Study The Mechanism For Impaired Embryo Implantation By Enhanced Sumoylation Of HOXA10

Object:Study the molecular mechanism for SUMOylation of HOXA10 impairing endometrial receptivity and embryo implantation.Methods:The physiological levels of SUMOylation-related proteins in proliferative and mid-secretory endometrium from normal fertile women and the pathological levels of SUMOylation-related proteins in mid-secretory endometrium from patients with recurrent implantation failure(RIF)after IVF-ET treatment were detected by Western Blot and immunohistochemical staining.The covalent binding of HOXA10 and SUMO1 molecules was clarified by coimmunoprecipitation follewed with Western Blot,and SUMO1 modification site was clarified by point mutation of lysine 164 of HOXA10 protein.The expression of SUMO1-HOXA10 was detected in Ishikawa cells treated with estrogen and progesterone.The expression level of SUMO1-HOXA10 fuesd protein in the mid-secretory endometrium of patients with RIF was detected via protein co-immunoprecipitation followed with Western Blot.The subcellular localization of HOXA10WT and SUMOylation defective mutant HOXA10K164R was detected by immunofluorescence and cytoplasmic protein separation technology.The effect of SUMO1 on the stability of HOXA10 protein was detected in cells treated with protein synthesis inhibitor cycloheximide.The molecular mechanism for SUMO1 regulating the stability of HOXA10 protein was determined by using proteasome inhibitor MG132.The effort of SUMOl modification on the transcriptional activity of HOXA10 was detected through luciferase reporter gene assay,and the underlying molecular mechanism was determined through the DNA-protein co-precipitation experiment.The SUMO-specific proteases(SENPs)which mediate the deSUMOylation of HOXA10 was identified by protein co-immunoprecipitation followed with Western Blot.In vitro embryos adhesion models were constructed by co-culture of BeWo cell blastocyst-like spheroids or mice blastocysts and endometrial Ishikawa monolayer cells,and the effect of HOXA10 SUMOylation on embryo implantation was examined in these two in vitro models.Results:In the first part,the level of SUMO1-modified proteins with molecular weight of～90kDa in the secretory phase was significantly decreased than in the proliferative phase(P<0.05),while the expression of SENP1(P<0.01)and SENP2(P<0.05)was significantly increased in the secretory phase compared with that in the proliferative phase.In the Ishikawa cells treated with estrogen and progesterone,the level of SUMO1-modified proteins with molecular weight of～90kDa were decreased in a time-dependent manner,but expression of SENP1 and SENP2 increased in a time-dependent manner.The expression of HOXA10 protein in the endometrium of patients with RIF was not significantly different from that in the normal control group,but the level of SUMO1-modified proteins was significantly increased in the endometrium from patients with RIF than in normal control group(P<0.01),both in the glandular epithelial cells and stromal cells.In the second part,HOXA10 was found to be covalently modified by SUMO1 at lysine 164,and combined stimulaiton of estrogen and progesterone significantly inhibited the expression of SUMO1-HOXA10 in Ishikawa cells.Both HOXA10WT and SUMOylation defective mutant HOXA10K164R located in the nucleus of Ishikawa cells.SUMO1 inhibited the protein stability of HOXA10 by promoting promotes ubiquitin-mediated degradation of HOXA10(P<0.05),and diminished HOXA10-mediated transactivation by decreasing its DNA-binding capacity(P<0.001).However,SUMO1 could not inhibit the protein stability and transcriptional activity of HOXA10K164R.Both SENP1 and SENP2 directly mediated the removal of SUMO1 from HOXA10 and promoted the protein stability of HOXA10.In the third part,we found that HOXA10 and SUMO1 coexpression significantly decreased Be Wo spheroid attachment compared with that of HOXA10 alone(P<0.05),but SUMO1 overexpression did not impair HOXA10K164R-promoted BeWo spheroid attachment.An in vitro model of mouse embryo attachment was used to further confirm that SUMO1 overexpression inhibited the HOXA10WT-promoted mouse embryo attachment significantly(P<0.01),but did not inhibit HOXA10K164R-promoted mouse embryo attachment.Overexpression of SENP2 could reverse the inhibitory effect of SUMO1-HOXA10 on embryo attachment(P<0.01).In particularly,there was abnormally enhanced expression of SUMO1-HOXA10 in the mid-secretory endometrium from patients with RIF compared with that in normal fertile women(P<0.05).Conclusions:SUMO1 modification significantly supressed the protein stability and transcriptional activity of HOXA10,and inhibited the HOXA10-promoted BeWo spheroids attachment rate and mouse blastocysts attachment stability in the in vitro embryo implantation models.SENP2-mediated deSUMOylation of HOXA10 could reverse the inhibitory effect of SUMO1-HOXA10 on embryo attachment.The abnormally elevated SUMO1 modified HOXA10 in RIF patients impairs the endometrial receptivity and embryo attachment by supressing the HOXA10 protein function,whicn may contribute to the embryo implantation failure.Detection SUMO1 modified HOXA10 can help assess the endometrial receptivity of patients with IVF-ET treatment,assist the determination of "window of implantation",provide the research foundation and open up new treatment directions for improving the embryo implantation rate of RIF patients.