Scientia Agricultura Sinica ›› 2026, Vol. 59 ›› Issue (7): 1564-1575.doi: 10.3864/j.issn.0578-1752.2026.07.014

• ANIMAL SCIENCE·VETERINARY SCIENCE • Previous Articles     Next Articles

The miR-362-3p Regulates the Proliferation and Steroid Hormone Synthesis of Mare Follicular Granulosa Cells by Targeting BMPR2

YUE XiaoYu(), ZHAO ShiChen, WANG Qin*()   

  1. College of Animal Science and Technology, China Agricultural University, Beijing 100193
  • Received:2024-11-22 Accepted:2026-02-25 Online:2026-04-08 Published:2026-04-08
  • Contact: WANG Qin

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Abstract:

【Background】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.【Objective】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.【Method】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. 【Result】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. 【Conclusion】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.

Key words: Mongolian mare, granulosa cells, miR-362-3p, BMPR2, proliferation, steroid hormones

Table 1

Primers used in RT-qPCR"

基因 Gene 引物序列Primer sequence（5′→3′）
BMPR2 F: GCAAGCACAAGCTCGAATCC
R: GAGTGAGGCCAGTGGTGTTT
Cyclin D1 F: GATGCCAACCTCCTCAACGA
R: GGAAGCGGTCCAGGTAGTTC
Cyclin B F: CGATACTCCGTCTCCAAGCC
R: AGCCAGTCAATCAGGATGGC
PCNA F: ATCAGCTCAAGTGGCGTGAA
R: TGCCAAGGTGTCCGCATTAT
CDK4 F: AAGTGGTGGGACAGTCAAGC
R: ACCACCACAGGTGTAAGTGC
CYP11A1 F: AGCTGGCATCTCCACAAAGA
R: TAAGCCAGCCATTGTCACCA
CYP19A1 F: AGCATGGTGTCCGAAGTTGT
R: GCTGGGACCTGGTATTGAGG
StAR F: CCCGAGACTTTGTGAGCGTA
R: CGCTCTGATGACACCCTTCT
3β-HSD F: TGCCACAATCTGACCGCATC
R: CTCCACCAACAGGCAGATGA
GAPDH F: GGACTCATGACCACGGTCCAT
R: TCAGATCCACAACCGACACGT
U6 F: CTCGCTTCGGCAGCACA
R: AACGCTTCACGAATTTGCGT

Fig. 1

Tissue expression profile of miR-362-3p"

Fig. 2

The relative expression of miR-362-3p in mare follicular GCs"

Fig. 3

The effect of miR-362-3p on relative mRNA expression of proliferation marker genes in mare follicular GCs"

Fig. 4

The effect of miR-362-3p on protein expression of proliferation marker genes in mare follicular GCs"

Fig. 5

The effect of miR-362-3p on the viability of mare GCs"

Fig. 6

The effect of miR-362-3p on newborn cells of mare GCs A: The proliferation of cells analyzed using Edu assay when miR-362-3p mimic, mimic NC, miR-362-3p inhibitor and inhibitor NC were transfected into mare follicular GCs for 48 h; B: Proportion of EdU-positive cells. DAPI: Nuclear staining; EdU: Labeled newborn cells; Merge: Merging results"

Fig. 7

The target genes of miR-362-3p predicted by TargetScan, miRDB, miRWalk and ENCORI"

Fig. 8

Binding sites between miR-362-3p and BMPR2 mRNA 3′-UTR A: The predicted binding results of miR-362-3p with the 6912th to 6918th bases of the 3′-UTR region of BMPR2; B: The predicted binding results of miR-362-3p with the 264th to 269th bases of the 3′-UTR region of BMPR2"

Fig. 9

The effect of miR-362-3p on BMPR2 expression A: Relative mRNA expression of BMPR2 after 48h of transfection of miR-362-3p mimic, mimic NC, miR-362-3p inhibitor and inhibitor NC into horse follicle GCs; B and C: Relative protein expression of BMPR2 after 48h of transfection of miR-362-3p mimic and mimic NC, miR-362-3p inhibitor and inhibitor NC into horse follicle GCs"

Fig. 10

Results of dual luciferase reporter assay"

Fig. 11

Effect of miR-362-3p on steroid hormone secretion of mare GCs"

Fig. 12

Effect of miR-362-3p on steroid hormone synthesis marker genes in mare GCs"

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