MiR-23a promotes autophagy of yak cumulus cells to alleviate apoptosis via the apoptosis signal-regulating kinase 1/c-Jun N-terminal kinase pathway

Abstract

The ultimate fate of Graafian follicles is ovulation or atresia which relies on the highly coordinated processes of apoptosis and autophagy in ovarian cells. Long non-coding RNA maternally expressed gene 3 (LncRNA MEG3), miR-23a, and apoptosis signal-regulating kinase 1 (ASK1) are factors associated with autophagy. However, whether these factors can regulate autophagy in cumulus cells (CCs) of yak is unclear. Here, miR-23a overexpression upregulated the LC3-II/LC3-I ratio and Beclin1 abundance while reducing p62 accumulation (p < 0.05). The monodansylcadaverine assay exhibited a marked increase in punctate green fluorescence, and the GFP-LC3B displayed increased yellow fluorescence (p < 0.05). The opposite effect was observed for miR-23a inhibitors. Furthermore, miR-23a overexpression downregulated the abundance of ASK1 mRNA and total ASK1 protein (t-ASK1), whereas miR-23a inhibitors up-regulated them (p < 0.05). The effects of miR-23a overexpression on ASK1 phosphorylated protein at serine 845 (P-845), total JNK (c-Jun N-terminal kinase) (t-JNK) and the JNK phosphorylated protein (p-JNK) were similar to those of t-ASK1 but elicited the opposite effect on ASK1 phosphorylated protein at serine 967 (P-967) (p < 0.05). We further demonstrated that ASK1 expression can be silenced by small-interfering RNA (siRNA), which had no significant effect on t-JNK abundance (p > 0.05) but significantly suppressed the p-JNK expression (p < 0.05). Silencing ASK1 significantly improved Beclin1 abundance and the LC3-II/LC3-I ratio, but decreased p62 abundance (p < 0.05). An increase in yellow GFP-LC3B puncta and green MDC staining puncta were observed (p < 0.05). Overexpression of LncRNA MEG3 significantly increased the expression of t-ASK1, P-845, and JNK and decreased the abundance of P-967 and miR-23a (p < 0.05). In addition, miR-23a upregulation reduced the number of the TUNEL-positive cells, and the addition of 8 mM 3-methyladenine (3-MA) reversed this downregulation (p < 0.05). Similar trends were observed for the Bax/Bcl2 ratio and cleaved-caspase3 abundance. In summary, miR-23a promotes autophagy by inhibiting ASK1 abundance, which reduces apoptosis of yak CCs. This effect can be inhibited by LncRNA MEG3, which has implications for decreasing abnormal Graafian follicular atresia and maintaining development.

Keywords: ASK1/JNK axis; Autophagy; Cumulus cells; LncRNA MEG3; miR-23a.