Talin1 Regulates Endometrial Adhesive Capacity Through The Ras Signaling Pathway

Aim and significance:To explore the influence of Talin1 on endometrial adhesive function and the potential molecular mechanism.First,we analyze the influence of Talin1 on endometrial adhesive function,and then search for downstream pathways through RNA-sequencing analysis.In the end,the molecular mechanism of Talin1 affecting endometrial adhesive function was explored.Methods:Part Ⅰ,in the first part,the m RNA and protein expression levels of Talin1 and IntegrinαVβ3 in recurrent implantation failure(RIF)and normal fertile control(NC)endometrium during LH+7 days were determined by Real-time quantitative polyme Rase Chain Reaction(RT-q PCR),western blot(WB),and Immunohistochemistry staining(IHC).Part Ⅱ,the adhesion model of human trophoblast spheres(JEG-3)to human endometrium epithelial cells(Ishikawa)was established.In Ishikawa,Talin1 was successfully silenced or overexpressed.The silenced or overexpressed Talin1(si Talin1 or PCDNA3.1-Talin1)was detected by RT-q PCR and WB methods,and the adhesive function of JEG-3 trophoblast spheroid to endometrial epithelial cells was observed.Part Ⅲ: Talin1 si RNA were transfected into the intrauterine cavity of CD1 mice on pregnancy day 2.The knockdown efficiency in the endometrium of mice was verified by WB and RT-q PCR methods.The number of embryo implantation on pregnancy day 8 and the number of offspring after parturition were calculated.Part Ⅳ: Next generation RNA sequence technology and bioinformatics analysis were used to find the downstream molecules and signaling pathways affected by Talin1.The effect of Talin1 on Ras signaling pathway was verified by RT-q PCR and WB methods.Part Ⅴ: First,Salirasib was used in Ishikawa cells and CD1 mice to verify the effect of Ras signaling pathway on the adhesive function of trophoblast spheroid JEG-3 to Ishikawa and the number of embryo implantation in mice.Then,in Ishikawa cells,rescue experiments confirmed that Talin1 regulated the adhesion of endometrial epithelial cells through the Ras signaling pathway.Result:1.Talin1 and IntegrinαVβ3 are located in endometrial luminal epithelial gland epithelium and mesenchymal cells.The m RNA and protein expression levels of talin1 and IntegrinαVβ3 in endometrium(LH+7)in the RIF group were lower than those in the control group.2.In Ishikawa cells,silencing or overexpression of Talin1 level can decrease or increase the adhesion ability of JEG-3 spheroid to endometrial epithelial Ishikawa cells.3.Talin1 was silenced in the uterine cavity of CD1 mice on pregnancy of day 2,which affected the number of embryos implantation and the number of offspring born after childbirth.4.The Next generation whole genome sequencing and bioinformatics analysis showed that silencing Talin1 affected the activity of Ras signaling pathway5.Salirasib reduced the ability of JEG-3 to adhere to endometrial epithelial cell Ishikawa.Intrauterine injection of Salirasib in CD1 mice on day 2 affected the number of embryos implanted.In Ishikawa cells,the inhibitory effect of Salirasib on the Ras signaling pathway and the reduced adhesion function of the JEG-3 trophoblastic spheres to Ishikawa could be rescued by Talin1 overexpression.Conclusion:This study confirmed the effect of Talin1 on the adhesive function of endometrial epithelial cells from the four dimensions of clinical tissue level,cell level,molecular level,and animal level.