Upregulation of miR-151a-5p in high DFI sperm induces DNA damage and mitochondrial dysfunction by targeting INPP4B and VAMP1.

OBJECTIVE: To explore the potential the role of miR-151a-5p in sperm dysfunction and its association with DNA fragmentation, mitochondrial dysfunction, and male infertility. METHODS: Sperm samples with high DNA fragmentation were collected, and miR-151a-5p expression was measured using quantitative polymerase chain reaction. GC-2 cells were transfected with miR-151a-5p mimics to assess its effects on DNA damage, apoptosis, mitochondrial function, and reactive oxygen species levels. RNA sequencing, RNA pull-down, and bioinformatics analyses were used to identify the direct target genes of miR-151a-5p. Dual-luciferase reporter assays confirmed the binding of miR-151a-5p to the 3' untranslated regions of INPP4B and VAMP1. Knockdown of these genes was performed to validate their roles in miR-151a-5p-induced effects. In vivo experiments were conducted by injecting miR-151a-5p mimics into mouse zygotes to examine the impact on embryo development. RESULTS: miR-151a-5p was significantly upregulated in sperm with high DNA fragmentation and negatively correlated with sperm motility and viability. Overexpression of miR-151a-5p in GC-2 cells was associated with increased DNA damage, apoptosis, mitochondrial dysfunction, and elevated reactive oxygen species. RNA sequencing and bioinformatics analyses suggested INPP4B and VAMP1 as direct targets of miR-151a-5p. Dual-luciferase assays confirmed that miR-151a-5p binds to their 3' untranslated regions, inhibiting their expression at both the messenger RNA and protein levels. Knockdown of INPP4B or VAMP1 partially reproduced the cellular changes observed with miR-151a-5p overexpression. In vivo, microinjection of miR-151a-5p mimics into mouse zygotes exerted only mild effects on embryo development, indicating a possible contribution of miR-151a-5p primarily through sperm dysfunction. CONCLUSIONS: miR-151a-5p may contributes to sperm dysfunction by targeting INPP4B and VAMP1, linking DNA fragmentation with mitochondrial and genomic instability. This study offers additional insights into the molecular mechanisms underlying male infertility.