Study On Function Of MiR-132-3p During The Initiation Of Mammalian Parturition

Labor or parturition is a process of inflammation and hormonal changes involving interactions between the myometrium and signals derived from placenta,fetal membranes and fetal.Qingping sows are an excellent local breed in Hubei province with a short gestation period,but the mechanism of the premature onset of parturition is still unclear.miRNAs could be involved in the maintenance of pregnancy and the process of initiation parturition by regulating the expression of transcription factors,uterine contraction-related genes,and hormone synthesis and metabolism-related enzymes in the myometrium.Our research group’s preliminary miRNA sequencing results revealed that miR-132-3p was upregulated in the placental tissues of Large White and Qingping sows with signs of labor onset.Research shows that miR-132-3p can participate in the process of various immune-related diseases by resisting or triggering inflammatory responses.The fetal membranes are compartments that the initially receive signals of fetal maturation,but it is not clear how miR-132-3p in the fetal membranes is involved in the initiation of parturition.Therefore,in this study,we propose to analyze the expression pattern of miR-132-3p in term labor and infection-induced preterm birth using a normal-gestation model and a preterm birth model constructed by lipopolysaccharide(LPS)induced mice;to predict target genes of miR-132-3p using online bioinformatics websites and investigate the role of miR-132-3p on inflammatory response and hormonal cascade using human amniotic epithelial cells(WISH);to verify its effect on parturition in vivo by injection of miR-132-3p agomir and detect the expression of miR-132-3p,its target genes and related signaling pathways in amnion membranes,chorion and placenta of Qingping and Large White sows at different gestation stages.To finally elucidate the molecular mechanism of the miR-132-3p mediated premature onset of parturition in Qingping sows.The main findings are as follows.1.Expression of miR-132-3p,labor-associated genes and signaling pathways in normal pregnancy model and LPS-induced preterm labor mouse modelRT-qPCR and Western blot results showed that miR-132-3p,interleukin-1β(IL-1β),interleukin-6(IL-6),interleukin-8(IL-8),cyclooxygenase 2(COX2),chemokine C-C motif ligand 5(CCL5),chemokine C-X-C motif ligand 16(CXCL16)expression(P < 0.05),and p38 and JNK phosphorylated protein levels were significantly increased in mouse amnion membranes at 18.5 days post coitum(dpc)relative to 15.5 dpc(P <0.01).Pearson correlation analysis showed miR-132-3p expression in mouse membranes were significantly positively correlated with that of IL-1β,IL-6,COX2,CCL5 and CXCL16 mRNA(P < 0.05),while it was not significantly correlated with IL-8 mRNA(P > 0.05).The expression of miR-132-3p,IL-1β,IL-6,COX2,CCL5,CXCL16,and p38 and JNK phosphorylated protein levels in amnion membranes were significantly higher in the LPS-induced preterm labor group compared with the nonlabor group(P < 0.05),suggesting that miR-132-3p may be involved in the process of labor onset.2.miR-132-3p promotes inflammatory and hormonal cascadeRT-qPCR,Western blot,Magnetic Luminex Assays,and ELISA analysis showed that miR-132-3p could promote the expression of inflammatory factors(IL-1β,IL-6,TNF-α),COX2,CC chemokines(IL-8,CCL5,CCL20,CCL22,CCL28),and CXC chemokine(CXCL10,CXCL11,CXCL16),and the secretion of prostaglandin E2(PGE2)in WISH cells relative to the control group(P < 0.05),indicating that miR-132-3p can promote inflammatory and hormonal cascade.3.DUSP9 is a target gene of miR-132-3pDUSP9 is a potential target gene for miR-132-3p by using online bioinformatics websites.The dual-luciferase reporter assay system verified that miR-132-3p could target DUSP9 3’ untranslated region(3’UTR).Overexpression or inhibition of miR-132-3p significantly inhibited or promoted DUSP9 mRNA and protein expression in WISH cells,respectively(P < 0.05).Immunohistochemical analysis showed that DUSP9 was expressed in mouse amnion epithelial cells and the abundance of DUSP9 mRNA and protein were significantly lower in mouse amnion membranes at 18.5 dpc relative to 15.5 d(P < 0.05);however,there was no significant difference in the expression level of DUSP9 mRNA and protein in amnion membranes of