Phosphorylated Proteomics Of Protein Spectrum And Axl And MTOR Related Regulation On The Pathogenesis Of Endometriosis

Objective: This study aims to explore specific proteins and phosphorylated sites related to the pathogenesis of endometriosis through the combined analysis of proteomics and phosphorylated proteomics,and verify their influence on the pathogenesis of endometriosis,in order to provide theoretical basis for the early diagnosis of endometriosis and the study of non-hormone therapeutic targets.Methods: 1)in this study,patients with endometriosis of the ovary were selected as the research object,5 patients with ectopic endometrium tissue samples and 5 patients with in situ endometrium tissue samples were selected as the control group,and the total protein was extracted and hydrolysis by protease,followed by LC-MS/MS high resolution mass spectrometry,TMT labeling,and peptide classification.High resolution mass spectrometer was used to detect the differential expression protein and obtain the original data.The phosphorylated peptide was labeled by TMT,and the phosphorylated peptide was enriched by Ti O2 method and IMAC method in series.The original mass spectrometry data were detected by high resolution mass spectrometer,and the differentially expressed proteins and differentially expressed protein phosphorylation modification sites were obtained by bioinformatics analysis.2)The characteristics of proteomic and phosphorylated proteomic data were observed from a global perspective.The combined analysis of the two omics was carried out.Statistical methods such as debackground analysis,PCA analysis,Venn and volcano map were used to analyze the differences between and within the groups of differential protein peptides and differential phosphorylated modified peptides in the two omics.The annotation and enrichment function differences of GO and KEGG were analyzed by histogram and cyclic graph.The kinases and substrates with the most significant differences were found by motif analysis,kinase-substrate prediction and kinase-substrate interaction PPI network analysis.KSEA analysis was used to predict kinase activity,and combined with the KEGG pathway analysis results,more important and valuable protein kinases,phosphorylation events and key signaling pathways were found.Then,a valuable signaling pathway and the protein kinases and substrates with the largest activity difference in this signaling pathway were selected,and the difference was verified by q RT-PCR method.3)The PI3K/AKT signaling pathway and Axl and mTOR protein kinase targets with research value were verified and functional studies were conducted based on the two sets of studies.The subjects were isolated and cultured as before,HEc ESCs,HEu ESCs and HEn ESCs,and identified by immunohistochemistry.To investigate the mechanism of PI3K/AKT signaling pathway affecting HESCs cell apoptosis by targeting upstream Axl kinase and mTOR kinase at the cellular level.The expression differences of HEc ESCs,HEu ESCs,HEn ESCs,Axl,mTOR,p-mTOR and their m RNA were verified by q RT-PCR.At the cell level,si RNA was selected to transfect Axl targets and rapamycin to regulate mTOR targets.Differences in the expression of Axl,mTOR and p-mTOR cyclin D1 were detected by WB and q RT-PCR.Results:1)A total of7575 different proteins were screened in the proteomic study of the three groups of samples.Compared with the ectopic endometrial group and the present endometrial group,the maximum number of different proteins was 535,of which 272 were up-regulated and263 were down-regulated.Secondly,there were 296 different proteins between the ectopic endometrial group and the normal endometrial group,among which 198 were up-regulated and 98 were down-regulated.There were 52 different proteins between the present endometrial group and the normal endometrial group,among which 12 were up-regulated and 40 were down-regulated.GO annotation and enrichment analysis results showed that the ectopic endometrial group and the normal endometrial group had the most significant differences in extracellular matrix tissues,growth plate cartilage,chondrocyte morphogenesis and elastic fiber assembly.In terms of molecular function,the difference of antigen binding,CXCR3 chemokine receptor binding and interleukin-8 receptor binding between the endometrial group and the normal endometrial group was the most significant.In terms of cell composition,the difference between the three groups was extracellular space.The immunoglobulin complex and hemoglobin complex were significantly different between the present endometrium group and the normal endometrium group,while the ectopic endometrium group had significant functional differences between the present endometrium group and the normal endometrium group,including extracellular matrix,Z-disk and membrane attack complex,respectively.KEGG annotation and enrichment analysis results showed that the pathways with the highest differences among the three groups were metabolic pathway,PI3K/AKT signaling pathway,complement and coagulation cascade,adhesion plaque,cancer pathway and systemic lupus erythematosus.The common differential signaling pathway among the three groups was metabolic pathway and PI3K/AKT signaling pathway.The subcellular localization distribution of different proteins between ectopic endometrium group and endometrium group and normal endometrium group was sorted by the size of proportion,which were nucleus,extracellular,plasma membrane and cytoplasm in order.The subcellular localization distribution of different proteins in the endometrium group and the normal endometrium group was sorted according to the size of the proportion,in order of extracellular,nuclear,cytoplasm and plasma membrane.2)Based on proteomic studies and phosphorylated proteomic studies,the analysis results showed that there were 2071 proteins in common between proteomics