MiR-375 Mediates CRH Signaling Pathway In Inhibiting E2 Synthesis In Porcine Granulosa Cell

miR-375 plays an important role in endocrine system such as regulating insulin and ACTH production in animals.It is reported that miR-375 is expressed in the ovary of chicken,but the function of miR-375 is still unclear.the functions of miR-375 and the related signaling pathway in porcine ovary.CRH is an important regulator of stress and inflammation.Studies have shown that CRH inhibits the synthesis of steroid hormones in ovarian cells.The aim of present study was to investigate the functions of miR-375 in the adult pocine ovary and its interaction with CRH signaling pathway.The main contents and results including the following aspects:1.In situ hybridization(ISH)and RT-qPCR results showed that miR-375 is highly expressed in all of the porcine granulosa cells,suggesting that miR-375 may play an important role in porcine granulosa cells.2.To investigate the function of miR-375 in porcine granulosa cell,the intracellular miR-375 was overexpressed and inhibited by miR-375 mimic or inhibitor.The results showed that miR-375 significant affects E2 production in cultured granulosa cell.However,the overexpression or inhibition of intracellular miR-375 has no significant influences on P4 synthesis,proliferation and apoptosis of granulosa cells.Further,we examined the key enzymes in the progress of steriod hormone synthesis.The results demonstrate that miR-375 surppresses E2 synthesis depending on Cyp19a1 expression.3.Our results showed that only Crhrl is expressed in the porcine ovary,while Crhr2 mRNA is not detected.These findings suggest that Crhr1 may be involved in a more important role in the regulation of ovarian functions.CRH enhanced the expression of miR-375 in porcine granulosa cell both in vitro and in vivo.The further results showed that the signaling pathway inhibitors including H89(PKA)and SB203580(p38 MAPK)blocked the stimulation effect of CRH on miR-375 expression.In addition,the Western blot results show that CRH can increase the phosphorylation of p38 MAPK.These results indicate that CRH up-regulates the expression of miR-375 through CRHRl-PKA-p38 MAPK signaling pathway.4.CRH significantly inhibited the Cypl9al expression and E2 production in porcine granulosa cells.The further results showed that miR-375 enhanced the inhibitory effect of CRH on Cyp19a1 expression and E2 synthesis,whereas overexpression of miR-375 inhibited the effect of CRH on Cypl9al expression and E2 synthesis.These results demonstrate that miR-375 mediates the CRH suppressed E2 synthesis signaling pathway.5.According to bioinformatics prediction,Sp1?Lhr1 and Usf1 are potential targets gene of miR-375.Dual luciferase reporter assay and Western blot results showed that Spl is the direct target gene of miR-375 in porcine granulosa cells.Further,the results demonstrate that Spl is a common downstream molecule of miR-375 and CRH,and miR-375 mediates CRH regulating E2 synthesis and Cyp19a1 expression by targeting Sp1.In addition,Dual luciferase report assay result showed that overexpression of Spl significantly enhanced Cypl9al promoter activity,indicating that Spl promotes the expression of Cyp19a1 by regulating its promoter activity.Taken together,we have shown that the signaling of CRH is transduced through CRHR1 and PKA-p3 8 MAPK,subsequently enhancing miR-3 75 expression,then targeting Sp1 and decreasing E2 synthesis in the porcine ovary.These results provide further insight into the roles and mechanisms of CRH functions in steroid hormone synthesis and provide potential molecule targets for treatment of steroid-related reproductive disorders.