DACT3 promotes ferroptosis of glioma cells through activating BMP4/Smad signaling pathway

Abstract

Objective To investigate the molecular mechanism of DACT3 promoting ferroptosis in human glioma cell lines U251 and U118. Methods Glioma U251 and U118 cells were transfected with lentiviral vector carrying DACT3 to establish a cell line with stable over-expression of DACT3, which was verified for the efficiency of transfection by real-time PCR and Western blotting. Then the above cells were divided into control group (Ctrl group), DACT3 overexpression group (DACT3 group) and DACT3 overexpression+ferroptosis inhibitor group (DACT3+ferrostatin-1 group). CCK-8 assay was applied to detect the cell survival rate, and iron ion, malondialdehyde (MDA) and glutatione (GSH) reagent kits were employed to determine the contents of iron ion, MDA and GSH in the cells. Western blotting was applied to measure the protein levels of GPX4, SLC7A11, TFR1, BMP4, and p-Smad. The above cells were further divided into Ctrl group, DACT3 group and DACT3+siBMP4 group (knockdown of BMP4). Above experiments were performed again for cell survival rate, contents of iron ion, MDA and GSH, and protein levels of above proteins. Results The expression of DACT3 at mRNA and protein levels was significantly higher in the DACT3 group than the Ctrl group. The DACT3 group had significantly reduced cell survival rate, increased contents of iron ions and MDA but decreased content of GSH, down-regulation of GPX4 and SLC7A11 and up-regulation of TFR1, p-Smad and BMP4 when compared with the Ctrl group (P < 0.05). However, ferrostatin-1 treatment and knockdown of BMP4 reversed the changes of the above indicators caused by DACT3 to a certain extent, with statistical significances (P < 0.05). Conclusion DACT3 promotes the ferroptosis of glioma U251 and U118 cells, which may be associated with the activation of BMP4/Smad signaling pathway. [

Keywords

• dact3
• glioma
• ferroptosis
• bmp4/smad signal pathway