TAp73 modulates proliferation and ferroptosis in mammary epithelial cells

Abstract

Introduction: TAp73, a transcriptionally active isoform of the p73 gene, is essential for epithelial tissue development. Ferroptosis, a regulated form of cell death characterized by lipid peroxidation and reactive oxygen species (ROS) accumulation, has been increasingly studied in recent years. However, its role in epithelial cells and the regulatory function of TAp73 in this context remain poorly understood.

Methods: We investigated the role of TAp73 in epithelial cell proliferation and ferroptosis using ectopic overexpression and RNA interference approaches. Cell proliferation was assessed through colony formation and DNA synthesis assays. Ferroptosis was induced using RSL3, and the effects were evaluated by measuring cell viability, ROS levels, and the expression of ferroptosis-associated genes PTGS2 and TFRC.

Results: TAp73 overexpression significantly increased p21 expression, suppressed colony formation and DNA synthesis, thereby inhibiting cell proliferation. In contrast, TAp73 knockdown reduced p21 levels and enhanced cell proliferation. RSL3 treatment induced a dose-dependent increase in cell death and ROS accumulation, confirming the susceptibility of epithelial cells to ferroptosis. Furthermore, TAp73 overexpression enhanced RSL3-induced ferroptosis by upregulating PTGS2 and TFRC, while TAp73 knockdown diminished their expression, reducing oxidative stress and lipid peroxidation.

Conclusion: TAp73 acts as a dual regulator of epithelial cell fate by inhibiting proliferation and promoting ferroptosis. These findings reveal a novel role for TAp73 in epithelial cell biology and suggest potential therapeutic targets for diseases involving epithelial cell death.

Keywords: TAp73; cell death; ferroptosis; mammary epithelial cells; proliferation.

FIGURE 1

Effect of ectopic expression of TAp73 on BMECs proliferation. (A) Relative expression levels of TAp73 in BMECs following TAp73 plasmid transfection. (B) Relative expression levels of p21 in BMECs following TAp73 plasmid transfection (C) A colony formation assay was performed following TAp73 plasmid transfection. (D, E) EdU assay was performed following TAp73 plasmid transfection. Three biological replicates and three technical replicates were performed for the qPCR analysis. The colony formation and EdU assays were conducted with six replicates. Data are presented as mean ± SD. *p < 0.05.

FIGURE 2

Effect of knockdown of TAp73 on BMECs proliferation. (A) Relative expression levels of TAp73 in BMECs following TAp73 siRNA transfection. (B) Relative expression levels of p21 in BMECs following TAp73 siRNA transfection. (C) A colony formation assay was performed following TAp73 siRNA transfection. (D, E) EdU assay was performed following TAp73 siRNA transfection. Three biological replicates and three technical replicates were performed for the qPCR analysis. The colony formation and EdU assays were conducted with six replicates. Data are presented as mean ± SD. *p < 0.05.

FIGURE 3

BMECs can undergo ferroptosis. (A) BMECs were treated with various concentrations of RSL3 (0, 5, 7.5, 10 μM) for 24 h, and representative microscopic images were taken to show cell morphology. (B) LDH content of BMECs cells treated with RSL3 (0, 5, 7.5, 10 μM) for 24 h. (C) Intracellular ROS levels were measured using the fluorescent probe DCFH-DA in BMECs cells treated with RSL3 (0, 3, 5 μM) for 24 h. (D, E) Relative expression levels of PTGS2 and TRFC in BMECs cells treated with RSL3 (0, 5, 7.5 μM) for 24 h. Three biological replicates and three technical replicates were performed for the qPCR analysis. The LDH, colony formation, and EdU assays were conducted with six biological replicates each. Data are presented as mean ± SD. *p < 0.05.

FIGURE 4

TAp73 is Involved in the Regulation of Ferroptosis in BEMCs. (A, B) Relative expression levels of PTGS2, and TRFC in BEMCs following TAp73 plasmid transfection. (C, D) Relative expression levels of PTGS2 and TRFC in BEMCs cells following si-TAp73 transfection. (E) BEMCs cells were treated with RSL3, si-TAp73, or their combination, and representative microscopic images were taken to show cell morphology. (F) Intracellular ROS levels were measured using the fluorescent probe DCFH-DA in BEMCs cells treated with RSL3, si-TAp73, or their combination. Three biological replicates and three technical replicates were employed in the qPCR analysis to ensure data reliability and reproducibility. Data are presented as mean ± SD. *p < 0.05.