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