The Interactional Regulation Analysis Of SOCS3,IL-6,STAT3 And Relevant Non-Coding RNA In Porcine Endometrium

The efficiency of swine embryo implantation in endometrium has important effects on litter size,the molecular regulation mechanism of swine implantation is still in the research phases.Based on the RNA-seq of different breeds’ and different pregnant days’ sows,we screened out critical signaling pathway(JAK/STAT)and candidate genes SOCS3,IL-6 and STAT3 by bioinformatics analysis.We performed long non-coding RNA prediction by software and filtered out three differentially expressed lnc RNA(lnc RNA-162621,lnc RNA-1024795,lnc RNA-1538549)and ssc-mi R-18 a which have the possibility of regulating JAK/STAT signaling pathway.Therefore,in order to revealing the regulation mechanism of swine embryo implantation,we explored the influence of SOCS3,lnc RNA-1538549(candidate lnc RNA)and ssc-mi R-18 a on JAK/STAT signaling pathway via RNA overexpression and interference technology.The main results are as follows:1.QRT-PCR was used to detect the relative m RNA expression of SOCS3,IL–6,STAT3 and lnc RNA(lnc RNA-162621,lnc RNA-1024795,lnc RNA-1538549)in Yorkshire and Meishan sows’ endometrium on different pregnany day.The result indicates that the expression tendency of above-mentioned genes is consistent with the result of RNA-seq.The expression of SOCS3 and IL-6 increased with the number of pregnancy days,there are significant differences between different varieties.The expression of STAT3 in day 15 pregnancy sows is peak expression.The result indicates that the expression tendency of lnc RNA-162621 and lnc RNA-1024795 is consistent with the result of RNA-seq.The expression of lnc RNA–1538549 is low expression in day 18 pregnancy sows and increases in day 32 pregnancy sows.The expression of lnc RNA–1538549 in day 32 pregnancy Yorkshire and Meishan sows is higher than day 18 pregnancy sows’.The expression of lnc RNA-162621 and lnc RNA-1024795 is without trend.Western Blot was used to detect the relative protein expression of SOCS3,IL–6,STAT3 and phosphorylated STAT3(p-STAT3)in Yorkshire and Meishan sows’ endometrium on different pregnany day.The result indicates that the tendency of the expression of SOCS3 and IL-6 in Meishan sows’ endometrium is consistent with RNA-seq results.The expression of STAT3 and p-STAT3 in day 32 pregnancy Yorkshire and Meishan sows’ endometrium are significantly higher than day 18 pregnancy sows’,and the expression of above-mentioned protein in Meishan sows’ are significantly higher than Yorkshire sows’ in the same pregnancy day.2.The locations of SOCS3,IL-6 and p-STAT3 were performed by immunohistochemical experiments in day 18,32 pregnant Yorkshire and Meishan sows’ uterus.It shows that three proteins expressed in endometrial epithelial cells,uterus gland cell and vascular endothelial cells mainly,besides p-STAT3 was located in myometrial muscle cells.The expression level of SOCS3 and IL-6 in uterus gland cell is different between Yorkshire and Meishan sows.3.QRT-PCR,Western Blot and ELISA were used to detect the influence of overexpression and interference of SOCS3 on IL-6 and STAT3,and then detect the influence of overexpression of IL-6 on SOCS3.The result indicates that SOCS3 inhibits the expression of IL-6,and inhibits STAT3 protein phosphorylation.IL-6 promotes the expression of SOCS3.4.QRT-PCR,Western Blot and ELISA were used to detect the influence of overexpression of lnc RNA-1538549 and ssc-mi R-18 a on the expression of IL-6 and STAT3.The result indicates that lnc RNA-1538549 promotes the expression of IL–6 and inhibits the expression of its upstream gene LYPD6.Ssc-mi R-18 a can regulate the expression of PIAS3.It also can reduce the level of STAT3 protein phosphorylation.