Up-regulation of miR-210 induced by a hypoxic microenvironment promotes breast cancer stem cells metastasis, proliferation, and self-renewal by targeting E-cadherin.

Breast cancer stem cells (BCSCs), a small subset of breast cancer cells with stem cell-like properties, are essential in tumor formation, metastasis, resistance to anticancer therapies, and cancer recurrence. MicroRNAs (miRNAs) are involved in tumorigenicity by regulating specific oncogenes and tumor-suppressor genes, and their roles in BCSCs are becoming apparent. A novel, 3-dimensional (3D), semisolid culture system was established to culture MCF-7 spheroid cells with high percentage of BCSCs. The differences in miRNA expression among the MCF-7 parental cells, BCSC-enriched MCF-7 spheroid cells, and CD44/CD24 MCF-7 cells were evaluated by miRNA microarray, and the high expression of miR-210 in MCF-7 spheroid cells and CD44/CD24 MCF-7 cells was verified by quantitative RT-PCR. MCF-7 cells were cultured in a hypoxic chamber to detect the effect of hypoxia on miR-210 expression and the stemness of the cells. The 3-(4,5-dimethylthiazol-2- yl)-2,5-diphenyl tetrazolium bromide (MTT), transwell, and sphere-formation assays were performed to detect the proliferation, migration, and self-renewal ability of miR-210-overexpressed MCF-7 cells and MCF-7 spheroid cells with miR-210 knocked down. The target of miR-210 was validated with a dual-luciferase reporter assay and Western blotting. In vivo xenograft assay and metastasis assay were performed to study the effects of miR-210 targeting E-cadherin on BCSCs growth and lung metastasis, and the tumors were assessed by immunohistochemistry and immunofluorescence. We developed a novel 3D, semisolid culture system to culture MCF-7 spheroid cells, which are enriched in BCSCs, and found, by performing miRNAs expression profiling, miR-210 was up-regulated in those cells compared with MCF-7 parental cells. High miR-210 expression was also detected in CD44/CD24 MCF-7 cells and human CD44/CD24 breast cancer cells, which was demonstrated to be partially due to the hypoxic microenvironment around BCSCs in MCF-7 spheroids or solid tumors. Ectopic expression of miR-210 in MCF-7 cells promoted their migration, invasion, proliferation, and self-renewal in both in vitro and in vivo studies. We further reported that miR-210 suppressed E-cadherin expression by targeting the open reading frame region of E-cadherin mRNA and by up-regulation of E-cadherin transcription repressor, Snail. Accordingly, E-cadherin overexpression compromises the migration, invasion, proliferation, and self-renewal ability of miR-210-overexpressed MCF-7 both in vitro and in vivo. These findings reveal a novel regulatory pathway centered on hypoxia-mediated miR-210 targeting of E-cadherin, which contributes to the properties and breast tumorigenesis of BCSCs.-Tang, T., Yang, Z., Zhu, Q., Wu, Y., Sun, K., Alahdal, M., Zhang, Y., Xing, Y., Shen, Y., Xia, T., Xi, T., Pan, Y., Jin, L. Up-regulation of miR-210 induced by a hypoxic microenvironment promotes breast cancer stem cells metastasis, proliferation, and self-renewal by targeting E-cadherin.