miR-139-5p靶向PAK5基因通过Wnt/β-catenin信号通路对胃癌细胞侵袭和迁移的影响

MiR-139-5p inhibits invasion and migration of gastric cancer cells by targeting PAK5 gene to block Wnt/β-catenin signaling pathway

背景胃癌(Gastric cancer,GC)是人类消化系统最常见的癌症类型,发生局部或全身转移是导致患者预后差的主要原因.微小RNA(microRNA,miRNA)是胃癌发生发展的重要调控因子.然而,miR-139-5p对胃癌细胞侵袭和转移的影响及机制尚不清楚.目的探究miR-139-5p靶向p21活化的激酶5(PAK5)基因调控Wnt/β-catenin信号通路对胃癌细胞侵袭和迁移的影响.方法采用实时荧光定量PCR(qRT-PCR)和Western blot分别检测永生化胃黏膜细胞GES1和不同胃癌细胞SGC-7901、AGS和BGC-823中miR-139-5p和PAK5的表达情况;在胃癌SGC-7901细胞中转染miR-139-5p mimics,qRT-PCR检测转染效果;Transwell侵袭和划痕实验检测过表达miR-139-5p对SGC-7901细胞侵袭和迁移能力的影响;双荧光素酶报告基因实验和Western blot检测miR-139-5p对PAK5的靶向调控关系;Western blot检测过表达miR-139-5p对Wnt/β-catenin信号通路激活的影响.结果胃癌细胞中miR-139-5p的表达水平明显低于正常胃黏膜细胞(P<0.05),而PAK5 mRNA和蛋白表达水平均明显高于正常胃黏膜细胞(P<0.05);转染miR-139-5p mimics能够上调SGC-7901细胞中miR-139-5p的表达;过表达miR-139-5p可明显抑制SGC-7901细胞侵袭和迁移能力;双荧光素酶报告基因实验和Western blot检测结果显示,miR-139-5p可靶向负调控PAK5的表达;过表达miR-139-5p后,SGC-7901细胞中Wnt3a、β-连环蛋白(β-catenin)、细胞周期蛋白D1(CyclinD1)蛋白表达均明显下调.结论miR-139-5p靶向PAK5并通过阻断Wnt/β-catenin信号通路抑制胃癌细胞侵袭和迁移能力.

BACKGROUND Gastric cancer(GC)is the most common type of cancer of the digestive system.Local or systemic metastasis is the main cause of poor prognosis.MicroRNAs(miRNAs)are an important regulatory factor in the development of gastric cancer.However,the effect and mechanism of miR-139-5p on the invasion and metastasis of gastric cancer cells are still unclear.AIM To explore the effect of miR-139-5p on the invasion and migration of gastric cancer cells and the underlying mechanism.METHODS Real-time fluorescent quantitative PCR(qRT-PCR)and Western blot were used to detect the expression of miR-139-5p and PAK5,respectively,in immortalized gastric mucosal cell line GES1 and gastric cancer cell lines SGC-7901,AGS,and BGC-823.MiR-139-5p mimic was transfected into gastric cancer SGC-7901 cells,and qRTPCR was used detect the transfection efficiency.Transwell invasion and scratch assays were used to detect the effect of overexpression of miR-139-5p on the invasion and migration of SGC-7901 cells.Dual luciferase reporter gene assay and Western blot were used to detect the targeted regulation of miR-139-5p on PAK5.Western blot was used to detect the effect of overexpression of miR-139-5p on the activation of Wnt/β-catenin signaling pathway.RESULTS The expression level of miR-139-5p in gastric cancer cells was significantly lower than that of normal gastric mucosal cells(P<0.05),and the expression of PAK5 mRNA and protein was significantly higher than that of normal gastric mucosal cells(P<0.05).Transfection of miR-139-5p mimic up-regulated the expression of miR-139-5p in SGC-7901 cells.Overexpression of miR-139-5p significantly inhibited the invasion and migration of SGC-7901 cells.The results of dual luciferase reporter gene assay and Western blot showed that miR-139-5p can target and negatively regulate the expression of PAK5.After overexpression of miR-139-5p,the expression of Wnt3a,β-catenin,and Cyclin D1 proteins in SGC-7901 cells was significantly downregulated.CONCLUSION MiR-139-5p inhibits the invasion and migration of gastric cancer cells by targeting the PAK5 gene to block the Wnt/β-catenin signaling pathway.