Knockdown of ALDH1A3 reduces breast cancer stem cell marker CD44 via the miR-7-TGFBR2-Smad3-CD44 regulatory axis

Inhibition of aldehyde dehydrogenase 1 family member A3 (ALDH1A3) has been revealed to lead to significant increase of microRNA (miR)-7 expression and decrease of CD44 expression in breast cancer stem cells (BCSCs), however the mechanism is not clear. The aim of the present study was to investigate the regulatory relationship between ALDH1A3, miR-7, and CD44 in BCSCs. The expression of ALDH1A3 was inhibited by small interfering RNA (siRNA or si), and the expression of miR-7 was detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Then, the ratio of CD44(+) cells was analyzed by flow cytometry in MDA-MB-231 cells. The dual-luciferase reporter system was used to demonstrate that miR-7 binds to transforming growth factor-β receptor 2 (TGFBR2) 3'UTR, and ChIP-PCR determined whether the transcription factor Smad3 binds to the upstream regulatory region of the CD44 promoter. The results revealed that siALDH1A3 downregulated ALDH1A3 and promoted miR-7 expression, which resulted in downregulation of CD44 expression. siALDH1A3 also downregulated the CD44 expression on the surface of MDA-MB-231 cells and inhibited the G2/M phase in BCSCs as analyzed by flow cytometry. In addition, lenti-miR-7 cells transfected with TGF-β1 + SB431542 revealed that lenti-miR-7 inhibited the TGF-β1 pathway by inhibiting Smad2/3/4 expression and, thus, downregulated CD44 expression. miR-7 was revealed to directly bind to the TGFBR2 3'UTR through dual-luciferase reporter assay, and Smad3, a transcription factor, through ChIP-PCR was demonstrated to bind to the upstream region of the CD44 promoter. These results demonstrated the existence of the ALDH1A3-miR-7-TGFBR2-Smad3-CD44 axis in MDA-MB-231 cells. RT-qPCR results of 12 breast cancer surgical specimens and SK-BR-3, MCF-7, and LD cell lines further confirmed the presence of the regulatory axis. In conclusion the findings from the present study demonstrated that the ALDH1A3-miR-7-TGFBR2-Smad3-CD44 regulatory axis was highly efficient in the inhibition of CD44 expression in BCSCs, and that the regulatory expression of ALDH1A3 and miR-7 may provide a strategy in the therapy of breast cancer.