LPS-induced preterm labor group compared with the non-labor group(P > 0.05).Taken together,DUSP9 was the direct target gene of miR-132-3p and could be negatively regulated by miR-132-3p,and DUSP9 might not be involved in the process of infection-induced preterm labor.4.miR-132-3p enhances the inflammatory response and PGE2 synthesis via targeting DUSP9-dependent p38 and JNK signaling pathways in amnionRT-qPCR,Western blot,Magnetic Luminex Assays,and ELISA analysis showed that inhibition of DUSP9 expression significantly contributed to the expression of inflammatory factors,CC chemokines,CXC chemokines,and COX2,and the secretion of PGE2 compared to the control group in WISH cells(P < 0.05),which counteracts the inhibitory effect of miR-132-3p inhibitor.In summary,miR-132-3p could promote the secretion of inflammatory factors,chemokines,and PGE2 via targeting DUSP9.Western blot analysis showed that the protein levels of phosphorylated p65,p38,and JNK were obviously increased when WISH cells were transfected with miR-132-3p mimic or DUSP9 si RNA compared to NC(P < 0.05),but downregulated by the treatment of miR-132-3p inhibitor relative to inhibitor NC(P < 0.05).The induction of inflammatory factors,chemokines,and COX2,and the secretion of PGE2 by DUSP9 si RNA was significantly attenuated by the treatment of p38 inhibitor(Adezmapimod,SB203580)and JNK inhibitor SP600125(P < 0.05),while the treatment of NF-κB inhibitor(Pyrrolidinedithiocarbamate ammonium,PDTC)showed no significant difference(P > 0.05).These results indicate that miR-132-3p regulates inflammatory response and PGE2 synthesis by targeting DUSP9 dependent on p38 and JNK signaling pathway,but not to NF-κB signaling pathway.5.Effect of intraperitoneal injection of pregnant mice with miR-132-3p agomir on parturition initiationAt day 15.5 of pregnancy,the pregnant mice were intraperitoneally injected with miR-132-3p agomir or agomir NC.miR-132-3p agomir injection significantly shortened the gestation days of pregnant mice compared with the agomir NC group(P< 0.01),without causing any maternal or fetal death.RT-qPCR and Western blot analysis found that a significant decrease in DUSP9 level and upregulation in the level of COX2 mRNA and protein as well as phosphorylated p38 and JNK protein levels in mouse amnion membranes upon miR-132-3p agomir injection(P < 0.05).These results confirmed that miR-132-3p in activating p38 and JNK signaling pathways by downregulating DUSP9 in amnion membranes,thus inducing COX2 expression for parturition initiation.6.Inflammatory and hormonal cascades in the amnion,chorion and placenta of Qingping sows at 111 d gestation are earlier than those of Qingping and Large White sows at 114 d gestation during late gestationRT-qPCR and Western blot analysis showed that miR-132-3p expression was higher in amnion membranes of Qingping sows without signs of labor onset on day 109 of gestation(PRE109Q)than that of Large White sows without signs of labor onset on day 112 of gestation(PRE112D)and Qingping sows without signs of labor onset on day 111 of gestation(PRE111Q)(P < 0.01).In the pregnant state,p38 phosphorylated protein level in PRE109 Q amnion and chorion,IL-6,CCL5(P > 0.05),and CXCL16(P< 0.05)in PRE109 Q placenta were significantly higher than in PRE112 D and PRE111 Q,but DUSP9 protein level was not significantly different(P > 0.05).In the laboring state,the expression level of COX2 protein in amnion membranes of Qingping sows with signs of labor onset on day 111 of gestation(PAR111Q)was higher than Large White sows with signs of labor onset on day 114 of gestation(PAR114D)and Qingping sows with signs of labor onset on day 114 of gestation(PAR114Q)(P < 0.05),but DUSP9 protein expression level was not significantly different in the amnion membranes(P >0.05).These results suggest that miR-132-3p promotes the premature onset of parturition through activation of inflammatory and hormonal cascade ahead of schedule in Qingping sows at 111 d gestation.In summary,(1)miR-132-3p promotes inflammatory responses and PGE2 synthesis in the amnion through the activation of p38 and JNK signaling pathways via targeting DUSP9,leading to labor.(2)miR-132-3p promotes the premature onset of parturition through activation of inflammatory and hormonal cascades ahead of schedule in short-gestation Qingping sows during late gestation.