MiR-296-5p Modulates COX2 Via Targeting SRF To Participate In Parturition

From pregnancy to labor is a process in which multiple factors coordinate with each other.The myometrium changes from quiescent to regularly contraction,which requires complex interactions between placenta,mother and fetus.Inflammation and hormones are the final common ways of labor initiation.Recent studies have found that miRNA is also involved in the regulation of labor initiation.Our research group has obtained the miRNA expression profiles from the placentas of Qingping sows and Large White sows of different gestation stages,from which the differentially expressed miR-296-5p was screened in this study.miR-296-5p is involved in the maintenance of pregnancy by promoting angiogenesis,and this study aims to explore the role of miR-296-5p in the initiation of labor.In order to explore whether miR-296-5p is involved in labor initiation,LPS was intraperitoneally injected into pregnant mice at 15.5 d of gestation to establish a mouse model of premature delivery,and the expression changes of labor marker genes c-fos and COX2,as well as the expression patterns of miR-296-5p and the potential target gene SRF were detected.The targeting relationship between miR-296-5p and SRF was verified by the dual-luciferase report assay.Overexpression and deletion of miR-296-5p were simulated in WISH cells,and the changes of SRF and COX2 were detected to verify whether miR-296-5p was involved in delivery.Furthermore,the targeting relationship of SRF to COX2 was verified by transfection of pcDNA3.1-SRF and interference fragments,proving that miR-296-5p regulates the expression of COX2 through targeted SRF.The effect of miR-296-5p and SRF on the secretion of PGE2,as well as the feedback loop mechanism of PGE2 on miR-296-5p,SRF and COX2 were detected.The main research results are listed below:1)The expression levels of labor maker genes c-fos and COX2 in the premature mouse model were significantly higher than those in the control group,and the expression level of miR-296-5p was decreased.Meanwhile,the expression level of SRF which is one potential target gene of miR-296-5p was increased,indicating that miR-296-5p may target SRF to participate in the initiation of labor.2)miR-296-5p mimics were used to co-transfect WISH cells with pmirGLO vectors of SRF 3'UTR or SRF 3'UTR mutant respectively.miR-296-5p mimics significantly inhibited the dual-luciferase activity of pmirGLO-SRF 3'UTR vector,but had no effect on mutant vector,proving the targeting relationship between miR-296-5p and SRF.3)WISH cells were transfected with miR-296-5p inhibitor,and the expressions of SRF and COX2 were significantly increased,indicating that miR-296-5p regulates the COX2 involved in labor initiation.Furthermore,WISH cells were transfected with pcDNA3.1-SRF,and the expression of COX2 was significantly increased,indicating that miR-296-5p regulates the expression of COX2 through targeted SRF.4)Hormone levels were used to detect the effects of miR-296-5p and SRF on PGE2 secretion,and it was found that both interference of miR-296-5p and overexpression of SRF promoted the release of PGE2.Addition of 1?M PGE2 to WISH cells provided a feedback loop regulation mechanism for miR-296-5p,SRF and COX2.Conclusion: Down-regulated miR-296-5p promotes the expression of COX2 through targeted SRF during the initiation of labor,thus increasing the secretion of PGE2,and PGE2 has a feedback loop regulation mechanism for miR-296-5p,SRF and COX2.