MiR-21 regulates epithelial-mesenchymal transition phenotype and hypoxia-inducible factor-1α expression in third-sphere forming breast cancer stem cell-like cells

Abstract

Cancer stem cells (CSCs) are predicted to be critical drivers of tumor progression due to their "stemness", but the molecular mechanism of CSCs in regulating metastasis remains to be elucidated. Epithelial-mesenchymal transition (EMT), hypoxia-inducible factor (HIF)-1α, and miR-21, all of which contribute to cell migration for metastasis, are interrelated with CSCs. In the present study, third-sphere forming (3-S) CSC-like cells, which showed elevated CSC surface markers (ALDH1(+) and CD44(+)/CD24(-/low)) and sphereforming capacity as well as migration and invasion capacities, were cultured and isolated from breast cancer MCF-7 parental cells, to evaluate the role of miR-21 in regulating the CSC-like cell biological features, especially EMT. EMT, which was assessed by overexpression of mesenchymal cell markers (N-cadherin, Vimentin, alpha-smooth muscle actin [α-SMA]) and suppression of epithelial cell marker (E-cadherin), was induced in 3-S CSC-like cells. Moreover, both of HIF-1α and miR-21 were upregulated in the CSC-like cells. Interestingly, antagonism of miR-21 by antagomir led to reversal of EMT, downexpression of HIF-1α, as well as suppression of invasion and migration, which indicates a key role of miR-21 involved in regulate CSC-associated features. In conclusion, we demonstrated that the formation of CSC-like cells undergoing process of EMT-like associated with overexpression of HIF-1α, both of which are regulated by miR-21.

Figure 1

Culture and identification of mammospheres. (a) Typical images of 1‐S, 2‐S, 3‐S, 4‐S. (b) The mammosphere forming efficiencies (MSFEs) were calculated and analysis based on 1000 single cells that were initially seeded. (c,d) Examples and analysis of ALDH ^bright cell subpopulation in indicated cells, by ALDEFLUOR assay. (e,f) Examples and analysis of CD44⁺/CD24^−/low phenotype cell subpopulation in indicated cells, by fluorescence‐activated cell sorting (FACS). (g) The protein levels of ALDH1 and CD44 in indicated cells by western blot analysis. (h) Immunofluorescence images of the P cells and 3‐S cells stained with antibody against ALDH1 (green), and cell nuclei were stained with 4′6′‐diamidino‐2‐phenylindole dihydrochloride (DAPI) (blue). (i) Immunofluorescence images stained with antibody against CD44 (green) and DAPI (blue).

Figure 2

Breast cancer stem cell (bCSC)‐like cells induced epithelial‐mesenchymal transition (EMT) phenotype. (a–c) The mRNA levels of mesenchymal markers (N‐cadherin, Vimentin and alpha‐smooth muscle actin [α‐SMA]) in 3‐S bCSC‐like cells were significantly overexpressed (P = 0.0007, P = 0.0016, and P = 0.0459, respectively), while (d) the mRNA level of epithelial marker (E‐cadherin) was significantly decreased (P = 0.0055), by real time reverse transcription‐polymerase chain reaction (RT‐PCR) analysis. (e) The relative protein levels of EMT markers were shown, by western blotting analysis.

Figure 3

Breast cancer stem cell (bCSC)‐like cells showed hypoxia‐inducible factor (HIF)‐1α and miR‐21 overexpression, as well as more invasion and migration properties. (a,b) The relative mRNA expression of HIF‐1α in 3‐S bCSC‐like cells were significantly upregulated (a; P = 0.0079), but not for HIF‐1β (b; P = 0.187). (c) The relative protein levels of HIF‐1α and HIF‐1β were shown, by western blotting analysis. (d) The relative mRNA expression of Glut‐1 in 3‐S bCSC‐like cells were significantly upregulated (P = 0.0017). (e) The relative expression of miR‐21 in 3‐S bCSC‐like cells were significantly upregulated (P = 0.0018). (f,g) Cell invasion and migration properties in bCSC‐like cells significantly increased (P < 0.0001 and P < 0.0001, respectively), by Transwell assay.

Figure 4

Antagonism of miR‐21 reversed epithelial‐mesenchymal transition (EMT) phenotype and hypoxia‐inducible factor (HIF)‐1α expression, as well as invasion and migration in breast cancer stem cell (bCSC)‐like cells. (a) Antagonism of miR‐21 was established in 3‐S bCSC‐like cells by transfected with hsa‐miR‐21 antagomir. (b,c) Antagomir reversed EMT phenotype (b) and HIF‐1α expression (c), but not for HIF‐1β (c), in bCSC‐like cells. The relative protein levels of EMT markers, HIF‐1α and HIF‐1β were shown. (d) Antagonism of miR‐21 significantly decreased cell invasion and migration properties in bCSC‐like cells (P < 0.0001 and P < 0.0001, respectively), by Transwell assays. (e) Antagonism of miR‐21 significantly decreased the mammosphere forming efficiency (MSFE) of bCSC‐like cells (P < 0.0001).