| Growth arrest specific protein 6(GAS6)plays an important role in the initiation and progress of tumors,and its signal transduction is involved in cell proliferation,adhesion and migration,but its related functions and mechanisms in endometriosis(EMs)are still unclear.In this study,we searched and downloaded the transcriptome datasets of endometriosis from GEO database for GEO online analysis,and differential genes were screened for GO clustering and KEGG pathway enrichment analysis.In endometriosis clinical samples,qRT-PCR was used to quantitatively validate the differential genes shared by more than three datasets.Immunohistochemistry and qRT-PCR were used to verify the expression patterns of potential target GAS6 and EMT markers,and immunofluorescence was used to co-label GAS6 and E-cadherin.Finally,GAS6 was overexpressed in HEC-1A cells,and the expression level of EMT markers and cell migration ability were examined by qRT-PCR,Western Blot and cell wound scratch assay.It is clear that the up-regulated expression of GAS6 mediates the EMT process and promotes cell migration to take part in the initiation and progress of endometriosis.The findings are as follows:1.Screening and analysis of differential genes in endometriosis.Four endometriosis related transcriptome datasets were screened out from the GEO database using "endometriosis" or "EMs" as keywords.Fortyseven differential genes were screened out by GEO online analysis and Venn map intersection.Go and KEGG enrichment analysis of the differential genes by metascape,and the results demonstrated that they were greatly enriched in biological processes for instance cell migration and related signal pathways like MAPK,PI3K-AKT,and tight junction.2.Quantitative verification of differential genes in endometriosis.The endometrium of ten women with no endometriosis and no definite disease was taken as the control group,and the ectopic endometrium of eleven patients with chocolate cyst of ovary as the case group,the qRT-PCR was used to quantitatively verify the nine differential genes shared by more than three datasets.It can be seen that the m RNA levels of the case control group were in line with the results of bioinformatics analysis.3.Expression patterns of GAS6 and EMT markers in endometriosis clinical samples.qRT-PCR and immunohistochemistry to test the expression patterns of GAS6 and EMT markers in clinical samples of endometriosis.It was confirmed that GAS6 was up-regulated in the case group compared with the control group(P < 0.05),and the sign of EMT in the case group,that is,the down-regulated expression of E-cadherin(P < 0.05)and the up-regulated expression of Vimentin(P < 0.01).In clinical samples of endometriosis,GAS6 and E-cadherin were co-labeled by immunofluorescence.The results proved that the expression of E-cadherin was low in the ectopic endometrium glandular epithelial cells of patients with endometriosis whose GAS6 was upregulated,suggesting that GAS6 may mediate the EMT process in endometriosis.4.The overexpression of GAS6 in HEC-1A cells mediates EMT to promote cell migration.HEC-1A cells were overexpressed with GAS6,qRT-PCR,Western Blot and cell wound scratch assay were used to test the expression model of EMT markers and the migration ability of cells.The results indicated that the up-regulated expression of GAS6(P < 0.01)can inhibit E-cadherin(P <0.05),promote the m RNA levels of N-cadherin(P < 0.01)and Vimentin(P< 0.01),and promote the migration of cells(P < 0.05).In conclusion,this study preliminarily revealed that GAS6 is highly expressed in endometriosis patients and may mediate the EMT process,promote the migration of endometrial epithelial cells,participate in the initiation and progress of endometriosis,and offer a potential target for clinical diagnosis and treatment of endometriosis. |
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