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

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，Annexin Ⅴ-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。

Abstract

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-FU-resistant 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； Annexin Ⅴ-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 of β-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 expression of SFRP1. Compared with miR-208a inhibitor+si-NC group， the proliferation activity of the cells in miR-208a inhibitor+si-SFRP1 group was increased （P<0.05）， the resistance index was increased， the apoptotic rate was decreased（P<0.05）， and the expression levels of β-catenin， P-gp， and ABCB1 proteins in the cells were increased （P<0.05）. Conclusion Downregulation of miR-208a can improve the resistance of the HT-29/5-FU cells to 5-FU by targeting and upregulating the SFRP1 expression， thereby inhibiting the transmission of the Wnt signaling pathway.

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

Key words

Colorectal neoplasm / MicroRNA-208a / Secreted crimp-related protein 1 / Wnt signaling pathway / 5-flurouracil / Drug resistance

Author summay

胡兵兵（1986-），男，湖南省衡阳市人，主治医师，医学硕士，主要从事胃肠肿瘤基础和临床方面的研究。