DNMT1 targets SRD5A2 to induce mitochondrial homeostasis and EMT in urothelial cells of hypospadias.

BACKGROUND: Hypospadias, a common congenital malformation of the urinary system, significantly impacts neonatal development. The enzyme Steroid 5 Alpha-Reductase 2 (SRD5A2), essential for androgen metabolism, is regulated epigenetically through methylation by DNA Methyltransferase 1 (DNMT1). This modification plays a critical role in cell differentiation and development. This study aims to reveal the molecular mechanisms by which DNMT1-mediated methylation of SRD5A2 affects the pathophysiology of hypospadias. METHODS AND RESULTS: Western blot was utilized to quantify DNMT1 and SRD5A2 expression in primary urethral epithelial cells derived from rats afflicted with hypospadias. The regulation of SRD5A2 expression by DNMT1 methylation was confirmed through Chromatin Immunoprecipitation (ChIP) assays. Additionally, the influence of SRD5A2 on epithelial-mesenchymal transition (EMT), mitochondrial homeostasis, and energy metabolism was elucidated by further in vitro experiments, highlighting its potential biological impacts on hypospadias. DNMT1 methylation significantly upregulates SRD5A2 expression in urethral epithelial cells from hypospadias rats. Knockdown of DNMT1 and SRD5A2 elevated the levels of proteins associated with the cell cycle and mitochondrial function, and genes related to energy metabolism. Concurrently, these alterations inhibited EMT, promoted cellular proliferation and migration, arrested the cell cycle in the G1/S phase, and reduced apoptosis. CONCLUSIONS: DNMT1 and SRD5A2 exhibit elevated expression in hypospadias, with DNMT1 enhancing the expression of key proteins involved in the cell cycle, mitochondrial function, and energy metabolism through the methylation of SRD5A2. This inhibition of EMT and modulation of cellular functions suggest that SRD5A2 is a viable therapeutic target for hypospadias. TRIAL REGISTRATION: Not applicable. CLINICAL TRIAL NUMBER: Not applicable.