Human subjects and serum sample collection
All manipulations were approved by the ethics committee (20180601 and 202003802) at Shenzhen Children’s hospital and written informed consent was acquired from the guardians of all donors. A total of 40 subjects, including 20 normal children (average age:1.90 years and the ratio of male/female: 11/9) and 20 KD patients (average age:1.80 years and the ratio of male/female: 11/9), were recruited from Shenzhen Children’s Hospital. Serum samples were separated by centrifugation at 1,000×g for 10 min and aliquots were stored at −80℃.The diagnostic criteria of KD were according to the Japanese Circulation Society Joint Working Groups performed in 2012.
HCAECs culture and treatment
HCAECs were brought form ScienCell (San Diego, CA, USA) and cultured with endothelial cell growth medium containing growth factors, supplements, and 10% fetal bovine serum at 37℃ in 5%CO2. Cells were then stimulated with culture medium containing 15% serum (15% (V/V)) from KD patients or healthy donors for 24h. The cells were then used in subsequent studies. Each measurement was performed in triplicate (n=3).
RNA isolation and RNA-seq analysis
After treatments, total RNA was extracted using a Trizol reagent (Illumina, San Diego, CA, USA) according to the manufacturer’s protocol and isolated RNA was used for following studies. The MGI Easy mRNA library was used to construct the mRNA library and an Illumina HiSeq-2000 was used for sequencing. Data with less than 17 bases will be eliminated. The data were compared with the human mRNA database (reference database: human_hg19-refMrna20150317as) by the FANse2 algorithm. After being standardized by the RPKM (read per kilo bases per million mapped reads) method, the differentially expressed genes were analyzed from two aspects of different multiple and significant level. The threshold value was set up as follows: |log2 (Fold Change) | > 1 and p value < 0.01. The DEGs was drawn into heat map using R language.
Bioinformatics analysis
To further elucidate the role of DEGs in KD-induced coronary artery endothelial cells,
GO (Gene Ontology) enrichment analysis was performed to predict the function of DEGs and KEGG (Kyoto encyclopedia of genes and genomes) pathway analysis were performed to determine the involvement of DEGs in different biological pathways. FDR <0.01 were selected as threshold values for GO and KEGG pathway enrichment. And P<0.05 were considered statistically significant.
Quantitative Real time-polymerase chain reaction (qPCR)
qPCR was conducted to measure the mRNA expression levels of SMAD1, CD34, PITX2, APLN, and GAPDH (used as the internal control), and primer information listed in the table 1. cDNA was synthesized from isolated RNA using the Prime Script RT Reagent Kit according to the manufacturer’s instructions. qPCR was performed using the SYBR Green PCR Master Mix (Applied Biosystems) in the ABI 7500 RT-PCR System (Applied Biosystems). Thermal cycling conditions were as follows: pre-denaturation for the 30s at 95℃, followed by 40 cycles and denaturation for 5s at 95℃, annealing for 30s at 60℃.
Statistical analysis
Results are presented as the mean ± standard deviation (SD). To confirm a significant difference between specific treatment groups, group comparisons were performed using the Student’s t-test. In all cases, P-value < 0.05 was considered a statistically significant difference.