miR-671-5p inhibits epithelial-to-mesenchymal transition by downregulating FOXM1 expression in breast cancer

Abstract

MicroRNA (miRNA) dysfunction is associated with a variety of human diseases, including cancer. Our previous study showed that miR-671-5p was deregulated throughout breast cancer progression. Here, we report for the first time that miR-671-5p is a tumor-suppressor miRNA in breast tumorigenesis. We found that expression of miR-671-5p was decreased significantly in invasive ductal carcinoma (IDC) compared to normal in microdissected formalin-fixed, paraffin-embedded (FFPE) tissues. Forkhead Box M1 (FOXM1), an oncogenic transcription factor, was predicted as one of the direct targets of miR-671-5p, which was subsequently confirmed by luciferase assays. Forced expression of miR-671-5p in breast cancer cell lines downregulated FOXM1 expression, and attenuated the proliferation and invasion in breast cancer cell lines. Notably, overexpression of miR-671-5p resulted in a shift from epithelial-to-mesenchymal transition (EMT) to mesenchymal-to-epithelial transition (MET) phenotypes in MDA-MB-231 breast cancer cells and induced S-phase arrest. Moreover, miR-671-5p sensitized breast cancer cells to cisplatin, 5-fluorouracil (5-FU) and epirubicin exposure. Host cell reactivation (HCR) assays showed that miR-671-5p reduces DNA repair capability in post-drug exposed breast cancer cells. cDNA microarray data revealed that differentially expressed genes when miR-671-5p was transfected are associated with cell proliferation, invasion, cell cycle, and EMT. These data indicate that miR-671-5p functions as a tumor suppressor miRNA in breast cancer by directly targeting FOXM1. Hence, miR-671-5p may serve as a novel therapeutic target for breast cancer management.

Keywords: EMT; FOXM1; breast cancer; miR-671-5p; tumor suppressor.

Figure 1. Expression of miR-671-5p in IDC vs. adjacent normal

Black bars depict TNBCs while gray bars are for non-TNBCs, in comparison to the normal in white. Down-regulation of miR-671-5p expression was present in 21 of 30 (70%) of IDC compared with their adjacent normal tissue (p < 0.05).

Figure 2. miR-671-5p targets FOXM1 in breast cancer cell lines

(A) Inverse correlated expression of miR-671-5p and its target FOXM1 in breast cancer cell lines. (B) Location of the miR-671-5p binding site at the 3′-UTR of FOXM1 corresponding to the miR-671-5p sequence. (C) Relative luciferase activity was measured in breast cancer cell lines co-transfected with 100 ng of DNA with pEZX-miR-671-5p or pEZX-scrambled control (mock), and 100 ng of pEZX-MT05-FOXM1 (wild type or mutant) by FuGENE reagent (Promega) for 48 h. The data were reported as mean ± S.D. for three independent experiments (*p < 0.05).

Figure 3. miR-671-5p negatively regulates FOXM1 expression in breast cancer cell lines

(A) qRT-PCR analysis of miR-671-5p and FOXM1 mRNA expression in breast cancer cell lines transfected with miR-671-5p mimic or mock (**p < 0.01, *p < 0.05). (B) Immunofluorescence analysis of FOXM1 protein expression in MDA-MB-231 cells transfected with miR-671-5p or mock. (C) Western blotting analysis of FOXM1 expression in breast cancer cell lines. FOXM1 expression was decreased more significantly in MDA-MB-231 (71%) and MCF-7 (60%) cells in comparison with SKBR3 (23%) cells when miR-671-5p was overexpressed.

Figure 4. miR-671-5p inhibits proliferation and decreases invasive ability of breast cancer cell lines

(A) FOXM1 protein expression was examined by Western blot after transfection of miR-671-5p mimic and inhibitor. (B) Effects of miR-671-5p on cell proliferation were determined by MTT assays. Proliferative activity was decreased after transfection of miR-671-5p mimic and increased after transfection of miR-671-5p inhibitor compared to the mock control in breast cancer cell lines. Values represent the mean ± S.D. for three independent experiments (**p < 0.01, *p < 0.05). (C) Transwell assays with Matrigel were performed for the invasion activity of breast cancer cells transfected with either miR-671-5p mimic or the mock control. Overexpression of miR-671-5p significantly reduces cell invasion in MDA-MB-231, Hs578T and SKBR3, but only slightly in MCF-7 and T47D. Invasion ability of the cells was displayed as a percentage of the absolute cell number (bottom). Results are displayed as mean data ± SE (**p < 0.01, *p < 0.05). Five fields of unit area on each membrane or whole membrane were counted for cell numbers, and the experiments were repeated three times in triplicate.

Figure 5. miR-671-5p induces S-phase arrest and inhibits EMT

(A) Schematic diagram of miR-671-5p precursor sequence in pEZX-MR04. (B) Overexpression of miR-671-5p (top) induces S-phase arrest by flow cytometry (middle) and downregulated CCNB1 expression (bottom) in miR-671-5p stable transfected MDA-MB-231 cells when compared with mock transfected cells using Western blot analysis. (C) Overexpression of miR-671-5p shifts MDA-MB-231 cells from EMT to MET phenotype. Top panel showing cell morphology was observed by microscopy in MDA-MB-231 cells transfected with mock and miR-671-5p. Mock transfected MDA-MB-231 cells displayed elongated, irregular fibroblastoid morphology whereas miR-671-5p transfected cells showed a more epithelioid appearance. The middle panel shows immunofluorescence staining of E-cadherin and vimentin in the indicated cells. The bottom panel shows the Western blot analysis of E-cadherin and vimentin protein levels in indicated cells.

Figure 6. Effect of miR-671-5p on sensitivity of breast cancer cell lines to UVC/Chemosensitivity

miR-671-5p or mock was stable transfected into MDA-MB-231 cell line. The stable transfected MDA-MB-231 cell line was further transfected with miR-671-5p inhibitor or mock. Cells were treated by cisplatin, 5-FU, epirubicin and UVC respectively. Cell sensitivity was measured by MTT assays. miR-671-5p overexpression significantly increased cell sensitivity to cisplatin, 5-FU and epirubicin. Results are displayed as mean data ± SE. **p < 0.01 and *p < 0.05 are considered statistically significant with comparison to the mock.

Figure 7. A schematic model for the regulation of miR-671-5p

miR-671-5p directly targets FOXM1. Down-regulation of FOXM1 could 1) inhibit GINS2 and promote cell proliferation; 2) inhibit CDK2 and enhance cell invasion and induce S-phase arrest; 3) inhibit CCNB1 to induce S-phase arrest; 3) inhibit MCM10 which is involved in S-phase arrest and EMT; 4) inhibit CCNB1 to induce S-phase arrest; 5) affect DNA repair gene(s) to function in chemotherapy.