Study on the expression patterns and biological functions of oar-miR-214_3p in the ovary and uterus of Liangshan Black Sheep

This study elucidates the expression dynamics and biological functions of oar-miR-214_3p in ovarian and uterine tissues of Liangshan Black Sheep across distinct stages of the oestrous cycle, providing novel insights into its regulatory mechanisms governing ovine reproductive physiology. The study employed RT-qPCR, northern blotting, FISH, dual-luciferase reporter assays, and ELISA to analyse the expression and biological functions of oar-miR-214_3p in the ovary and uterus of Liangshan Black Sheep across different stages of the oestrous cycle (proestrus, oestrus, metoestrus, and dioestrus phases). RT-qPCR and northern blotting revealed that oar-miR-214_3p is expressed in the ovarian and uterine tissues, with significant variations across different stages. Compared with the dioestrus and metoestrus phases, oar-miR-214_3p expression was significantly increased during the proestrus and oestrus phases (P < 0.05), with the highest levels observed during oestrus (P < 0.05). FISH analysis indicated that oar-miR-214_3p is primarily localised in the cytoplasm. We constructed wild-type and mutant vectors for mammalian target of rapamycin(mTOR) and Semaphorin 4D (Sema4D). After intervention for 48 h in granulosa cell cultures, RT-qPCR analysis of mTOR and Sema4D expression revealed that the mimic and inhibitor groups suppressed and promoted the expression of these target genes, respectively. The control and NC groups showed stable expression levels with no significant differences. Dual-luciferase reporter assay confirmed that mTOR and Sema4D are oar-miR-214_3p target genes. ELISA revealed that the mimic and inhibitor treatment groups promoted and suppressed oestrogen secretion, respectively. These findings confirm that oar-miR-214_3p, primarily localised in the cytoplasm, regulates ovarian follicle development, ovulation, oestrogen secretion, and the oestrous cycle via its target genes, mTOR and Sema4D. The study provides valuable insights into improving sheep reproductive performance through molecular breeding techniques. These findings have significant clinical and practical applications for enhancing reproductive efficiency.