Downregulation of microRNA-96-5p protects TM3 cells against zearalenone toxicity via targeting ATG9A.

Male infertility factor accounts for ~50% of all infertility cases, and traditional treatments for male infertility are limited. The association between the dysfunction of Leydig cells and hypospermatogenesis is essential for developing novel treatment methods for male infertility. It was previously stated that elevated expression of microRNA (miR)-96-5p was associated with the toxicological response of Leydig cells to treatment with zearalenone (ZEN). However, the exact role of miR-96-5p in Leydig cells remains to be illustrated. The mouse Leydig cell line TM3 was used in the present study to investigate the role of miR-96-5p. ZEN was used to induce cell injury in TM3 cells. Cell Counting Kit-8 assay and the Ki67 staining method were used to evaluate cell viability. Reverse transcription-quantitative PCR was used to determine the expression levels of miR-96-5p. In addition, a dual luciferase assay was used to investigate the target of miR-96-5p. Annexin V/propidium iodide staining was performed to detect cell apoptosis. Western blot analysis was used to detect the expression levels of certain proteins. Finally, monodansylcadaverine (MDC) and LC3 staining were applied for monitoring the level of autophagy. ZEN inhibited the proliferation of TM3 cells in a dose-dependent manner. In addition, the level of miR-96-5p were significantly increased in ZEN-treated TM3 cells. Meanwhile, inhibition of miR-96-5p could reverse ZEN-induced decrease in viability in TM3 cells. Moreover, ZEN notably inhibited autophagy in TM3 cells and this phenomenon was reversed by the application of the miR-96-5p inhibitor. Autophagy related 9A (ATG9A) was identified as the biological target of miR-96-5p. The results derived from MDC and LC3 staining demonstrated that downregulation of miR-96-5p expression levels protected TM3 cells against ZEN toxicity by regulating autophagy. Inhibition of miR-96-5p expression protected TM3 cells against ZEN via targeting ATG9A. Therefore, miR-96-5p may serve as a potential biomarker for male infertility.