KD is the leading cause of acquired heart disease in children, given its coronary artery complications[9, 10], and there is still no effective therapy to prevent or treat the occurrence of coronary artery damage due to the limited information. It has been shown that coronary artery endothelial dysfunction plays an important role in the development of coronary artery damage under KD condition [11, 6]. Therefore, it is meaningful to figure out the changes in endothelial cells with or without KD condition, which might provide novel indicators and potential therapeutic targets for KD. In the present study, DEGs following treatment of coronary artery endothelial cells with KD plasma were investigated by RNA-sEq. Moreover, the biological functions of these DEGs were also analyzed by bioinformatics analysis to unravel the molecular mechanisms underlying KD.
The results of RNA-seq showed that 102 DEGs were significantly expressed, including 59 up-regulated and 43 down-regulated, in the KD group compared with the control group. And these genes are enriched in multiple biological processes and pathways, including cellular response to cytokines, cytokines-mediated signaling pathways, regulation of immune cells migration and chemotaxis, immune responses, cytokine-cytokine receptor interaction, chemokine signaling pathway, and TNF signaling pathway. And these biological processes and pathways is associated with vascular inflammation and activation of immune systems, which are two major contributors of the development of KD [12, 13].Therefore, genes relevant to above mentioned biological processes and pathways expressed in KD serum stimulated coronary artery endothelial cells were expected and partly verified by previous studies. For instance, the severity of TNF-mediated vascular inflammation and concurrent coronary artery damage were found in KD patients [14].
To validate the results of RNA-seq, six DEGs (SMAD1, SMAD6, CD34, PITX2, CXCL1, and APLN), which are significantly differentially expressed in KD group compared with control group and are closely associated with cardiovascular system, were analyzed by qPCR. SMADs family consists of 10 members (SMAD1-10) and activated by transforming growth factor (TGF)-β signaling pathway. TGF-β pathway is involved in inflammation and tissue remodeling mediated by endothelial cells, and immune activation, which support the importance of TGF-βpathway in KD occurrence and outcomes[15, 16].CD34 positive cells with enhanced adhesive and homing properties facilitates immune cells homing to the inflammatory sites and mediates inflammatory and immune responses [17].CXCL1, as the chemokine ligand, presents on endothelial surface and mediates immune responses by recruitment of neutrophils and monocytes [18, 19].APLN(apelin) is a vasoactive peptide and an endogenous ligand for APJ receptors. APLN/APJ receptor system plays a vital role in regulation of vascular function, including vascular tone, angiogenesis, proliferation, and permeability [20, 21].In addition, APLN/APJ system also mediates the anti-inflammatory effects of 1,25(OH)2D3 by reduction of pro-inflammatory mediators and adhesion molecules in LPS-stimulated macrophages [22]. PITX2 plays an important role in regulating the differentiation of endothelium and angiogenesis [23, 24].Although these studies provide insights into these biomarkers involved in regulation of cardiovascular and immune system, it is yet unknown whether these alterations can be considered as potential indicators for prevention and treatment of coronary artery damage in KD, which needs further investigations. Besides, some differences in levels of gene regulation between the RNA-seq and PCR data were noted. For example, the down-regulated expressions of SMAD6 and CXCL1 in RNA-seq results were shown to be reregulated in qPCR results, which were possible due to primer specificity, alternatively spliced forms of the genes, RNA integrity, and/or PCR conditions[25].