Improvement effect of down-regulation of miR-208a on 5-FU resistance in colorectal cancer cells through targeting SFRP1 for mediating Wnt signaling pathway
Improvement effect of down-regulation of miR-208a on 5-FU resistance in colorectal cancer cells through targeting SFRP1 for mediating Wnt signaling pathway
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
维普
万方数据