CDKN2B Inhibits Vascular Smooth Muscle Phenotypic Switching in Corpus Spongiosum Surrounding the Urethral Plate in Hypospadias

<b>Objective:</b> Phenotypic switching of vascular smooth muscle cells (VSMCs) in the corpus spongiosum may contribute to abnormal urethral development in hypospadias, but the underlying molecular regulators remain unclear. This study aimed to identify hub genes associated with VSMCs phenotypic switching in the corpus spongiosum using RNA sequencing and Weighted Gene Co-expression Network Analysis (WGCNA), and to functionally characterize the top candidate gene <i>CDKN2B</i>. <b>Methods</b>: Corpus spongiosum tissue samples were collected from seven patients with proximal hypospadias and five patients with urethral stricture (control group). The expression of the VSMCs contractile markers Calponin 1 and α-SMA, and the secretory marker OPN, was evaluated by qRT-PCR and Western blotting to assess VSMCs phenotypic state. RNA sequencing and Weighted Gene Co-expression Network Analysis (WGCNA) were performed to identify hub genes, which were then validated by qRT-PCR. Primary VSMCs were isolated from corpus spongiosum tissue and transduced with lentiviral vectors to either suppress or overexpress <i>CDKN2B</i>. Changes in VSMC marker expression and in key signaling pathways associated with phenotypic switching-specifically <i>TGF/Smad</i> and <i>SRF/MYOCD</i>-were analyzed using qRT-PCR and Western blotting. <b>Results</b>: In hypospadias tissue, the decreased expression of α-SMA and Calponin 1, together with increased OPN, indicated a shift in VSMCs from a contractile to a secretory phenotype. RNA-seq and WGCNA identified 11 differentially expressed genes, among which <i>CDKN2B</i> showed a marked downregulation in hypospadias samples. In control VSMCs, <i>CDKN2B</i> inhibition led to reduced α-SMA and Calponin 1, elevated OPN, and suppressed activity of <i>TGF/Smad</i> and <i>SRF/MYOCD</i> signaling. Conversely, <i>CDKN2B</i> overexpression in VSMCs from hypospadias samples restored α-SMA and Calponin 1 expression, decreased OPN, and enhanced <i>TGF/Smad</i> and <i>SRF/MYOCD</i> pathway activation. <b>Conclusions</b>: VSMCs in the corpus spongiosum surrounding the urethral plate in hypospadias undergo a transition from a contractile to a secretory phenotype. <i>CDKN2B</i> emerges from unbiased transcriptomic screening as a key hub gene and functions as a critical regulator of this process, maintaining the contractile phenotype by modulating canonical <i>TGF/Smad</i> and <i>SRF/MYOCD</i> signaling. The <i>CDKN2B-TGF/Smad</i> axis may represent a central pathway linking VSMC phenotypic switching to abnormal vascular remodeling in hypospadias.