SOX9与Smad4在不同角膜基质细胞中的表达及相互作用OA

Objective To investigate the differential expression and interaction of SRY-box transcription factor 9(SOX9)and Smad family member 4(Smad4)in resting-state keratocytes(KCs),corneal fibroblastic cells(CFs),and cor-neal myofibroblasts(CMFs),and to provide experimental evidence for the mechanism and targeted therapy of corneal fi-brotic disorders.Methods Bioinformatic methods were used to analyze the GSE287915 dataset in the Gene Expression Omnibus database,screen for differentially expressed genes,and focus on SOX9.Quantitative real-time reverse transcription polymerase chain reaction(qRT-PCR)was performed to verify the expression of SOX9 in the mouse KCs(m-KCs),mouse CFs(m-CFs),and mouse CMFs(m-CMFs).The Gene Transcription Regulation Database(JASPAR)was used to predict the potential binding sites between SOX9 and Smad4.qRT-PCR was applied to detect the expression levels of Smad4 in the said three types of cells,and finally,co-immunoprecipitation(Co-IP)was used to verify the interaction between SOX9 and Smad4 proteins.Results Bioinformatic analysis showed that compared with KCs,SOX9 was significantly highly expressed in CFs and CMFs(both P<0.05).The qRT-PCR detection results revealed that the expression levels of Smad4 and SOX9 followed the trend:m-KCs group<m-CFs group<m-CMFs group,with statistically significant differences among the three groups(all P<0.001).The JASPAR database predicted that there were potential binding sites between SOX9 and the Smad4 promoter region.Co-IP results demonstrated that when SOX9 and Smad4 antibodies were used as the capture antibodies,the binding amount of Smad4 and SOX9 in the m-CMFs group was significantly higher than that in the m-CFs group and the m-KCs group(both P<0.05),while no obvious binding was detected in the m-KCs group.Conclusion The expression lev-els of SOX9 and Smad4 in in vitro cultured m-KCs,m-CFs,and m-CMFs are elevated with the degree of cellular activation and fibrosis progression.Moreover,a specific interaction between them has been identified in fibrotic phenotype m-CFs and m-CMFs induced in vitro.The upregulation of their expression and their protein-protein interaction may contribute to the fi-brosisn-related activation of keratocytes in vitro,providing an experimental basis for further mechanism studies on corneal fibrotic diseases.