MiR-199a-5p confers tumor suppressive role by inhibiting stemness characteristics in triple negative breast cancer

Abstract

OBJECTIVES: Triple negative breast cancer (TNBC) is defined by lack of expression in estrogen (ER) and progesterone receptors (PR) as well as human epidermal growth factor receptor 2 (HER2), which accounts for 10-15% of all breast cancers. TNBC is a more aggressive histological subtype with worse prognosis when compared to other subtypes due to limited therapeutic targets for hormonal and HER2 receptors. With limited treatment options and ineffective targeted therapy, there is an urgent need for the development of novel therapeutic targets to treat this malignant form of breast cancer. METHODS: Focused on the previously identified TNBC biomarker miR-199a-5p by our group, cells were transfected with 199a-5p mimic for functional studies. MTT and wound healing assay were performed to investigate the cell proliferation and migration ability. Immunofluorescence staining was introduced to study TWIST1 and E-cadherin expression in miR199a-5p stable transfected cells. Aldehyde dehydrogenase (ALDH) expression level was compared between breast cancers and normal control cases’ plasma by using real-time RT-PCR. We further used ALDH activity assay to evaluate the stem cell population. In vivo study was also carried out in nude mice to study tumorigenicity. RESULTS: Cell proliferation assay showed that overexpression of miR-199a-5p significantly inhibited cell growth (p = 0.0008). Wound healing assay indicated the ectopic expression of miR-199a-5p can inhibit cell migration compared to control group. In miR-199a-5p stable transfected MDA-MB-231 cell line, increased translocation of Twist 1 protein from nucleus to cytoplasm and increased expression of E-cadherin was detected by immunofluorescence staining. ALDH activity assay result indicated that the proportion of ALDH(BR+) cells decreased significantly from 17.21% to 8.95% in the miR-199a-5p transfected cells when compared to the control group (p = 0.0279). Consistent with in vitro study, the well-recognized breast cancer stem-cell marker, ALDH1A, was significantly upregulated in the plasma of breast cancer patients than in healthy controls. Moreover, in vivo study also revealed that stable MDA-MB-231 cell line with ectopic miR199a-5p expression resulted in smaller volume of tumors in nude mice. CONCLUSIONS: Our data implicate that miR-199a-5p plays a tumor suppressive role in the pathogenesis of TNBC, which may due to the inhibition of stemness characteristics in breast cancer cells. These findings suggest that miR-199a-5p involved in TNBC stemness-related features and thus provide insights in development of the novel therapeutic strategies for this highly malignant breast cancer.