Downregulation of miR-322 promotes apoptosis of GC-2 cell by targeting Ddx3x.

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.

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.

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.