AKT3 and related molecules as potential biomarkers responsible for cryptorchidism and cryptorchidism-induced azoospermia

BACKGROUND: Cryptorchidism is a common congenital malformation strongly related to future oligospermia and male infertility. Normally functioning early-stage spermatogonia are vital to ensure fertility. The present study aimed to identify new differentially expressed genes (DEGs) associated with signaling pathways related to spermatogonial stem cell (SSC) maintenance during early spermatogenesis. METHODS: GEO2R was used to screen for genes differentially regulated in cryptorchidism using mRNA expression profiling data in the GEO database. DAVID was used to perform GO and KEGG enrichment analysis of DEGs to analyze their functions. A protein-protein interaction (PPI) network of DEGs was constructed using the STRING database. The hub genes in the PPI networks were identified using Maximal Clique Centrality (MCC) in Cytohubba, and the top 50 genes were displayed as hub genes using Cytoscape software. Then, the miRNAs targeting hub genes were predicted using miRWalk and an mRNA-miRNA interaction network was constructed using Cytoscape. We took the intersection of these target miRNAs and the differentially expressed miRNAs identified from a non-coding RNA sequencing dataset, GSE149084. Furthermore, the intersected miRNAs and their predicted target genes were validated in the testicular tissue of rats with cryptorchidism. RESULTS: A total of 474 DEGs were identified, most of which were annotated to the PI3K-AKT-mTOR signaling pathway. Hub genes related to the pathway were predicted to be targeted by 27 miRNAs. Further miRNA mining revealed that miRNA-7-5p and miRNA-519d-3p were both dysregulated in cryptorchidism patients. Further, we found that these two miRNAs were predicted with high confidence to share a common target gene, AKT3. In the testicular tissue of rats with cryptorchidism, miRNA-519d-3p was upregulated while miRNA-7-5p and AKT3 were downregulated. We also found that AKT3 plays an essential role in regulating SSC state through the PI3K-AKT-mTOR signaling pathway and that AKT3 is one of the key genes related to SSC self-renewal, proliferation, and differentiation. CONCLUSIONS: The PI3K-AKT-mTOR signaling pathway functions in SSC maintenance, and alterations in this pathway may explain defects in spermatogenesis. AKT3-related miRNAs, including hsa-miR-7-5p and hsa-miR-519d-3p, might be responsible for cryptorchidism and cryptorchidism-induced azoospermia and serve as potential biomarkers.