The Mechanism Study Of Increased Crotonylation Modification Of EGFR In Cumulus Cells Mediated By Ep300 Is Essential For The Human Oocyte Matuaration In Vitro

Objective: The purpose of this study was to investigate the role of Ep300 in the regulation of crotonylation of EGFR during maturation of cumulus cells and its related molecular mechanisms.Methods:1)To detect the effect of Ep300 on proliferation and apoptosis of cumulus cells:We used siRNA technology to knock down the expression of Ep300 and specific activator CTPB to activate Ep300,and Dot Blot to detect the level of total Kcr in cells.Secondly,in the case of low expression or activation of Ep300,Annexin V-FITC/PI staining and Western Blot detection of apoptotic markers fox-01 a and fox-03 a were used to verify whether Ep300 inhibited apoptosis.Meanwhile,CCK8 assay was used to detect whether Ep300 promoted cell proliferation.2)Screening and validation of substrates of Ep300:The intersection of the target proteins modified by Kcr showed that EGFR could be modified by Kcr and might be the substrate of Ep300.First,recombinant EGFR(r-EGFR)was co-incubated with Kcr-CoA to verify the presence of Kcr modification of EGFR,and Co-immunoprecipition(Co-IP)to verify the presence of Kcr modification of endogenous EGFR.Secondly,the Co-IP and siRNA experiments were performed to verify whether Ep300 binds to EGFR and Kcr modifies EGFR.Finally,the effects of Nacr & siEp300 Mediated-Kcr modification on EGFR and P-EGFR expression levels were preliminarily explored.3)To explore the molecular mechanism of up-regulation of EGFR and P-EGFR expression by Kcr of EGFR and its influence on downstream pathways.First,the molecular mechanism of crotonylation to up-regulate EGFR expression was verified:(1)Verification of transcription level:RT-PCR was used to detect the expression of EGFR under different conditions(Nacr vs cont.).(2)Synthesis level verification: HEK 293T(Nacr vs.cont.)was treated with protein-synthesis inhibitor cyclohexanilimide(CHX),and the expression of EGFR was detected by Western Blot.(3)Degradation level verification: HEK 293T(Nacr vs.cont.)was treated with proteasome inhibitor MG-132,and the expression of EGFR was detected by Western Blot.Second,to verify the molecular mechanism of crotonylation to up-regulate P-EGFR expression:Combined with the analysis of Uniprot database,it was found that the Kcr site of EGFR was also one of the ATP-binding sites.Therefore,we co-cultured r-EGFR and different concentrations ofKcr-CoA,and cultured them with ATP equivalent TNP-ATP to detect the binding ability of r-EGFR and TNP-ATP.To further detect the effect of crotonylation on the binding of endogenous EGFR to ATP:HEK 293 T was treated with different treatments(Nacr vs cont.),and IP EGFR was incubated with TNP-ATP 4 h later,to detect the binding capacity of EGFR and TNP-ATP.Finally,the influence of Ep300-mediated crotonylation on the downstream EGFR signaling pathway was detected.Nacr and siEp300 were applied in the cumulus cells and HEK 293 T,respectively,and the expressions of P-ERK,ERK,P-AKT and AKT in the downstream molecules of EGFR were detected by Western Blot.Results:1)After silencing Ep300,Kcr expression decreased and apoptotic markers fox-01 A and fox-03 A increased.CTPB,the specific activator of Ep300,increased the expression of Kcr and decreased the apoptotic markers fox-01 A and fox-03 A after the activation of Ep300.Annexin V-FITC/PI and CCK8 showed that Ep300 promoted the proliferation of cumulus cells and HEK 293 T and inhibited apoptosis.2)According to the database analysis,EGFR may be the substrate of Ep300.Firstly,both endogenous and exogenous EGFR can be modified by Kcr,and the Kcrmodification of EGFR is mediated by Ep300,and crotonylation can up-regulate the expression of EGFR and p-EGFR.3)It was verified that crotonylation could inhibit EGFR degradation and up-regulate EGFR expression.At the same time,the binding ability of EGFR and ATP was enhanced,thereby up-regulating the expression of p-EGFR.Further experiments confirmed that crotonylation of EGFR can activate the expression of p-ERK and p-AKT in its downstream pathways.Conclusion: Ep300 mediates crotonylation of EGFR,up-regulates the expression of EGFR and p-EGFR in cumulus cells,activates EGFR pathway to promote oocyte maturation in vitro,and provides a new strategy for improving the oocyte maturation in vitro.