miR-32-5p靶向CEBPA调控EGFR/β-catenin信号通路影响骨髓瘤细胞的增殖、凋亡和侵袭

miR-32-5p targeting CEBPA regulating EGFR/β-catenin signaling pathway and affecting proliferation,apoptosis and invasion of myeloma cells

目的:探究miR-32-5p靶向CCAAT增强子结合蛋白α(CCAAT/enhancer binding proteinsα,CEBPA)调控表皮生长因子/β联蛋白(EGFR/β-catenin)信号通路影响骨髓瘤细胞增殖、凋亡和侵袭的机制。方法:qRT-PCR、Western blot检测细胞中miR-32-5p、CEBPA的表达水平;转染慢病毒至骨髓瘤细胞U266,MTT检测细胞增殖情况,流式细胞仪检测细胞凋亡水平;生物信息学预测miR-32-5p的靶基因,双荧光素酶报告基因、Western blot验证miR-32-5p与CEBPA的关系及在骨髓瘤细胞中的作用,Western blot检测miR-32-5p与CEBPA的表达对EGFR/β-catenin信号通路的影响。结果:与正常浆细胞比较,骨髓瘤细胞株H929、U266、IM-9、RPMI-8226中miR-32-5p表达水平显著增高,CEBPA mRNA和蛋白水平显著降低(P<0.05);与NC、anti-miR-con组比较,anti-miR-32-5p组miR-32-5p mRNA表达量显著降低,细胞凋亡率显著升高,24 h、48 h、72 h的OD值显著降低,细胞侵袭数目显著减少(P<0.05);TargetScan生物信息学预测显示,miR-32-5p与CEBPA 3'UTR存在结合位点,双荧光素酶报告实验、Western blot进一步证实miR-32-5p可与CEBPA直接结合负向调控CEBPA的表达;与NC、pcDNA-control组比较,pcDNA-CEBPA组CEBPA表达量显著升高,24 h、48 h、72 h的OD值显著降低,凋亡率显著升高,细胞侵袭数目显著减少(P<0.05);与anti-miR-con组比较,anti-miR-32-5p组CEBPA蛋白表达量显著升高,EGFR、p-EGFR、β-catenin蛋白表达量显著降低;与anti-miR-32-5p+si-con组比较,anti-miR-32-5p+si-CEBPA组CEBPA蛋白表达量显著降低,EGFR、p-EGFR、β-catenin蛋白表达量显著升高(P<0.05)。结论:骨髓瘤细胞的增殖、凋亡和侵袭受miR-32-5p和CEBPA的双重调控,miR-32-5p可靶向CEBPA调控EGFR/β-catenin信号通路影响骨髓瘤细胞的增殖、凋亡和侵袭。

Objective:To explore the effect of microRNA-32-5p targeting CCAAT enhancer binding protein alpha on the regulation of epidermal growth factor/β-catenin signaling pathway in affecting proliferation,apoptosis and invasion of myeloma cells.Methods:qRT-PCR and Western blot were used to detect the expression ofmicroRNA-32-5p and CEBPA in human colorectal cancer U266 cells.MTT was used to detect cell proliferation and flow cytometry was used to detect cell apoptosis.Bioinformatics was used to predict the target gene of microRNA-32-5p.Double luciferase reporter gene and Western blot were used to verify the relationship between microRNA-32-5p and CEBPA,and the role of microRNA-32-5p in myeloma cells.The effects of the expression of microRNA-32-5p and CEBPA on EGFR/β-catenin signaling pathway were detected.Results:Compared with normal bone marrow cells,the expression levels of microRNA-32-5p in myeloma cell lines H929,U266,IM-9 and RPMI-8226 were significantly increased,and the levels of CEBPA gene and protein were significantly decreased(P<0.05).Compared with NC and anti-miR-con,the expression of miR-32-5p in anti-miR-32-5p group was significantly decreased,the apoptotic rate was significantly increased.The OD values at 24 h,48 h and 72 h were significantly decreased,and the number of cell invasion was significantly decreased.Target Scan bioinformatics predicted that there was a binding site between microRNA-32-5p and CEBPA 3'UTR.Double luciferase reporter assay and Western blot further confirmed that microRNA-32-5p could directly bind to CEBPA and negatively regulate CEBPA expression.Compared with NC and pcDNA-control,the expression of CEBPA gene in pcDNA-CEBPA group increased significantly,and OD values decreased significantly at 24 h,48 h and 72 h(P<0.05).Compared with anti-miR-con group,anti-miR-32-5p group showed a significant increase in CEBPA protein expression and a significant decrease in EGFR,p-EGFR andβ-catenin protein expression.Compared with anti-miR-32-5p+si-con group,anti-miR-32-5p+si-CEBPA group showed a significant decrease in CEBPA protein expression and a significant increase in EGFR,p-EGFR andβ-catenin protein expression(P<0.05).Conclusion:The proliferation,apoptosis and invasion of myeloma cells are regulated by both microRNA-32-5p and CEBPA.MicroRNA-32-5p can target CEBPA to regulate EGFR/β-catenin signaling pathway,which affects the proliferation,apoptosis and invasion of myeloma cells.