下调miR-208a通过靶向SFRP1介导Wnt信号通路对结直肠癌细胞5-FU耐药的改善作用  

Improvement effect of down-regulation of miR-208a on 5-FU resistance in colorectal cancer cells through targeting SFRP1 for mediating Wnt signaling pathway

作　　者：胡兵兵 罗康宁 彭肃 周煜中 陈茂良 刘昌化 HU Bingbing;LUO Kangning;PENG Su;ZHOU Yuzhong;CHEN Maoliang;LIU Changhua(Department of Vascular Hernia Pediatric,Second Affiliated Hospital,Hengyang Medical School,University of South China,Hengyang 421001,China;Department of Gastrointestinal Surgery,Second Affiliated Hospital,Hengyang Medical School,University of South China,Hengyang 421001,China)

机构地区：[1]南华大学衡阳医学院附属第二医院血管疝儿外科,湖南衡阳421001 [2]南华大学衡阳医学院附属第二医院胃肠外科,湖南衡阳421001

出　　处：《吉林大学学报（医学版）》2024年第4期947-955,共9页Journal of Jilin University：Medicine Edition

基　　金：湖南省科技厅创新型省份建设专项科普专题项目(2022ZK4289);湖南省卫健委科研项目(202204013770,202204014806)。

摘　　要：目的:探讨下调微小RNA-208a(miR-208a)对结直肠癌细胞5-氟尿嘧啶(5-FU)耐药的影响,阐明其相关分子机制。方法:采用实时荧光定量PCR(RT-qPCR)法检测结直肠癌5-FU耐药细胞株HT-29/5-FU及其亲本HT-29细胞中miR-208a和分泌型卷曲相关蛋白1(SFRP1)mRNA表达水平。以HT-29/5-FU细胞为研究对象,将miR-208a抑制物(miR-208a inhibitor)质粒及其阴性对照质粒(inbibitor-NC)和SFRP1小干扰质粒(si-SFRP1)及其阴性对照质粒(si-NC)分别或同时转染至HT-29/5-FU细胞中,联合5-FU处理,将细胞分为空白组、inhibitor-NC组、miR-208a inhibitor组、miR-208a inhibitor+si-NC组和miR-208a inhibitor+si-SFRP1组。MTT法检测各组细胞增殖活性并计算耐药指数,AnnexinⅤ-FITC/PI双染法结合流式细胞术检测不同浓度5-FU作用后各组细胞凋亡率,Western blotting法检测各组细胞中SFRP1、β-连环蛋白(β-catenin)、P-糖蛋白(P-gp)和ATP结合盒B亚家族成员1转运蛋白(ABCB1)蛋白表达水平。双荧光素酶报告基因实验验证miR-208a与SFRP1的靶向关系。结果:与HT-29细胞比较,HT-29/5-FU细胞中miR-208a表达水平升高(P<0.05),SFRP1 mRNA表达水平降低(P<0.05)。与inhibitor-NC组比较,miR-208a inhibitor组细胞增殖活性降低(P<0.05),耐药指数降低,细胞凋亡率升高(P<0.05),细胞中β-catenin、P-gp和ABCB1蛋白表达水平降低(P<0.05)。双荧光素酶报告基因实验提示SFRP1是miR-208a靶基因,且miR-208a可负向调控SFRP1的表达。与miR-208a inhibitor+si-NC组比较,miR-208a inhibitor+si-SFRP1组细胞增殖活性升高(P<0.05),耐药指数升高,细胞凋亡率降低(P<0.05),细胞中β-catenin、P-gp和ABCB1蛋白表达水平升高(P<0.05)。结论:下调miR-208a可通过靶向上调SFRP1表达抑制Wnt信号通路的转导,进而改善HT-29/5-FU细胞对5-FU的耐药。Objective:To discuss the effect of downregulating microRNA-208a(miR-208a)on the resistance of the colorectal cancer cells to 5-fluorouracil(5-FU),and to clarify its related molecular mechanism.Methods:Real-time fluorescence quantitative PCR(RT-qPCR)method was used to detect the expression levels of miR-208a and secreted frizzled-related protein 1(SFRP1)mRNA in the 5-FUresistant colorectal cancer cell line HT-29/5-FU and its parent HT-29 cells.The HT-29/5-FU cells were transfected with miR-208a inhibitor plasmid and its negative control plasmid(inhibitor-NC),and SFRP1 small interfering RNA(si-SFRP1)and its negative control plasmid(si-NC),either separately or in combination,followed by treatment with 5-FU.The cells were divided into inhibitor-NC group,miR-208a inhibitor group,miR-208a inhibitor+si-NC group,and miR-208a inhibitor+si-SFRP1 group.MTT assay was used to detect the proliferation activities of the cells and the resistance indexes were calculated;AnnexinⅤ-FITC/PI double staining and flow cytometry were used to detect the apoptotic rates of the cells after treated with different concentrations of 5-FU;Western blotting method was used to detect the expression levels of SFRP1,β-catenin,P-glycoprotein(P-gp),and ATP-binding cassette subfamily B member 1(ABCB1)proteins in the cells in various groups;dual-luciferase reporter gene assay was used to validate the targeting relationship between miR-208a and SFRP1.Results:Compared with HT-29 cells,the expression level of miR-208a in the HT-29/5-FU cells was increased(P<0.05),and the expression level of SFRP1 mRNA was decreased(P<0.05).Compared with inhibitor-NC group,the proliferation activity of the cells in miR-208a inhibitor group was decreased(P<0.05),the resistance index was decreased,the apoptotic rate was increased(P<0.05),and the expression levels ofβ-catenin,P-gp,and ABCB1 proteins in the cells were decreased(P<0.05).The dual-luciferase reporter gene assay results showed that SFRP1 was a target gene of miR-208a and miR-208a could negatively regulate the e

关 键 词：结直肠肿瘤 微小RNA-208a 分泌型卷曲相关蛋白1 WNT信号通路 5-氟尿嘧啶 耐药性 

分 类 号：R735.1[医药卫生—肿瘤]