proliferation of human malignant glioma(HMG)U-87 MG cells and the mechanism of signaling pathway.Methods The mRNA and protein expression levels of miR-30a-5p and OVOL2 in HA1800 and U-87 MG cell lines were detected by quantitative polymerase chain reaction(qPCR)and Western blotting,respectively.The proliferation ability of U-87 MG cells with miR-30a-5p silencing or OVOL2-overexpression was detected by a cell counting kit-8(CCK-8)assay,respectively.The mRNA and protein expression levels of OVOL2 in miR-30a-5p-inhibited U87-MG cells were detected by qPCR and Western blotting.The binding sequence between OVOL2 mRNA 3'untranslated regions(3'UTR)and miR-30a-5p was predicted by Targetscan,and the targeted binding relationship was identified by double luciferase reporter gene assay.The proliferation level of U-87 MG cells with down-regulated miR-30a-5p and inhibition of OVOL2 expression was tested by CCK-8.The protein expression of total-β-catenin(t-β-catenin)and phosphorylated-β-catenin(p-β-catenin)in U-87 MG cells with down-regulated miR-30a-5p or up-regulated OVOL2 expression was detected by Western blotting.Results Compared to HA1800 cells,the expression of miR-30a-5p in U-87 MG cells was increased,and that of OVOL2 was decreased.Both down-regulated miR-30a-5p expression and up-regulated OVOL2 expression could inhibit the proliferation of U-87 MG cells.The miR-30a-5p could regulate the proliferation of U-87 MG cells by targeting OVOL2.OVOL2 knockdown could reverse the inhibition of the proliferation of U-87 MG cells by down-regulating the expression of miR-30a-5p.Down-regulating the expression of miR-30a-5p or up-regulating the expression of OVOL2 inhibited the level of wingless integrated(Wnt)/β-Catenin signaling pathway in U-87 MG cells.Conclusion MiR-30a-5p/OVOL2 axis regulates the proliferation of HMG cells through Wnt/β-Catenin signaling pathway,and OVOL2 is supported to be an novel molecular target for the treatment of HMG.