MiRNA Expression Analysis And MiR-144 Functions Research During Labor Of Large White And Qingping Sows

Labor onset is a very complex physiological process,and its mechanism is poorly understood.Many factors are involved in the onset of labor,such as various hormones and inflammatory mediators during labor,which remain a dominant research topic in this field.Recent studies have reported that miRNA play a role in the regulation of genes that influence uterine quiescence/contractility during pregnancy and labor.We obtained the miRNA expression profile from the placentas of four groups of sows: Qingping sows pregnancy 112 d with signs of labor onset(QS),Qingping sows pregnancy 114 d with signs of labor onset(QL),Large White sows pregnancy 114 d with signs of labor onset(LL)and Large White sows pregnancy 112 d without signs of labor onset(LN).Compared with in LN,obtained differentially expressed miRNA,potential target genes were analysed using GO terms.Research miR-144 functions during labor of Large White and Qingping sows.Eight main results are listed below:1.In Large White and Qingping sows by high throughput sequencing obtained 4,131,656 and 5,687,285 small RNA raw reads,accounting for 60%~93% of raw reads were high-quality reads.2.Compared with the LN placenta,9 up-and 10 down-regulated miRNAs were found in the LL placenta,miR-144 modulate synthesis of PGE2,13 up-and 10 down-regulated miRNAs were found in the QL placenta,and 14 up-and 7 down-regulated miRNAs were found in the QS placenta.3.miRNA putative targets were analysed using GO terms.For the miRNA target genes,the annotation cluster with the highest score was involved in hormone stimulus and immune response.4.miRNA/mRNA networks in LN/LL were constructed using the Cytoscape software.We found that mi R-345-5p and miR-326 correlate with targets HNF4 A,Fos and SMAD3,whereas miR-193a-3p,miR-664-3p and miR-122 correlate with target SMAD3.5.Analysis of mouse amniotic tissue at 15.5 and 18.5 d results showed that miR-144,Fos,COX2 and PGE2 were increased.LPS treatment promoted miR-144,Fos and COX2 expression in the amnion.These findings suggest that up-regulation of the expression of miR-144,Fos and COX2 may play a role in labor.6.Overexpression Fos of WISH cells was led to an increase of mi R-144 and COX2.We found the pGL3-promoter-700 was promoter region.By using EMSA and ChIP assay indicated that Fos was recruited to the miR-144 promoter.7.We transfected WISH cells with miR-144 mimics and pmirGLO plasmid of Fos 3’UTR or COX2 3’UTR,which resulted in significant down-regulation of the luciferase activity of both the Fos 3’UTR and COX2 3’UTR.Transfecting the WISH cells with miR-144 mimics significantly decreased the levels of Fos and COX2 as well as the concentration of PGE2.We transfected WHIS cells with either miR-144 mimics/COX2 target protector/Fos target protector or miR-144 mimics/COX2 target protector/Fos target protector negative control(NC).The result showed that the protein levels of COX2 increased,thus implying that miR-144 can indirectly target COX2 via Fos.We transfected WHIS cells with Fos overexpression/Fos target protector/COX2 target protector or Fos overexpression/Fos target protector/COX2 target protector NC.The result showed that the protein levels of COX2 protein increased,as did PEG2 synthesis,thus indicating that miR-144 can partly inhibit COX2 protein via the up-regulation of Fos.8.WISH cells were treated with 10 nM E2,and mice were administered an amniotic sac injection of E2.E2 injection,which led to a moderate increase in the expression of miR-144,Fos and COX2 in both the amnion and WISH cells.Conclusion:(1)This is the first comprehensive report about the miRNA signatures in the placenta,their targets,and their transcriptional and hormonal regulation.(2)mi R-144 and targets,Fos and COX2,modulate synthesis of PGE2 in the amnion during labor.