Study The Mechanism Of CAPN7 Impaired Embryo Implantation Via Degradating HOXA10

Object:The aim of this study was to assess the role of CAPN7 in the impaired embryo implantation.Methods:The expression of CAPN7 and HOXA10 were analyzed by Western Blot after treatment with estrogen and progesterone in Ishikawa cells.The expression of CAPN7,HOXA10 and ITGB3 in endometrium of EMT patients were also analyzed by qRT-PCR,Western Blot and immunohistochemical staining.Western Blot and qRT-PCR were also used to detect the expression of HOXA10 and its downstream genes ITGB3 in adenovirus-mediated CAPN7 enhanced Ishikawa cells.Co-immunoprecipitation and immunofluorescence assay demonstrated the interaction and localization between CAPN7 and HOXA10.The mouse uterus on 4.5dpc were collected to detect the CAPN7 expression.Degradation assay in vitro,Luciferase and DNA-binding assay demonstrated that CAPN7 could degrade HOXA10 in vitro and prevented HOXA10 binding conserve sequence(TTAT)within the promoter region of ITGB3 and transcriptionally regulate their expression.Moreover,the in vitro embryo adhesion model was employed to explain the HOXA10-mediated function of increased CAPN7 in Ishikawa cells.Furthermore,the CAPN7 inhibitor was used to confirm the inhibition function of HOXA10.Results:In the mouse uterus of 4.5dpc,the CAPN7 expression was significantly decreased(decreased about 50%,p<0.01).The adenovirus-mediated enhanced expression of CAPN7 in the mouse uterus inhibited the embryo implantation on the 4.5dpc and the blastocyte were flushed with PBS.In the secretive-phase endometrium from the infertile endometriosis(EMT)patients,CAPN7 expression was aberrantly high(increased about 1.5 times,p<0.001),negatively related with the expression of HOXA10 and ITGB3 which were downregulated.In the Ishikawa cells,the repression of CAPN7 expression was observed by 12h after treatment with estrogen and progesterone.In contrast,HOXA10 expression was rapidly induced after in vitro stimulation.In addition,CAPN7 and HOXA10 physiologically combined with each other in the Ishikawa cells and both of them localized mainly in the nucleus of Ishikawa cells.Enhanced expression of CAPN7 in the Ishikawa cells decreased HOXA10 expression and its transcriptional activity(decreased about 40%,p<0.05),leading to the downregulation of HOXA10-mediated target gene ITGB3 expression.At the same time,CAPN7 impaired embryo implantation in the BeWo spheroids adhesion assay(decreased about 60%,p<0.05).Mechanistically,CAPN7 degraded HOXA10 in vitro and impaired HOXA10 protein stability,preventing the binding of HOXA10 to the conserve sequence(TTAT)within the promoter region of ITGB3.Moreover,the selected inhibitor ALLN reversed the effect of CAPN7 on HOAX10.Conclusion:CAPN7 expression was significantly decreased during the window of embryo implantation in the mouse.In the infertile EMT endometrium,the CAPN7 expression was aberrantly high,while HOXA10 expression was decreased.Moreover,CAPN7 expression was decreased after treatment with estrogen and progesterone,which indicated that CAPN7 was a negative regulator of embryo implantation.The aberrantly increased CAPN7 in the endometrial of infertile EMT patients impaired embryo adhesion by degrading HOXA10 expression and transcriptionally repressing HOXA10 activity.In addition,selected CAPN7 inhibitor reversed CAPN7 degradation of HOXA10 and saved HOXA10 function.The research on the mechanism of CAPN7-regulating embryo implantation could provide the theoretical basis for improving the success rate of embryo implantation in women with EMT who are seeking for IVF-ET.