吉林大学学报(医学版) ›› 2024, Vol. 50 ›› Issue (4): 947-955.doi: 10.13481/j.1671-587X.20240408

• 基础研究 • 上一篇    下一篇

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

胡兵兵1,罗康宁2(),彭肃2,周煜中2,陈茂良2,刘昌化2   

  1. 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
  • 收稿日期:2023-08-03 出版日期:2024-07-28 发布日期:2024-08-01
  • 通讯作者: 罗康宁 E-mail:lkn1987622@126.com
  • 作者简介: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。
  • 基金资助:
    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)

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

Bingbing HU1,Kangning LUO2(),Su PENG2,Yuzhong ZHOU2,Maoliang CHEN2,Changhua LIU2   

  1. 1.Department of Vascular Hernia Pediatric,Second Affiliated Hospital,Hengyang Medical School,University of South China,Hengyang 421001,China
    2.Department of Gastrointestinal Surgery,Second Affiliated Hospital,Hengyang Medical School,University of South China,Hengyang 421001,China
  • Received:2023-08-03 Online:2024-07-28 Published:2024-08-01
  • Contact: Kangning LUO E-mail:lkn1987622@126.com

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。

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

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.

Key words: Colorectal neoplasm, MicroRNA-208a, Secreted crimp-related protein 1, Wnt signaling pathway, 5-flurouracil, Drug resistance

中图分类号: 

  • R735.1