MicroRNA-34a-5p induces cell cycle arrest and leads to spermatogenesis failure by targeting CDC25A

Non-obstructive azoospermia (NOA) is among the most severe type of male infertility, which is characterized by the absence of sperm in the ejaculate of affected individuals. The underlying causes of NOA remain largely unidentified, leading to a dearth of effective clinical interventions. This study aimed to explore a potential biomarker for NOA, and to elucidate the underlying mechanisms involved. The RNA-sequencing of the testis tissue revealed that miR-34a-5p increased, while CDC25A mRNA decreased in NOA patients compared to obstructive azoospermia (OA) controls. RT-qPCR confirmed that miR-34a-5p was upregulated in the seminal plasma of NOA patients compared to healthy individuals, demonstrating its potential as a discriminatory marker for distinguishing between NOA and healthy individuals. The dual-luciferase assay demonstrated that miR-34a-5p directly targets CDC25A and inhibits the cell proliferation of TM4, GC-1 and GC-2 cells by arresting the cell cycle at the G1 phase. Both in vitro and in vivo experiments, the overexpression of miR-34a-5p led to a reduction in the level of CDC25A and CCNB1, accompanied by an elevation in the level of CDK1 phosphorylation. In mice overexpressing miR-34a-5p, the testicular size, testicular organ coefficient, total epididymal sperm count and motility were significantly decreased, the structure of seminiferous tubules was disrupted, and the abnormal tubules was significantly increased. In summary, miR-34a-5p causes cell cycle arrest by targeting CDC25A, inhibiting the expression of CDC25A and CCNB1, preventing the dephosphorylation of CDK1, which ultimately leads to male spermatogenic failure. The miR-34a-5p level in seminal plasma may have potential value for the diagnosis of NOA.