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Up‐regulation of miR‐210 induced by a hypoxic microenvironment promotes breast cancer stem cell 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 sternness 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 selfrenewal 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 cell metastasis, proliferation, and self‐renewal by targeting E‐cadherin. FASEB J. 32, 6965–6981 (2018). www.fasebj.org