miR-361 enhances sensitivity to 5-fluorouracil by targeting the FOXM1-ABCC5/10 signaling pathway in colorectal cancer

Abstract

Colorectal cancer (CRC) is one of most common malignancies worldwide. 5-fluorouracil (5-FU) is a mainstay of CRC treatment, particularly in patients with advanced stages of the disease; however, 5-FU-based chemotherapy is not always effective and may result in progression of the disease. The present study investigated several candidate microRNAs (miRs) in parental and 5-FU-resistant HCT116 and HT29 cells, and identified miR-361 as a novel regulator of chemosensitivity. Overexpression of miR-361 enhanced the 5-FU susceptibility of parental and resistant HCT116 and HT29 cells in vitro. Impaired colony formation capacity and increased cell apoptosis (as determined via flow cytometry) was observed in resistant HCT116 and HT29 cells. Furthermore, forkhead box M1 (FOXM1) was identified as a target gene of miR-361 using a dual-luciferase reporter assay, western blotting and reverse transcription-quantitative PCR. Additionally, FOXM1 knockdown improved the cytotoxicity of 5-FU in resistant CRC. ATP binding cassette subfamily C members 5 and 10 (ABCC5/10) were found to be downstream effectors of miR-361. In conclusion, miR-361 increased chemosensitivity, at least in part, via modulation of FOXM1-ABCC5/10. miR-361 may serve as a potential therapeutic target for patients with CRC.

Keywords: 5-fluorouracil; ATP binding cassette subfamily C; chemosensitivity; colorectal cancer; forkhead box M1; microRNA-361.

Figure 1.

miR-361 is upregulated in 5-FU-resistant HCT116 and HT29 cells. (A) Parental and 5-FU-resistant HCT116 and HT29 cells were treated with 5-FU at the indicated concentrations for 48 h and subjected to a CCK-8 assay. Cell viability was presented as the relative value normalized to the 0 µg/ml group (n=6; **P<0.01, parental vs. resistant cells; ^##P<0.01, the viability of resistant cells at 20 µg/ml vs. 0 µg/ml). (B) Relative differential expression of candidate miRNAs between parental and 5-FU resistant HCT116 and HT29 cells (n=3; *P<0.05, **P<0.01, vs. parental cells). (C) Overexpression of miR-361 sensitizes parental and resistant HCT116 and HT29 to 5-FU. The parental and 5-FU-resistant HCT116 and HT29 cells were transfected with an miR-361 mimic or NC, exposed to 5-FU at the indicated concentrations for 48 h and subjected to a CCK-8 assay (n=6; **P<0.01). miR/miRNA, microRNA; 5-FU, 5-fluorouracil; CCK-8, Cell Counting Kit-8; NC, negative control; Res, resistant.

Figure 2.

miR-361 inhibits colony formation and activates caspase 3/7 in resistant HCT116 and HT29 cells. (A) Colony formation ability of resistant miR-361-overexpressing cells and NC cells (n=3; **P<0.01). (B) Caspase 3/7 activities of resistant miR-361 overexpressing cells and NC cells were measured via a caspase 3/7 Glo Luc assay (n=3; **P<0.01). miR, microRNA; NC, negative control; Res, resistant; 5-FU, 5-fluorouracil.

Figure 3.

miR-361 induces cell apoptosis in resistant HCT116 and HT29 cells. Annexin V staining of resistant miR-361-overexpressing (A) HCT116 and (B) HT29 cells. Q1: Dead cells, Q2: Late apoptotic cells, Q3: Living cells, Q4: Early apoptotic cells. The histogram indicated the percentage of early, late and total apoptotic cells with different concentrations of 5-FU. The total apoptotic cells include early and late apoptotic cells (n=3, **P<0.01 vs. NC cells). miR, microRNA; 5-FU, 5-fluorouracil; NC, negative control; Res, resistant.

Figure 4.

miR-361 is a direct regulator of FOXM1 in resistant colorectal cancer cells. (A) Schematic representation of the luciferase reporter constructs. The phosphoglycerate kinase promoter in the reporter construct drives the constitutive transcription of a chimeric mRNA containing the firefly luciferase coding sequence fused to the wide-type or mutated FOXM1 3′UTR. (B) FOXM1 expression in parental and 5-FU resistant colorectal cancer cells. (C) Expression levels of miR-361 in resistant HCT116 and HT29 cells transfected with miR-361 mimic or NC (n=3; **P<0.01 vs. NC cells). (D) mRNA and (E) protein levels of FOXM1 in resistant HCT116 and HT29 cells overexpressing miR-361 and NC (n=3; **P<0.01). (F) Relative activity of the luciferase gene fused with the wild-type or mutant FOXM1 3′UTR in resistant HCT116 and HT29 cells. Data were normalized to Renilla luciferase activity (n=3; **P<0.01) miR, microRNA; FOXM1, forkhead box M1; UTR, untranslated region; 5-FU, 5-fluorouracil; NC, negative control; Res, resistant; RLU%, Percentage of relative luminescence; WT, wild type; Mut, mutant.

Figure 5.

Inhibition of FOXM1 suppresses colony formation and induces apoptosis through downregulation of ABCC5/10. (A) mRNA and (B) protein levels of FOXM1 were downregulated by siRNA knockdown. (C) Knockdown of FOXM1 resulted in impaired colony formation (D) Knockdown of FOXM1 in resistant cells increased caspase 3/7 activity (n=3; **P<0.01). (E) Protein levels of ABCC5/10 in parental, resistant cells, miR-361-overexpressing and FOXM1 knockdown cells. (F) Reduced expression of miR-361 in colorectal cancer cells contributes to 5-FU chemoresistance by activating the FOXM1-ABCC5/10 signaling pathway. FOXM1, forkhead box M1; ABCC, ATP binding cassette subfamily C; si(RNA), small interfering (RNA); NC, negative control; miR, microRNA; 5-FU, 5-fluorouracil; Res, resistant.