MiR-124调节Wnt/β-catenin信号通路对脑胶质瘤细胞增殖、凋亡和侵袭的影响

Effects of miR-124 on the Proliferation,Apoptosis and Invasion of Glioma Cells by Regulating Wnt/β-catenin Signaling Pathway

目的探讨miR-124调节Wnt/β-catenin信号通路对脑胶质瘤细胞增殖、凋亡和侵袭的影响。方法2019年7月至2021年6月收集接受胶质瘤切除手术和颅内减压手术患者的胶质瘤组织(胶质瘤组)和正常脑组织(正常对照组)各30例,另购买的体外培养人脑胶质瘤细胞株U251、U87MG、LN229、A172以及正常人脑星形胶质细胞(NHA),用qRT-PCR法检测miR-124和WNT2B相对表达量。利用脂质体2000转染试剂盒对U251细胞进行转染并分组:对照组、miR-NC组、miR-124 mimics组、miR-124 mimics+pcDNA组、miR-124 mimics+pc-WNT2B组。用CCK-8法检测各组U251细胞增殖能力;Transwell法测定各组U251细胞侵袭能力;流式细胞仪检测各组U251细胞凋亡;Western blot法检测各组U251细胞中细胞周期蛋白D1(cyclin D1)、基质金属蛋白酶-2(MMP-2)、半胱氨酸天冬氨酸蛋白酶-3(caspase-3)、β-连环蛋白(β-catenin)、C-myc蛋白水平;双荧光素酶报告实验检测miR-124与WNT2B的作用关系。结果miR-124在胶质瘤组肿瘤组织和各细胞系中表达降低,WNT2B表达升高(P<0.05),选择miR-124相对表达量最低的U251细胞进行转染实验。miR-124过表达后,U251细胞A值(48 h和72 h)、侵袭细胞数、cyclin D1、MMP-2、β-catenin、C-myc蛋白水平均显著降低(均P<0.05),凋亡率及caspase-3蛋白水平升高(均P<0.05)。miR-124靶向下调WNT2B表达;上调WNT2B可激活Wnt/β-catenin信号通路蛋白表达,并逆转过表达miR-124对U251细胞发挥的上述作用(P<0.05)。结论miR-124可能通过靶向下调WNT2B抑制Wnt/β-catenin信号通路,进而抑制脑胶质瘤细胞的增殖、侵袭并促进凋亡。

Aim To investigate the effects of miR-124 on the proliferation,apoptosis and invasion of glioma cells by regulating the Wnt/β-catenin signaling pathway.Methods From July 2019 to June 2021,30 cases of glioma tissue(as a glioma group)and 30 cases of normal brain tissues(as a normal control group)from the patients undergoing glioma resection and intracranial decompression surgery in our hospital were collected.The human glioma cells strains U251,U87MG,LN229,A172 and normal human brain astrocytes(NHA)cultured in vitro were purchased,real-time fluorescent quantitative PCR(qRT-PCR)method was used to detect the relative expression of miR-124 and WNT2B.U251 cells were transfected with liposome 2000 transfection kit and divided into different groups:a control group,a miR-NC group,a miR-124 mimics group,a miR-124 mimics+pcDNA group,a miR-124 mimics+pc-WNT2B group.CCK-8 method was used to detect the proliferation ability of U251 cells in each group.Transwell method was used to determine the invasion ability of U251 cells in each group.Flow cytometry was used to detect the apoptotic ability of U251 cells in each group.Western blot method was used to detect the levels of cyclin D1,matrix metalloproteinase-2(MMP-2),cysteine aspartic protease-3(caspase-3),β-catenin,C-myc protein.Double luciferase reporter assay was used to detect the relationship between miR-124 and WNT2B.Results The relative expression of miR-124 was decreased in tumor tissue of glioma group and cell lines,while the expression of WNT2B was increased(P<0.05),and the U251 cells with lowest relative expression of miR-124 were used for transfection experiments.After miR-124 overexpressed,the U251 cell A value(48 h and 72 h),the number of invasive cells,the protein levels of cyclin D1,MMP-2,β-catenin,and C-myc were significantly reduced(P<0.05),the apoptosis rate and caspase-3 protein level were increased(P<0.05).WNT2B expression was down-regulated by miR-124.Upregulation of WNT2B activated the protein expression of Wnt/β-catenin signaling pathway and reversed the above effects of overexpression of miR-124 on U251 cells(P<0.05).Conclusion miR-124 may inhibit the Wnt/β-catenin signaling pathway by targeting down-regulation of WNT2B,thereby inhibiting the proliferation and invasion of glioma cells and promoting apoptosis.