Pik3r1 Can Affect Endometrial Decidual Process In Mice Through PI3K-AKT-mTOR Pathway

Objective:As a key link in successful pregnancy,the successful completion of embryo implantation requires a functionally complete blastocyst to worksynergistically with the endometrium in a tolerant state.The decidualization of endometrial stromal cells is an important part of embryo implantation,but its mechanism is not yet clear.This article aims to investigate the role of pik3r1 in the process of endometrial decidualization in mice and the related molecular mechanisms.Methods:(1)The normal pregnancy model of mice was established,on the 4th day of pregnancy,the uterine horn injection of PI3 K inhibitor LY294002,the effect of PI3 K on embryo implantation was observed in the 6th day of pregnancy.(2)The normal pregnancy model of mice was established,the protein expression of pik3r1 in endometrial tissue of D1-D7 was detected by immunohistochemistry and Western Blot.(3)Establishment of artificially induced decidualization model in mice,the protein level of pik3r1 was detected by Western Blot.(4)The primary mouse endometrial stromal cells were isolated and cultured,and the purity of the primary endometrial stromal cells was observed by immunofluorescence to detect the expression of the stromal cell marker vimentin.Treatment of cells with siRNA transfection of pik3r1 and PI3 K inhibitor LY294002,the protein level of pik3r1 before and after transfection was detected by Western Blot,and the transfection effect was observed,the cell cycle and apoptosis levels were detected by flow cytometry to study the effect of pik3r1 on endometrial stromal cells.(5)The primary mouse endometrial stromal cells were isolated and cultured,after pik3r1 siRNA transfection and PI3 K inhibitor LY294002 treatment of cells,E2 and P4 were added to induce decidualization of endometrial stromal cells.Quantitative PCR was used to detect mRNA levels of dtprp,a marker of stromal cell decidualization,the effects of silencing pik3r1 expression and inhibition of PI3 K activity on decidualization of endometrial stromal cells were observed.(6)The protein levels of PCNA and BAX/BCL-2 were detected by Western Blot,and the effects of silencing pik3r1 expression and inhibiting PI3 K activity on endometrial stromal cells in decidualization were observed.Western blot was used to detect the protein levels of PI3KdownstreamrelatedsignalmoleculesAKT/p-AKT/mTOR/p-mTOR/P70S6K/p-P70S6 K.Themolecularmechanism of pik3r1 affecting decidualization of endometrial stromal cells was initially revealed.Results:(1)After PI3 K inhibition,the number of mouse embryo implantation was significantly lower than that of the control side.(2)The expression of pik3r1 in the endometrium of mouse early pregnancy had spatio-temporal differences.Its protein is localized in endometrial stromal cells,but it is low in decidual cells,and its expression is decreased with the decidualization of stromal cells.(3)The expression of pik3r1 was significantly reduced in the artificially induced decidualized endometrium compared with the control side.(4)Down-regulation of pik3r1 expression and inhibition of PI3 K activity increased the apoptosis of endometrial stromal cells,the percentage of S phase in the cell cycle decreased,and the expression of PCNA protein decreased.(5)Down-regulating the expression of pik3r1 and inhibiting PI3 K activity can significantly reduce the mRNA level of dtprp,a molecular marker of decidualization.(6)Down-regulation of pik3r1 expression and inhibition of PI3 K activity both inhibited PCNA protein expression,up-regulated BAX protein expression,and down-regulated BCL-2 protein levels;meanwhile,the downstream signaling molecules AKT/mTOR/P70S6 K phosphorylation levels decreased.Conclusion:Pik3r1 promotes stromal cell proliferation and inhibits cell apoptosis through the PI3K-AKT-mTOR signaling pathway.It plays an important role in the process of mouse uterine endometrial decidualization and participates in the regulation of embryo implantation.