PMC

Potential miRNA-mRNA interactions from miRmap, TargetScan and miRDB databases. miRmap (selection criteria: miRmap score >99.0), TargetScan and miRDB (selection criteria: miRDB score >99.0) were used to identify genes with potential miRNA-mRNA interactions of the miRNAs with >2-fold changes. The intersection of three databases revealed two potential miRNA-mRNA interactions: hsa-miR-129-5p/CDC42EP3 and hsa-miR-330-3p/HELZ. miRNA, microRNA.

Changes in miRNAs and mRNAs in hypoxic AC16 cardiomyocytes. miRNA/miR, microRNA; TNS1, tensin 1; BNIP3L, B-cell lymphoma 2-interacting protein 3 like; STC1, stanniocalcin 1; TFRC, transferrin receptor; METTL7A, methyltransferase like 7A.

Heatmap of the differential expression of genes specifically associated with apoptosis, cell proliferation inhibition, and cell cycle arrest in hypoxic, vs. normoxic AC16 cardiomyocytes. The upregulated and down-regulated genes are shown in red and green, respectively.

Top 20 biological processes of the DEGs in hypoxic AC16 CMs. The top 20 biological processes of DEGs in hypoxic AC16 CMs are presented in a pie chart. numbers represent numbers of genes in the biological process. The top four biological processes of these genes were cellular response to hypoxia (12 genes), brown fat cell differentiation (seven genes), positive regulation of apoptotic process (13 genes), and response to hypoxia (nine genes). CMs, cardio-myocytes; DEGs, differentially expressed genes.

Enrichment analysis of KEGG pathways from differentially expressed genes in hypoxic AC16 CMs. In the graph, the top 20 KEGG pathways (details listed in ) were plotted with −log₁₀ (P-value), vs. fold enrichment. Higher fold enrichment values and −log₁₀ (P-value) indicate that the pathway is expressed at a higher level and is more reliable. Legionellosis, regulation of lipolysis in adipocytes, glycolysis/gluconeogenesis pathways were significantly enriched in hypoxic AC16 CMs. KEGG, Kyoto Encyclopedia of Genes and Genomes; CMs, cardiomyocytes.

Investigating hypoxia-related changes in AC16 CMs. (A) Flowchart showing the methods for investigating various genetic expression changes in hypoxic AC16 CMs using next-generation sequencing and bioinformatics approaches. (B) AC16 CMs (5×10⁵) were incubated in either a normoxic (control) or hypoxic condition for 24 h, and analyzed using a flow cytometer following staining with fluorescein isothiocyanate-conjugated Annexin V and propidium iodide. Increased apoptosis was noted in the hypoxic AC16 CMs. miRNA, microRNA; CMs, cardiomyocytes; DAVID, Database for Annotation, Visualization and Integrated Discovery; GEO, Gene expression omnibus; KEGG, Kyoto Encyclopedia of Genes and Genomes; IPA, Ingenuity^® Pathway Analysis.

Potential miRNA-mRNA interactions in hypoxic AC16 CMs. (A) Volcano plot of −log₁₀ (P-value), vs. log₂ (fold change) shows differentially upregu-lated and downregulated genes in hypoxic, vs. normoxic AC16 CMs; genes with −log₁₀ (P-value) >1.3 and ≥2-fold changes are plotted in red (upregulated) and green (downregulated). (B) RNA-sequencing heatmap analysis showing differentially expressed genes (total 459 genes) with fold changes >2.0. (C) Genes Venn diagram shows that 280 genes were upregulated and 179 genes were downregulated in hypoxic AC16 CMs, vs. normoxic AC16 CMs; the miRNA Venn diagram showed that there were 85 downregulated miRNAs and 99 upregulated miRNAs in hypoxic AC16 CMs, vs. normoxic AC16 CMs; the Targets Venn diagram revealed the putative gene targets of the miRNAs with >2-fold changes predicted with miRmap database with threshold of miRmap scores >99.0. The intersection Venn diagram between Genes and Targets revealed 24 genes with potential miRNA-mRNA interactions. (D) Small RNA-sequencing heatmap analysis showing differentially expressed miRNAs (total 184 miRNAs) with fold changes >2. miRNA, microRNA; CMs, cardiomyocytes.

GEO database analysis of genes with potential miRNA-mRNA interactions. The 24 genes with potential miRNA-mRNA interactions, including (A) upregulated and (B) downregulated genes in hypoxic cardiomyocytes are listed in . They were further validated using related array data from the GEO database. The gene expression levels in cardiac microvascular endothelial cells were extracted from the GEO database (GEO accession: GDS3483), and the expression levels of the genes were compared between cells treated with hypoxia for different lengths of time (3, 24 and 48 h) and normoxic cells. ^*P<0.05; **P<0.01; ^***P<0.001. miRNA, microRNA; GEO, Gene Expression Omnibus.

Networks analysis of the 24 differentially expressed genes with potential miRNA-mRNA interactions in hypoxic AC16 CMs. The network analyses of the 24 differentially expressed genes with potential miRNA-mRNA interactions in hypoxic AC16 CMs were investigated using Ingenuity^® Pathway Analysis software. (A) In network 1, 14 targeted genes (DIO2, SLC2A5, APOL6, HDAC11, STC1, MME, CHAC1, APLN, TFRC, CD4, HIF3A, BCL2, DMTN, and SLC6A8) were associated with free radical scavenging, small molecule biochemistry, and carbohydrate metabolism. (B) In network 2, seven targeted genes (SLC7A11, ACVRL1, BNIP3L, MME, METTL7A, TNS1, and GPR68) were associated with cancer, organismal injury and abnormalities, and cell death and survival. In network 3, only CAMK1D was associated with carbohydrate metabolism, small molecule biochemistry, and digestive system development and function. CMs, cardiomyocytes; miRNA, microRNA. Diamonds, horizontal ovals, vertical ovals, squares, rectangles, trapezoids, and inverted triangles represent enzymes, transcription regulators, transmembrane receptors, cytokines, ligand-dependent nuclear receptors, transporters, and kinases, respectively. Solid and dashed lines represent direct and indirect action, respectively. Red and green symbols represent upregulated and downregulated molecules, respectively.