Background
Aberrant DNA damage of germ cells, which impairs spermatogenesis and lowers fertility, is an important factor contributing to male infertility. MicroRNAs (miRNAs) play a significant role in the expression and regulation of multiple genes during spermatogenesis. Our previous study found much lower miR-424 (murine homologue miR-322) levels in the seminal plasma of infertile patients with high DFI(DNA Fragmentation Index)than in the fertile group. However, the mechanism by which miR-322 regulates germ cells during spermatogenesis remains unknown.
Conclusion
MiR-322 plays a key role in promoting GC-2 cell apoptosis by directly regulating Ddx3x expression. MiR-424 downregulation in infertile men may induce spermatogenic cell apoptosis and sperm DNA damage by directly acting on the target gene locus Ddx3x, resulting in male infertility.
Methods
In this study, we successfully established a GC-2 cell model of miR-322 downregulation resulting in impaired spermatogenesis. And the cell viability were measured using Cell Counting Kit-8 (CCK-8; Dojindo, Japan) and MTT (Sigma Aldrich, USA). Immunofluorescence assay was used to detect cell damage and the expression of apoptosis-related proteins were measured using real-time quantitative PCR and Western blot analysis. Target genes were predicted and verified by online database retrieval and Dual-luciferase reporter gene assay.
Results
We observed evident decreases in the cell viability of GC-2 cells along with remarkable increases in apoptosis after miR-322 inhibition. While the expression of apoptosis-related genes, including Bax and caspases 3, 9, and 8 greatly increased in GC-2 cells after miR-322 downregulation, that of the anti-apoptotic Bcl-2 gene decreased. Ddx3x was found to be the direct target of miR-322. MiR-424 was then detected in the seminal plasma of infertile patients with high DFI(DNA Fragmentation Index); this miRNA was down-regulated but Ddx3x was upregulated in the infertile group.