CUX1 Modified by m⁶A Methylation Drives Benign Prostatic Hyperplasia via the PI3K/AKT/mTOR Signaling Pathway

Benign prostatic hyperplasia (BPH) is a common urological condition in elderly men, primarily characterized by abnormal proliferation of prostatic epithelial and stromal cells. The most common internal alteration of eukaryotic mRNA, N⁶-methyladenosine (m⁶A), is essential for controlling tissue hyperplasia and cancer. However, it is still unclear exactly how m⁶A alteration controls the pathophysiology of BPH.

To identify key molecules associated with BPH, we performed differential expression analysis on the BPH transcriptome dataset and screened potential METTL16-related candidate genes using gene correlation analysis. Subsequently, we validated the expression characteristics of candidate molecules in clinical BPH tissues. The expression and modification status of the candidate gene were validated in BPH cells using qRT-PCR, MeRIP-qPCR, and Western blotting. Functional assays, including CCK-8, colony formation, and flow cytometry, were conducted to evaluate the biological effects. Mechanistic investigations were carried out through ChIP-qPCR, dual-luciferase reporter assays, and mRNA stability analysis.

Bioinformatics analysis combined with clinical validation revealed that METTL16 and CUX1 were significantly upregulated in BPH, and the expression of CUX1 and METTL16 was positively correlated. Immunohistochemical analysis further confirmed that both METTL16 and CUX1 were highly expressed in clinical BPH tissues. Both CUX1 and METTL16 were markedly overexpressed in BPH-1 cells, and CUX1 mRNA exhibited elevated m⁶A modification. Knockdown of METTL16 significantly reduced the m⁶A enrichment on CUX1 transcripts and impaired their stability. Functionally, silencing CUX1 inhibited BPH-1 cell proliferation and promoted apoptosis. Mechanistically, CUX1 activated the PI3K/AKT/mTOR signaling pathway by directly binding to the PIK3CA promoter and increasing its transcriptional activity. The negative effects of METTL16 knockdown on BPH cell proliferation and pathway activation may be successfully reversed by CUX1 overexpression, according to rescue tests.

This study reveals that METTL16 promotes prostatic cell proliferation by stabilizing CUX1 mRNA through m⁶A modification, leading to activation of the PI3K/AKT/mTOR signaling pathway. These results point to METTL16 and CUX1 as candidate molecules for upcoming epigenetic-based therapies and offer fresh insights into the molecular mechanisms driving BPH.