Microarray analysis of the time-dependent expression profiles of long non-coding RNAs in the progression of vein graft stenotic disease

Abstract

Long non-coding RNAs (lncRNAs) have been reported to be involved in various biological processes, including cell proliferation and apoptosis. However, the expression profiles of lncRNAs in patients with vein graft restenosis remain unknown. In the present study, the time-dependent expression profiles of genes in vein bypass grafting models were examined by microarray analysis. A total of 2,572 lncRNAs and 1,652 mRNAs were identified to be persistently significantly differentially expressed. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis was performed to investigate the functions of these lncRNAs. A total of 360 lncRNAs and 135 protein-coding genes were predicted to be involved in the vascular remodeling process. Co-expression network analysis revealed the association between 194 lncRNAs and seven associated protein-coding genes, including transforming growth factor-β1, Fes, Yes1 associated transcriptional regulator, sphingosine-1-phosphate receptor 1, Src, insulin receptor and melanoma cell adhesion molecule. Moreover, reverse transcription-quantitative PCR results supported those of the microarray data, and overexpression of AF062402, which regulates the transcription of Src, stimulated the proliferation of primary vascular smooth muscle cells. The findings of the present study may facilitate the development of novel therapeutic targets for vein graft restenosis and may help to improve the prognosis of patients following coronary artery bypass grafting.

Keywords: co-expression network; coronary artery bypass grafting; long non-coding RNAs; mRNAs; microarray analysis; vein graft stenotic disease.

Figure 1

Establishment of an external jugular vein carotid bypass rat model. (A) Exposure of the external jugular vein; (B) Exposure of the carotid artery; (C) The external jugular vein was anastomosed to the carotid artery; (D) The blood flow in the graft was patent.

Figure 2

Overview of significantly differentially expressed lncRNAs. (A) Heatmaps showing lncRNAs that were differentially expressed between groups. Scatter and volcano plots illustrate variations in lncRNA expression between different time points (0 vs. days 7, 14 and 28). (B) Chromosomal and (C) length distribution of differentially expressed lncRNAs. (D) Time series analysis revealed the temporal expression patterns of differentially expressed lncRNAs. Each box represents the time-dependent expression profile of a lncRNA. Upper value refers to the number of profiles; lower value indicates P-value of the profiles in each box. (E) Pie chart illustrating the classification of differentially expressed lncRNAs based on genomic location relative to neighboring or overlapping genes. (F) Time-dependent change in the number of differential expressed lncRNAs at three post-operative timepoints. lncRNAs, long non-coding RNAs.

Figure 3

‘GO’ and ‘KEGG’ pathway enrichment analysis of lncRNA-associated protein-coding genes. (A) Significant BP, MF and CC terms in ‘GO’ enrichment analysis. (B) ‘KEGG’ enrichment analysis of significantly enriched pathways. P<0.05. GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; lncRNAs, long non-coding RNAs; BP, biological process; MF, molecular function; CC, cell component.

Figure 4

Biological processes associated with vascular remodeling and the interaction network between selected lncRNAs and associated protein-coding genes. (A) Time-dependent change in the number of differentially expressed lncRNAs predicted to be involved in ‘cell adhesion’, ‘migration’, ‘cell proliferation’, ‘MAP kinase activation’, ‘platelet activation’, ‘chemotaxis’, ‘WNT protein secretion’, ‘stimulation responses’ and ‘growth factor activity’. (B) Co-expression network between selected lncRNAs and associated protein-coding genes. Orange and blue circles indicate upregulated and downregulated lncRNAs, respectively. Green circles indicate protein-coding genes overlapped with mRNAs. lncRNA, long non-coding RNA.

Figure 5

Reverse transcription-quantitative PCR validation and Cell Counting Kit-8 assay. (A) Comparison of the expression levels of AF062402, BC091437 and BC166461 and associated protein-coding genes. (B) Expression of AF062402 in the Lentivirus-AF062402 group was significantly higher than that in the blank and Lentivirus-NC groups. Overexpression of AF062402 stimulated the proliferation of vascular smooth muscle cells. ^****P<0.0001. NC, negative control; Mcam, melanoma cell adhesion molecule.