Integrated analyses of lncRNAs microarray profiles and mRNA–lncRNA coexpression in smooth muscle cells under hypoxic and normoxic conditions

Figure 1. LncRNA and mRNA expression profiles in hypoxic and normoxic MOVAS cells (A). Detection of HIF1-α expression using Western blotting assay. Scatter plots were used to distinguish the differentially expressed (B) lncRNA and (C) mRNA between hypoxic and normoxic groups, respectively. (D) and (E) Volcano plots of lncRNAs and mRNA expression levels between the hypoxic and normoxic groups, respectively. The dendrogram demonstrates the relationships of the lncRNA and mRNA expression profiles between the hypoxia and normoxia MOVAS cell samples, The red dots represent up-regulated lncRNAs and mRNAs, and the green dots represent the down-regulated lncRNAs and mRNAs, respectively (P<0.05; fold change > 2.0).

Figure 2. Heat map of differentially expressed lncRNAs and mRNAs of MOVAS cells and validation of the microarray results by RT-qPCR Hierarchical clustering analyses of (A) lncRNAs and (B) mRNAs. C1–C4 represent normoxic samples; H1–H4 represent hypoxic samples. The color scale on the right illustrates the relative expression level of lncRNAs and mRNAs across all samples. The red shades indicate high relative expression, and the green shades indicate low relative expression. (C) and (D) The levels of up-regulated lncRNAs and down-regulated lncRNAs were detected using RT-qPCR. All data are representative of three independent experiments.

Figure 3. Gene ontology and Kyoto Encyclopedia of Genes and Genomes pathways analyses of differentially expressed mRNAs (A) The top 30 dysregulated genes from GO enrichment analyses that are related to biological processes (BP), cellular components, (CC) and molecular functions (MF). (B) The top 30 dysregulated genes from the KEGG pathway enrichment analyses correlated with cellular processes, metabolism, and environmental information processes, (C) and (D) Venn diagram of the common differentially expressed genes which are correlated with atherosclerosis according to GO and KEGG pathway enrichment analyses with the overlap of 16 genes (ten up-regulated genes; six down-regulated genes).

Figure 4. Construction of the lncRNA–mRNA coexpression network Coexpression network analyses were performed to characterize the 87 differentially expressed lncRNAs and ten differentially expressed mRNAs (six up-regulated and four down-regulated) based on the microarray results using Cytoscape 3.5.1 software. Red circular nodes indicate up-regulated mRNAs, and green circular nodes indicated down-regualted mRNAs. Yellow rhombus nodes denote up-regulated lncRNAs, whereas blue rhombus nodes designate down-regulated lncRNAs.

Figure 5. Effect of NONMMUT002434 knockdown on MOVAS cells invasion, proliferation (A) The expression of NONMMUT002434 after siRNA transfection. (B) CCK-8 proliferation, n = 5 per group, scramble group versus si-NONMMUT002434 group. (C) The migratory and invasive ability of MOVAS cells after knockdown of NONMMUT002434, assessing by transwell assay. Scale bar, 100 μm in each graph. The right graph represents quantitation of invaded cells. (D). Representative images showing EdU fluorescence staining to detect the newly synthesized DNA. Blue fluorescence shows cell nuclei and green fluorescence represents cells with DNA synthesis. Scale bar, 100 μm in each graph. The right graph shows the percentage of EdU-positive cells, calculating using ImageJ software, scar bar = 100 um. Data are expressed as mean ± S.D.; * P<0.05; **P<0.01; ***P<0.001. Similar results were obtained from experiments with three replicates.