and phosphorylated proteomic,5504 proteomic specific proteins,and 53 phosphorylated proteomic specific proteins.The number of differentially phosphorylated proteins in ectopic endometrial group and normal endometrial group was up to 1701,of which 607 were up-regulated and 1094 were down-regulated.Serine/arginine repeat matrix protein 2 was the common differential phosphorylated protein in the ectopic endometrium group,the present endometrium group and the normal endometrium group.The common differential phosphorylated homologous protein of ectopic endometrium group and normal endometrium group was extension protein A compared with the present endometrium group,respectively.The common differential phosphorylated proteins in the ectopic endometrial group,the present endometrial group and the normal endometrial group were kinase A anchored protein 12 and sorbic SH3 domain protein 2.The results of KEGG enrichment analysis showed that the main signaling pathways of the three groups were nuclear cytoplasmic transport,adhesion plaque,olfactory conduction,spliceosome,primary melaningeneration,long-term enhancement,vascular smooth muscle contraction,thyroid hormone,hepatocellular carcinoma and tumor small molecule RNA.The results of subcellular localization analysis of differential proteins between the three groups showed that the difference of differential proteins and differential phosphorylated proteins in the plasma membrane was the most significant between the ectopic endometrial group and the normal endometrial group.The difference of differential phosphorylated proteins between the ectopic endometrial group and the normal endometrial group showed two different key proteins,namely myosin light streptokinase and smooth muscle and myosin 11.It may be related to endometriosis migration,implantation and invasion.The kinase-substrate prediction analysis was used to compare the top 10 kinases with the number of different substrates between the three groups.The common kinases in the three groups were PRKACA,MAPK3,CDK1,CSNK2A1,CDK2,MAPK1,AKT,AKT1,PRKACA and PRKACD.MAPK and AKT kinases are involved in mTOR signaling pathway.KSEA analyzed the difference of kinase activity among the three groups.The comparison between the ectopic endometrial group and the normal endometrial group showed that the kinases with the most significant difference and up-regulated activity were AKT1,AKT,MAPKAPK2,MAPK12,etc.,and the kinases with the most significant difference and down-regulated activity were CDK2,CDK1,CDK7 and MTOR,etc.The common kinases down-regulated in activity are AKT,MAPK and mTOR,all of which interact with the PI3K/AKT signaling pathway.Axl kinase is an important regulatory point upstream of the PI3K/AKT signaling pathway.PI3K/AKT signaling pathway,mTOR kinase and Axl kinase are selected as further research targets for verification and functional studies.3)The m RNA expression levels of Axl and mTORm RNA in normal endometrial stromal cells were significantly different from those in ectopic endometrial stromal cells(P<0.05).Compared with blank control group,m RNA expression levels of Axl and cyclin D1 in rapamycin group and Axl-si RNA group were significantly different(P < 0.05).Compared with rapamycin group,Axl m RNA expression level in Axl-si RNA group was significantly different(P<0.05).The m RNA expression levels of Axl and cyclin D1 were significantly different in rapamycin group(P<0.05).Compared with negative control group,Axl and cyclin D1 m RNA expressions in Axl-si RNA group were significantly different(P < 0.05).Compared with rapamycin group,Axl m RNA expression level in Axl-si RNA group was significantly different(P<0.05).The m RNA expression levels of Axl and cyclin D1 were significantly different in rapamycin group(P<0.05).Compared with negative control group,Axl and cyclin D1 m RNA expressions in Axl-si RNA group were significantly different(P<0.05).Compared with blank control group,the relative expression level of mTOR m RNA in rapamycin group was significantly different(P<0.05).Compared with blank control group,rapamycin group and negative control group,Axl-si RNA group showed the most significant difference in data(P<0.05).There was no significant difference in the expression of mTOR protein among the four groups.Compared with blank control group,the protein expressions of P-mTOR and cyclin D1 in rapamycin group and Axl-si RNA group were significantly different(P<0.05),and compared with negative control group were significantly different(P<0.05)Conclusion: Based on the combined analysis of clinical samples of endometriosis by LC-MS/MS high resolution mass spectrometry,TMT proteomics and phosphorylated proteomics,a series of differential proteins,signaling pathways and the most active kinases and substrates related to the pathogenesis of endometriosis were successfully screened out.The PI3K/AKT signaling pathway,mTOR kinase and upstream Axl kinase were verified.Axl and mTOR kinase were highly expressed in both ectopic endometrial stromal cells and in the endometrium,which verified the omics differences and confirmed that Axl and mTOR were involved in the occurrence of endometriosis.Axl is involved in the activation and proliferation of ectopic endometrial stromal cells by mediating the PI3K/Akt signaling pathway.mTOR may be involved in the occurrence of endometriosis with Axl through other mechanisms other than the PI3K/AKT signaling pathway.Regulation of Axl and mTOR kinases can promote the apoptosis of endometrial stromal cells.Axl and mTOR can become one of the research ideas of the pathogenesis of endometriosis and non-hormone target drugs,which needs to be further verified in vivo.Further exploration of the results of omics analysis can screen out more valuable targets,and provide more ideas for exploring the early screening and diagnosis of endometriosis and the study of non-hormone target drugs.