miR-129-5p调控Wnt5a抑制胆囊癌细胞增殖和转移能力的机制研究

A Study on the Mechanism of MiR-129-5p Regulating Wnt5a to Inhibit theProliferation and Metastasis of Gallbladder Cancer Cells

目的研究微小RNA-129-5p(miR-129-5p)靶向调控Wnt5a抑制胆囊癌细胞增殖和转移能力的作用及机制。方法采用RT-PCR检测胆囊癌细胞NOZ、SGC-996、GBC-SD和正常胆管上皮细胞系HIBEpic中miR-129-5p的表达水平。选择miR-129-5p表达较低的胆囊癌细胞进行miR-129-5p mimic转染,分为对照组(NC组)和miR-129-5p过表达组(miR-129-5p mimic组),采用MTT实验检测各组细胞的增殖能力,Transwell实验检测各组细胞的转移能力。Targetscan7.1预测软件及双荧光素酶报道基因试验检测miR-129-5p对Wnt5a基因的靶向调控作用。RT-PCR检测胆囊癌细胞NOZ、SGC-996、GBC-SD及NC组、miR-129-5p mimic组中Wnt5a mRNA的表达水平。胆囊癌细胞分为NC组、miR-129-5p mimic组、miR-129-5p过表达与Wnt5a敲减质粒共转染组(miR-129-5p mimic+sh-Wnt5a组)和miR-129-5p过表达与Wnt5a过表达质粒共转染组(miR-129-5p mimic+oe-Wnt5a组),MTT实验检测各组细胞的增殖能力,Transwell实验检测各组细胞的转移能力。Western blot检测各组细胞中Wnt/β-catenin信号通路相关蛋白β-catenin、cyclinD1、cMYC和MMP2蛋白的表达。结果与正常胆管上皮细胞系HIBEpic相比,miR-129-5p在胆囊癌细胞系中的表达降低,在胆囊癌细胞GBC-SD中的表达最低。与NC组相比,miR-129-5p mimic组胆囊癌细胞增殖和转移能力均降低。Targetscan7.1预测软件、双荧光素酶报道基因及qRT-PCR试验表明miR-129-5p靶向调控Wnt5a。与HIBEpic相比,Wnt5a在胆囊癌细胞系中的表达增加。与NC组相比,miR-129-5p mimic组、miR-129-5p mimic+sh-Wnt5a组和miR-129-5p mimic+oe-Wnt5a组胆囊癌细胞增殖和转移能力均降低,其中miR-129-5p mimic+sh-Wnt5a组最低,miR-129-5p mimic+oe-Wnt5a组最高。与NC组相比,miR-129-5p mimic组、miR-129-5p mimic+sh-Wnt5a组和miR-129-5p mimic+oe-Wnt5a组胆囊癌细胞β-catenin、cyclinD1、cMYC和MMP2蛋白表达均降低,其中miR-129-5p mimic+sh-Wnt5a组最低,miR-129-5p mimic+oe-Wnt5a组最高。结论miR-129-5p靶向调控Wnt5a表达抑制胆囊癌细胞增殖和转移能力。

Objective To study the ability of microRNA-129-5p(microRNA-129-5p,miR-129-5p)to regulate Wnt5a(wingless-type MMTV integration site family,member 5A)and to inhibit the proliferation and metastasis of gallbladder cancer cells and its potential mechanisms.Methods RT-PCR was conducted to detect the expression level of miR-129-5p in gallbladder cancer cells NOZ,SGC-996,GBC-SD and normal bile duct epithelial cell line HIBEpic.We selected gallbladder cancer cells with low miR-129-5p expression for miR-129-5p mimic transfection,and divided them into the control group(NC group)and miR-129-5p overexpression group(miR-129-5p mimic group).MTT assay was performed to detect the proliferation ability of each group of cells,and Transwell method was used to detect the metastasis ability.Targetscan 7.1 prediction software and dual luciferase reporter gene test were used to detect the targeted regulation of miR-129-5p on Wnt5a gene.RT-PCR was conducted to detect the expression level of Wnt5a mRNA in gallbladder cancer cells NOZ,SGC-996,GBC-SD and NC groups,as well as the miR-129-5p mimic group.Gallbladder cancer cells were divided into the NC group,miR-129-5p mimic group,miR-129-5p overexpression and Wnt5a knockdown plasmid co-transfection group(miR-129-5p mimic+sh-Wnt5a group)and miR-129-5p overexpression and Wnt5a overexpression plasmid co-transfection group(miR-129-5p mimic+oe-Wnt5a group).MTT assay was used to detect the proliferation ability of each group of cells,and the Transwell method was used to detect the transfer ability.Western-blot was used to detect the expression of Wnt/β-catenin signaling pathway related proteinsβ-catenin,cyclinD1,cMYC and MMP2 protein in each group of cells.Results Compared with the normal bile duct epithelial cell line HIBEpic,the expression of miR-129-5p in the gallbladder cancer cell line was reduced,and the expression in the gallbladder cancer cell GBC-SD was the lowest.Compared with the NC group,the proliferation and metastasis ability of gallbladder cancer cells in the miR-129-5p mimic group were reduced.Targetscan7.1 prediction software,dual luciferase reporter gene and qRT-PCR test showed that miR-129-5p regulated Wnt5a.Compared with HIBEpic,the expression of Wnt5a in gallbladder cancer cell lines were increased.Compared with the NC group,the proliferation and metastasis ability of gallbladder cancer cells in the miR-129-5p mimic group,miR-129-5p mimic+sh-Wnt5a group,and miR-129-5p mimic+oe-Wnt5a group were reduced,and the miR-129-5p mimic+sh-Wnt5a group was the lowest,while the miR-129-5p mimic+oe-Wnt5a group was the highest.Compared with the NC group,expressions ofβ-catenin,cyclinD1,cMYC and MMP2 proteins of gallbladder cancer cells in the miR-129-5p mimic group,miR-129-5p mimic+sh-Wnt5a group and miR-129-5p mimic+oe-Wnt5a group were all decreased,among which the miR-129-5p mimic+sh-Wnt5a group was the lowest,while the miR-129-5p mimic+oe-Wnt5a group was the highest.Conclusion miR-129-5p targets the expression of Wnt5a to inhibit the proliferation and metastasis of gallbladder cancer cells.