过表达微小RNA-877-5p通过靶向叉头框转录因子M1调控胃癌细胞活力和凋亡

Over-expression of miR-877-5p regulates the viability and apoptosis of gastric cancer cells by targeting FOXM1

目的探讨微小RNA-877-5p(miR-877-5p)对胃癌细胞活力、凋亡的影响及其分子机制。方法本研究时间为2020年1—7月。胃癌和正常胃黏膜上皮细胞株购自美国典型培养物保藏中心。实时定量基因扩增荧光检测(qPCR)检测胃癌细胞株HGC-27、SUN-1、AGS和正常胃黏膜上皮细胞株GES-1中miR-877-5p和叉头框转录因子M1(FOXM1)信使核糖核酸(mRNA)表达。建立miR-877-5p过表达或抑制FOXM1表达细胞株,观察其在HGC-27细胞的活力、凋亡中的作用。MTT法检测细胞的活力,流式细胞术检测细胞凋亡,蛋白质印迹法(Western blotting)检测FOXM1、细胞周期蛋白D1(cyclinD1)、细胞周期依赖性激酶抑制因子p21、p27、B细胞淋巴瘤/白血病-2(Bcl-2)、Bcl-2相关X蛋白(Bax)、活化的含半胱氨酸的天冬氨酸蛋白水解酶3(cleaved-caspase 3)蛋白表达。TargetScan预测结合双荧光素酶报告实验分析miR-877-5p和FOXM1的靶向关系。共转染miR877-5p模拟物和FOXM1过表达载体(pcDNA-FOXM1),研究FOXM1过表达对miR-877-5p过表达诱导的HGC-27细胞增殖和凋亡的影响。结果与正常胃黏膜上皮细胞GES-1比较,胃癌细胞HGC-27、SUN-1、AGS中的miR-877-5p表达下调(1.00±0.08比0.34±0.03,0.51±0.05,0.44±0.04,P<0.05),FOXM1 mRNA和蛋白表达上调(1.00±0.09比2.41±0.23,2.58±0.24,2.26±0.23,P<0.05)。miR-877-5p过表达显著降低HGC-27细胞48 h、72 h的细胞活力(P<0.05),明显提高细胞凋亡率、p21、p27、Bax、cleavedcaspase3蛋白的水平(P<0.05),显著减少cyclinD1、Bcl-2蛋白表达量(P<0.05)。抑制FOXM1表达显著降低48 h、72 h的细胞活力(P<0.05),提高细胞凋亡率、p21、Bax蛋白水平(P<0.05),减少cyclinD1、Bcl-2蛋白表达量(P<0.05)。miR-877-5p靶向调控FOXM1的表达。FOXM1过表达后,miR-877-5p过表达对HGC-27细胞增殖、cyclinD1、Bcl-2蛋白表达的抑制作用被逆转,其对细胞凋亡、p21、Bax蛋白表达的促进作用也被逆转。结论过表达miR-877-5p通过靶向调控FOXM1表达抑制胃癌细胞的活力,并诱导细胞凋亡。

Objective To investigate the effect of microRNA-877-5p(miR-877-5p)on the viability and apoptosis of gastric cancer cells and its molecular mechanism.Methods This study was conducted from January to July 2020.Gastric cancer and normal gastric mucosal epithelial cell lines were purchased from the American Type Culture Collection.qPCR was used to detect the expressions of miR-877-5p and Forkhead box M1(FOXM1)transcription factor messenger ribonucleic acid(mRNA)in gastric cancer cell lines HGC27,SUN-1,AGS and normal gastric mucosal epithelial cell line GES-1.The miR-877-5p over-expressing or inhibiting FOXM1 expressing cell line was established,and their role in the proliferation and apoptosis of HGC-27 cells was observed.The cell viability was detected by MTT assay,apoptosis was detected by flow cytometry,and the protein levels of FOXM1,cyclinD1,cycle-dependent kinase inhibitor(p21,p27),B cell lymphoma/leukemia-2(Bcl-2),Bcl-2 associated X protein(Bax),cleaved cysteinyl aspartate specific proteinase 3(cleaved-caspase 3)were determined by Western blotting.TargetScan together with the dual luciferase reporter assay was used to analyze the targeting relationship between miR-877-5p and FOXM1.miR-877-5p mimic and FOXM1 overexpression vector(pcDNAFOXM1)were co-transfected to study the effect of FOXM1 over-expression on the viability and apoptosis of HGC-27 cells induced by miR-877-5p over-expression.Results Compared with normal gastric mucosal epithelial cell line GES-1,the expressions of miR-877-5p in the gastric cancer cell lines HGC-27,SUN-1 and AGS were down-regulated(1.00±0.08 vs.0.34±0.03,0.51±0.05,0.44±0.04,P<0.05),and the FOXM1 mRNA and protein levels were up-regulated(1.00±0.09 vs.2.41±0.23,2.58±0.24,2.26±0.23,P<0.05).Over-expression of miR-877-5p significantly decreased the cell viability of HGC-27 cells at 48 h and 72 h(P<0.05),evidently increased the apoptotic rate,p21,p27,Bax,cleaved-caspase3 protein levels(P<0.05),and reduced the levels of cyclinD1 and Bcl-2 protein(P<0.05).Inhibition of FOXM1 expression obviously reduced cell viability at 48h and 72h(P<0.05),increased apoptotic rate,p21 and Bax protein levels(P<0.05),and decreased cyclinD1 and Bcl-2 protein levels(P<0.05).miR-877-5p targeted to regulate the expression of FOXM1.Over-expression of FOXM1 reversed the inhibitory effect of miR-877-5p over-expression on the proliferation of HGC-27 cells,the protein expressions of cyclinD1 and Bcl-2,and the promotion of apoptosis,p21 and Bax protein levels.Conclusion Over-expression of miR-877-5p inhibits the viability of gastric cancer cells and induces apoptosis by targeting FOXM1 expression.