IER3 Involved In Initiation Of Parturition Through NF-κB Pathways Regulation In Decidual Stromal Cells

Research objectives:We investigate the expression and pathway of IER3 in decidual stromal cells（DSCs）,in order to reveal its role in mechanism of parturition.Reaserch methods:（1）Through the single cell transcriptome sequencing of decidua before and after labor,we analyzed the different cell populations and related gene expression changes in decidua after parturition.（2）The decidua of pregnant women in term delivery（TL group）and not in term delivery（TNL group）were collected,combined with normal and lipopolysaccharide（LPS）inflammatory pregnant mouse models.We usd RT-q PCR,Western Blot,immuno-histochemistry and immuno-fluorescence techniques to verify and explore the expression levels of significantly up-regulated gene IER3,key molecules of NF-κB pathway iκB and P65,and its downstream target gene（such as IL-1,IL-6,IL-8,CCL2,CCL4）in DSCs.（3）Human primary DSCs were extracted and cultured in vitro.The expression levels of IER3 and key molecules iκB and P65 of NF-κB pathway were studied by LPS,PDTC intervention and si-IER3transfection.At the same time,human primary smooth muscle cells were extracted,and monocytes were separated by CD14（+）magnetic beads.And then we explored the expression levels of downstream target genes（such as IL-1,IL-6,IL-8,CCL2,CCL4）and phenotypic changes through cell co-culture,ELISA,immuno-fluorescence staining and other techniques.（4）We used t test,one-way ANOVA,χ²test and correlation analysis to analyze the differences between groups.Reaserch results:（1）Single cell transcriptome sequencing results of decidua before and after labor:IER3 was significantly upregulated in DSCs after labor（Log₂Fc=2.24836255）,and its downstream parturition-related genes,such as IL-1,IL-6,IL-8,PTGDS and MMP2 were also upregulated in different cells.At the same time,the rank of NF-κB pathway was in top 10 of KEGG enrichment analysis.（2）Location and expression of IER3 in term decidua:IER3 is mainly located at DSCs.And the IER3 m RNA and protein expression levels were significantly higher after delivery than before（P<0.05）.And the m RNA expression in human TL group,TNL group,mouse TL group,TNL group,LPS control group and experimental group were as follows:0.98±0.13,3.09±0.46,1.39±0.12,2.11±0.15,0.74±0.07,1.27±0.08;the protein expression 0.79±0.06,1.01±0.06,0.69±0.06,1.16±0.12,0.85±0.10,1.32±0.06.The m RNA expression levels of downstream target genes IL-1,IL-6,IL-8,IL-17,TNFα,CCL2 and CCL4 were synchronously up-regulated.（3）Results of IER3 and its pathway in DSCs:1)Molecular changes of IER3 and NF-κB pathway in DSCs:IER3was mainly expressed in the nucleus by immunofluorescence.The expression level of IER3 was LPS concentration and time dependent,and its m RNA and protein levels peaked at LPS 100ng/ml in 3 h and 6 h.The phosphorylation levels of key molecules of NF-κB pathway such as iκB and P65 increased synchronously,and the correlation coefficients were0.92（P=0.0268）,0.96（P=0.01）,respectively.The expression levels of IER3,iκB and P65 phosphorylation were down-regulated synchronously with PDTC 20μM for 48 h,and the correlation coefficients were 0.92（P=0.0266）,0.98（P=0.0039）.2)Effects of IER3 knockdown on NF-κB pathway in DSCs:Knockdown of IER3 resulted in downregulation of the phosphorylation levels of iκB and P65.The knockdown efficiency of IER3 was 60%,the phosphorylation levels of iκB and P65 were down-regulated almostly40%and 30%,and p65 nuclear entry activation was down-regulated about 91%（P<0.05）.3)Effect of IER3 knockdown on downstream target genes in DSCs:LPS supplementation in DSCs increased the secretion of downstream inflammatory cytokines such as IL-1,IL-6,IL-8,IL-17,TNFαand chemokines such as CCL2,CCL4,CCL5,CCL7,while knockdown of IER3 decreased their expression（P<0.05）.4)Effects of DSCs IER3 knockdown on monocyte migration and smooth muscle cell contraction:In DSCs,LPS-treated supernatants were co-cultured with monocytes,and it was found that the chemotaxis of monocytes could be promoted,while IER3 knockdown reduced the chemotaxis of monocytes by 37%（P<0.05）.Similarly,LPS-treated supernatant of DSCs cells promoted the contraction of smooth muscle cells,while IER3 knockdown inhibited the contraction（P<0.05）.Reaserch conclusions:In decidual stromal cells,the expression of IER3 regulated by NF-κB pathway and IER3 can regulate P65 positively triggering cascade upregulation of downstream inflammation and chemokine expression,and then participating in the initiation of parturition.14 Figures,9 Tables,66 References...