Differential genes screening
Two sets of gene expression data GSE57338 and GSE141910 were downloaded from the GEO database. A total of 48 different genes were obtained from the GSE57338 dataset, including 25 up-regulated genes and 23 down-regulated genes, and 902 differential genes were collected from the GSE141910 dataset composed of 622 up-regulated genes and 280 down-regulated genes. (Figure 1 A-F). The two datasets had 679 samples, so the selected genes were fully representative.
Enrichment of differentially expressed genes by GO, KEGG pathway, and REACTOME analysis
A total of 43 stable differentially expressed genes were obtained from the two datasets which were in accordance with up and down regulated patterns in the GSE57338 and GSE 141910 datasets (Figure 2A). After GO enrichment analysis of 43 genes, it was found that the function of BP mainly focused on the adhesion of extracellular or epidermal cells, myocardial hypertrophy and regulation, the bone morphogenetic protein (BMP) signal pathway, inflammatory response, and blood pressure regulation. The cell component of CC was mainly enriched in extracellular collagen, blood particles, and other molecular components. MF was mainly enriched in extracellular matrix structural components, oxygen binding, and endopeptidase activity. (Figure 2C-E) Through KEGG enrichment analysis, the genes were mainly enriched in gap junctions, apoptosis, phagosomes, the cGMP-PKG signaling pathway, and tight junctions (Figure. 2B), which was consistent with the GO enrichment analysis. For the reactome analysis, DEGs were mainly enriched in interferon signal interleukins, asparagine-N-linked glycosylation, neutrophil degranulation, and regulation of Toll-like receptor (TLR) by endogenous ligand (Figure 2F-G).
PPI network based on DEGs and identification of tissue-specific genes by BioGPS
The PPI network of DEGs was constructed according to the STRING database, and individual differential gene nodes were deleted. The PPI network contained 25 differentially expressed genes (Figure 3A). The tissue-specific analysis of the DEGs showed that 37 of the 43 genes were tissue-specific expression genes (Table 3 and Figure 3B), among which 6 genes were specifically expressed in cardiovascular-related tissues or cells, natriuretic peptide A (NPPA), hemoglobin subunit beta (HBB), eukaryotic translation initiation factor 1A Y-linked (EIF1AY), serpin family A member 3 (Serpina3), hyaluronan and proteoglycan link protein 1(HAPLN1) and CD163 (Figure 3C).
Table 3. specific expressed genes identified by BioGPS
Tissue-specific differentially expressed genes
Non-tissue specific differentially expressed genes
MGST1, SLCO4A1, MXRA5, SERPINE1, SFRP4, OGN, ASPN, ECM2, SMOC2, IL1RL1, MYOT, LYVE1, IFI44L, AOX1, MNS1, PHLDA1, LUM, FCN3, CYP4B1, HAPLN1, VSIG4, FRZB, PI16, ANKRD2, RNASE2, METTL7B, NPPA, CD163, COL14A1, PLA2G2A, SERPINA3, MYH6, HBB，EIF1AY, MME，SFRP1，USP9Y
PDE5A, ADAMTS4, ADAMTS9, FNDC1, FREM1, HMGCS2
Identification of hub genes and validation of important gene expression levels with independent datasets
Four hub genes (NPPA, HBB, CD163, and HAPLN1) were obtained by the intersection of 25 genes constituting the PPI network and 6 genes specifically expressed in the cardiovascular system (Figure 3C) The main functions of the NPPA gene were related to muscular hypertrophy, myocardial adaptation, and blood pressure regulation. The main functions of the HBB and HAPLN1 genes were oxygen binding and regulation of the extracellular matrix. The four selected key genes of HF were all significantly differentially expressed in the validation dataset. The expression of the NPPA, HBB, and HAPLN1 genes in patients with HF was significantly up-regulated. The up-regulation range of the NPPA gene was the largest, logFC=3.907, and CD163 was down-regulated in patients with HF, logFC = 1.108 (Figure 3D-G)
Diagnostic efficacy analysis based on the four hub genes of HF
Except for CD163, all the other three genes demonstrated good diagnostic results, and the diagnostic accuracy of the HBB gene was as high as 93.14%. The area under the curves (AUCs) of NPPA and HAPLN1 were estimated to 0.8734 and 0.8414, respectively, which demonstrates a similar diagnostic value between NPPA and HAPLN1. The AUC value of CD163 was 0.2286, which may discriminate HF patients from healthy people (Figure 4A-D).
ceRNA network analysis
The ceRNA network was constructed for the four key genes of HF (the mRNA and miRNA interaction relationship was obtained based on the public database, as well as the miRNA-lncRNA and miRNA-circRNA interactions). A ceRNA network was constructed for the four selected four key genes. It was found that each gene was regulated by multiple miRNAs, which suggests a new direction for the further treatment of HF by regulating their upstream genes (Figure 4E-H). A previous study revealed that the long non-coding RNAs (lncRNAs) SNHG16 and XIST were significantly upregulated in mouse hypertrophic hearts and phenylephrine (PE)-stimulated cardiomyocytes [6, 7], therefore, we speculate that SNHG16-miR-23-3p/ miR-103-5p / HAPLN1 and XIST -miR-23-3p/miR-103-5p/HAPLN1 might be potential RNA regulatory methods of HF.
Construction of HF model in mice
Eight weeks after AB surgery, the mice had developed obvious HF. After AB surgery, the mice manifested an obvious cardiac pathological remodeling phenotype, with increased HW/BW, LW/BW, and HW/TL ratios (Figure 5A-C). AB mice also exhibited deteriorated cardiac function compared with sham-operated mice by echocardiographic examination with LVEDd, LVESd, FS, and EF (Figure 5D-G,M). The average CSA after AB surgery by H&E staining also confirmed the adverse pathological changes of AB surgery on cardiac remodeling (Figure 5 H and N). Fibrosis was evaluated by picrosirius red staining and AB surgery had resulted in obvious cardiac interstitial fibrosis (Figure 5 I and O). Based on these results, the successful establishment of AB-induced HF in mice was confirmed.
Detection of mRNA level of DEGs in a mouse model of HF
As shown in Figure 5I-L, the mRNA level of ANP was significantly higher than that of the sham-operated group which proved the successful establishment of the HF model. The level of HAPLN1 was upregulated, and CD163 was down-regulated in the HF group under pressure-overload induced heart failure.