Integrated bioinformatic analysis and experiments reveal EFEMP1 as a novel aging-related signature gene in calcific aortic valve disease

The risk of developing calcific aortic valve disease (CAVD) increases two-fold with each decade of life. This study aims to identify and validate critical aging-related gene involved in the pathological process of CAVD. In this study, the gene expression profile of CAVD patients was downloaded from Gene Expression Omnibus (GEO) database. Integrated bioinformatic analysis such as weighted gene co-expression network analysis (WGCNA), differential analysis and single cell RNA sequence (scRNA-seq) analysis was used to screen differentially expressed aging-related genes (DEAGs). EGF-like fibulin extracellular matrix protein 1 (EFEMP1) was identified by both bulk and scRNA-seq analysis. The expression signature of the EFEMP1 was validated at the tissue and cellular levels using both public and experimental data. EFEMP1 was knocked down using small interfering RNA to investigate its role in the osteogenic differentiation of valvular interstitial cells (VICs). A total of 699 differentially expressed genes (DEGs) were obtained from the combined datasets, and WGCNA identified 6 co-expression modules, of which one hub module (brown module) was most correlated with CAVD. By intersecting the DEGs, hub module genes and aging-related genes, we obtained 16 DEAGs, and a close mutual interaction between these DEAGs was detected. Among the 16 DEAGs, 12 DEAGs were up-regulated, while 4 DEAGs were down-regulated. To ascertain the expression profiles of the 16 DEAGs in VICs, we analyzed another two GEO datasets, and found that only EFEMP1 and IL6 were consistently up-regulated. The EFEMP1 was mainly expressed in VICs based on scRNA-seq analysis and up-regulated in the VICs from animal models of CAVD. The results of public data analyses were further verified by experimental data, the EFEMP1 expression was up-regulated in the calcified aortic valve samples detected by immunohistochemical and immunofluorescent staining at tissue level. Consistent with the changing trends of osteogenic markers such as ALP, RUNX2, and BMP2, the expression level of EFEMP1 was up-regulated in VICs induced by osteogenic medium detected by qPCR and western blotting and the expression levels of osteogenic markers were decreased in VICs after EFEMP1 knockdown. Public and experimental data revealed that EFEMP1 was up-regulated in CAVD at both tissue and cellular levels. Furthermore, functional experiment demonstrated that EFEMP1 knockdown alleviated the osteogenic differentiation of VICs.