The proliferation of granulosa cells (GCs) in mare follicles is a key developmental step in the formation of dominant follicles that meet ovulation criteria. MicroRNAs (miRNAs) are considered key regulatory factors that can modulate GC function by affecting downstream gene expression. In a previous study, our team found that the expression of miR-362-3p varied significantly during different estrous cycles in follicles of Mongolian mare, but the regulatory mechanism of miR-362-3p in ovarian development remained unclear.

The aim of this study was to investigate whether miR-362-3p regulates the proliferation of mare ovarian follicle GCs and steroid hormone synthesis through BMPR2, so as to improve the molecular regulatory network of mare follicle development.

9 tissue samples from Mongolian mare were collected, including heart, liver, spleen, lung, kidney, longissimus dorsi muscle, subcutaneous fat, mammary gland, and ovary. Reverse transcription-quantitative PCR (RT-qPCR) was used to construct a multi-tissue expression profile of miR-362-3p in mare. The effects of miR-362-3p on the proliferation of mare ovarian follicle GCs and steroid hormone synthesis were investigated by using RT-qPCR, Western blot, CCK-8, EdU, and ELISA. The target genes of miR-362-3p were predicted by using TargetScan, miRDB, miRWalk, and ENCORI databases, and combined with our team's previous transcriptome sequencing data to determine the target gene BMPR2. RT-qPCR and Western blot were employed to investigate the effects of overexpression and interference of miR-362-3p on BMPR2 mRNA and protein expression. The wild-type and mutant vectors of BMPR2 were constructed, and a dual luciferase reporter assay was used to verify the targeting relationship between miR-362-3p and BMPR2.

Compared with tissues such as spleen and muscle, miR-362-3p had the highest expression levels in the liver and ovary tissues of mares. Compared with the control group, after overexpression of miR-362-3p, the expression levels of proliferation marker gene mRNA and protein were significantly lower, cell viability was significantly decreased, and the number of newborn cells was significantly reduced. The results were opposite after interference with miR-362-3p. Importantly, miR-362-3p could significantly influence the expression of steroid hormone synthesis-related genes mRNA, thereby inhibiting the secretion of E₂ and promoting the secretion of P₄. Bioinformatics prediction results showed that there was a binding site between miR-362-3p and the 3′-UTR region of BMPR2. During cell proliferation, overexpression of miR-362-3p significantly downregulated the mRNA and protein expression levels of BMPR2, while interference with miR-362-3p produced opposite results. The dual luciferase reporter assay confirmed the binding site between miR-362-3p and BMPR2.

miR-362-3p could inhibit the proliferation of mares ovarian follicle GCs by targeting BMPR2 to reduce its expression level and influence P₄ and E₂ synthesis, thereby regulating the growth and development of horse follicles. This study provided a theoretical basis for further understanding the molecular regulatory mechanisms of mare follicle development.