miR-206 regulates 5-FU resistance by targeting Bcl-2 in colon cancer cells

Abstract

Introduction: Previous studies have found that miRNAs play a key role in drug resistance. Multiple reports show that miRNAs act as regulators in colorectal cancer (CRC) cells, but the role of miR-206 in CRC is still not well understood. The current study aimed to explore the potential function of miR-206 in 5-FU resistance.

Methods: To indentify the role of miR-206 in 5-FU resistance, the expression of miR-206 was examined by real-time polymerase chain reaction (RT-PCR) in 5-FU-resistant (FR) CRC (HCT116/FR and RKO/FR) and their parental cell lines. miR-206 mimic was transfected to 5-FU-FR CRC, and the 5-FU sensitivity was detected by MTS and flow cytometry. Using miRNA target prediction software, we found that miR-206 could target the 3' untranslated region (3'UTR) sequence of Bcl-2.

Results: miR-206 was found to be downregulated in 5-FU-FR CRC in comparison with their parental cell lines, suggesting its crucial relevance for colon cancer biology. Downregulation of miR-206 promoted drug resistance and decreased apoptosis of parental cells, while overexpression of miR-206 promoted drug cytotoxicity and apoptosis of HCT116/FR cells. We also identified miR-206 targeting Bcl-2 directly in CRC, which is required for miR-206 mediated-5-FU resistance.

Conclusion: Our results show that miR-206 targets Bcl-2 to mediate chemoresistance, proliferation, and apoptosis in CRC. This study provides a novel promising candidate for colon cancer therapy.

Keywords: 5-FU; Bcl-2; colorectal cancer; miR-206; resistance.

Figure 1

Generation of 5-FU-resistant HCT116 and RKO cells. Notes: (A) The indicated cell lines were treated with increasing concentrations of 5-FU for 72 hours. Cell proliferation was determined by MTS assay. (B) The indicated cell lines were treated with 40 μg/mL 5-FU for 48 hours. Apoptosis was analyzed by Annexin V/PI staining followed by flow cytometry. (C) The indicated cell lines were treated with 40 μg/mL 5-FU for 48 hours. Cell cycle distribution was detected by flow cytometry. Results in (A–C) are expressed as means ± SD of three independent experiments. **P<0.01. Abbreviations: 5-FU, 5-fluorouracil; PI, propidium iodide; SD, standard deviation.

Figure 2

miR-206 expression in 5-FU-resistant cells. Notes: (A) Relative miR-206 level in HCT116 parental cells and 5-FU-resistant cells was detected by real-time PCR. (B) Relative miR-206 level in RKO parental cells and 5-FU-resistant cells was detected by real-time PCR. Results are expressed as means ± SD of three independent experiments. **P<0.01. Abbreviations: 5-FU, 5-fluorouracil; PCR, polymerase chain reaction; SD, standard deviation.

Figure 3

Effect of miR-206 in 5-FU-resistant cells. Notes: (A) HCT116/FR and RKO/FR cells were transfected with miR-206 mimic or mimic-NC for 24 hours. Relative miR-206 level was detected by real-time PCR. (B) HCT116/FR cells were transfected with miR-206 mimic or mimic-NC for 24 hours, and then treated with increasing concentrations of 5-FU for 72 hours. Cell proliferation was determined by MTS assay. (C) RKO/FR cells were transfected with miR-206 mimic or mimic-NC for 24 hours, and then treated with increasing concentrations of 5-FU for 72 hours. Cell proliferation was determined by MTS assay. (D) HCT116/FR and RKO/FR cells were transfected with miR-206 mimic or mimic-NC for 24 hours, and then treated with 40 μg/mL 5-FU for 48 hours. Apoptosis was analyzed by Annexin V/PI staining followed by flow cytometry. Results are expressed as means ± SD of three independent experiments. *P<0.05; **P<0.01. Abbreviations: 5-FU, 5-fluorouracil; NC, negative control; PCR, polymerase chain reaction; PI, propidium iodide; SD, standard deviation.

Figure 4

Role of miR-206 in 5-FU resistance. Notes: (A) HCT116 and RKO cells were transfected with miR-206 inhibitor or inhibitor-NC for 24 hours. Relative miR-206 level was determined by real-time PCR. (B) HCT116 and RKO cells were transfected with miR-206 inhibitor or inhibitor-NC for 24 hours, and then treated with increasing concentrations of 5-FU for 72 hours. Cell proliferation was determined by MTS assay. (C) HCT116 and RKO cells were transfected with miR-206 inhibitor or inhibitor-NC for 24 hours, and then treated with 40 μg/mL 5-FU for 48 hours. Apoptosis was analyzed by Annexin V/PI staining followed by flow cytometry. Results are expressed as means ± SD of three independent experiments. *P<0.05; **P<0.01. Abbreviations: 5-FU, 5-fluorouracil; NC, negative control; PCR, polymerase chain reaction; PI, propidium iodide; SD, standard deviation.

Figure 5

Bcl-2 as a direct target gene of miR-206 in colon cancer. Notes: (A) The predicted binding site of miR-206 in the 3′-UTR of Bcl-2. (B) The relative luciferase activity of HCT116 cells was detected after Bcl-2-3′-UTR-WT or mut was cotransfected with miR-206 mimic and mimic-NC. (C) HCT116 and RKO cells were transfected with miR-206 mimic or mimic-NC for 24 hours, Bcl-2 mRNA was detected by real-time PCR (left), and Bcl-2 protein level was analyzed by Western blotting. (D) HCT116 and RKO cells were transfected with miR-206 inhibitor or inhibitor-NC for 24 hours, mRNA of Bcl-2 was detected by real-time PCR (left), and Bcl-2 protein level was analyzed by Western blotting. (E) HCT116 and RKO cells transfected with miR-206 inhibitor with or without Bcl-2 siRNA, and Bcl-2 expression were analyzed by Western blotting. Then, the cells were treated with increasing concentrations of 5-FU for 72 hours. Cell proliferation was determined by MTS assay. Results in (B–D) and (E) are expressed as means ± SD of three independent experiments. **P<0.01. Abbreviations: 5-FU, 5-fluorouracil; mut, mutation; NC, negative control; PCR, polymerase chain reaction; SD, standard deviation; WT, wild type.

Figure 6

miR-206 mediates 5-FU resistance in vivo. Notes: (A) Mice were treated with 5-FU, miR-206 mimic, or their combination. Tumor volume was calculated after treatment (n=6 in each group). *P<0.05; **P<0.01. (B) Paraffin-embedded sections of tumor tissues from mice were analyzed by TUNEL staining. Abbreviation: 5-FU, 5-fluorouracil.