Inhibition of Rab25 correlates with proliferation, apoptosis, and migration of foreskin fibroblasts via the β1- integrin/EGFR pathway in hypospadias

Abstract

Abstract Hypospadias is a common congenital abnormality of the penile. Abnormal spatiotemporal regulation of critical genes interfering with urethral development leads to hypospadias. This study investigated the role of Rab25 in hypospadias. The expression levels of various molecules in foreskin tissues and fibroblasts were confirmed using PCR, immunohistochemistry, and western blotting. Foreskin fibroblasts of silencing Rab25 were constructed by lentivirus transfection. Using CRISPR/Cas9 technology, Rab25−/− mice were generated. The biological functions of Rab25 were investigated using Cell Counting Kit-8 solution, flow cytometry, and wound scratch assays. The genital tubercles (GTs) of male wild-type (WT) and Rab25−/− fetal mice were collected on gestation day (GD) 18.5, and a scanning electron microscope (SEM) was used to visualize the external morphology of GT. We observed that the foreskin tissues of patients with hypospadias expressed less Rab25, β1-integrin, and EGFR. Moreover, Rab25 inhibition downregulated the β1-integrin and EGFR expressions, restrained proliferation and migration, and promoted apoptosis in foreskin fibroblasts. Abnormal GT morphology with incomplete fusion of the urethral fold and expanded distal cleft was observed in Rab25−/− fetal mice. The distal mild hypospadias phenotype is reproduced in Rab25−/− fetal mice. It suggests that Rab25 plays an important role in the hypospadias via the β1-integrin/EGFR pathway.