miR-149-5p通过MSH5/Wnt信号通路调控胶质瘤细胞恶性生物学行为

miR-149-5p Regulates Malignant Biological Behavior of Glioma Cells Through MSH5/WNT Signaling Pathway

目的探索miR-149-5p调控MSH5/Wnt信号通路影响胶质瘤细胞恶性生物学行为的具体作用机制。方法RT-qPCR检测miR-149-5p在胶质瘤组织和细胞系A172、U251、HS683、H4中的表达。CCK-8检测细胞活力,EDU染色检测细胞增殖,Transwell小室检测细胞的迁移和侵袭,流式细胞术检测细胞周期和细胞凋亡。双荧光素酶报告基因实验验证miR-149-5p和MSH5的靶向关系。Western blot检测MSH5、GAPDH、GSK3β、β-catenin、AXIN2的蛋白表达。结果miR-149-5p在胶质瘤组织(P<0.0001)和细胞系中表达降低(P<0.0001),过表达miR-149-5p可显著抑制A172细胞的增殖(P<0.01)、迁移(P<0.05)和侵袭(P<0.01)、细胞周期(P<0.01),并诱导凋亡(P<0.01),敲降miR-149-5p则具有相反的作用。双荧光素酶报告基因实验证明miR-14-5p靶向MSH5。敲降miR-149-5p可逆转敲降MSH5对A172细胞恶性生物学行为的作用。过表达MSH5通过抑制GSK3β、激活β-catenin/AXIN2通路,促进A172细胞的增殖、迁移和侵袭和细胞周期,并抑制细胞凋亡。结论miR-149-5p通过靶向上调MSH5,抑制AXIN2表达激活β-catenin/AXIN2通路,从而抑制间质瘤细胞的恶性生物学行为。

Objective To explore the mechanism of miR-149-5p regulating the malignant biological behavior of glioma cells through MSH5/Wnt signaling pathway.Methods RT-qPCR was used to detected the expression of miR-149-5p in glioma tissues and cell lines A172,U251,HS683 and H4.CCK-8 was used to detected cell viability,and EDU staining was performed to measure cell proliferation,Transwell assay was used to detected cell migration and invasion,and flow cytometry examined cell cycle and apoptosis.The dual luciferase reporter gene experiment verified the targeting relationship between miR-149-5p and MSH5.The protein expressions of MSH5,GAPDH,GSK3β,β-catenin and AXIN2 were detected by Western blot.Results The expression of miR-149-5p was decreased in glioma tissues(P<0.0001)and cell lines(P<0.0001).Overexpression of miR-149-5p significantly inhibited the proliferation(P<0.01),migration(P<0.05),invasion(P<0.01),and cell cycle(P<0.01)of A172 cells,and induced cell apoptosis(P<0.01).However,knocking down miR-149-5p had the opposite effect.Dual luciferase assay verified that miR-149-5p targeted MSH5.Knockdown of miR-149-5p reversed the effect of knocking down MSH5 on the malignant biological behavior of A172 cells.Overexpression of MSH5 promoted the proliferation,migration and invasion of A172 cells,cell cycle,and inhibited apoptosis by inhibiting GSK3βand activatingβ-catenin/AXIN2 pathway.Conclusions miR-149-5p inhibited the AXIN2 expression and activated theβ-catenin/AXIN2 pathway by targeting up-regulation of MSH5,thereby inhibited the malignant biological behavior of stromal tumor cells.