Comprehensive analysis of m6A modification lncRNAs in high glucose and TNF-α induced human umbilical vein endothelial cells

Abstract

N6-methyladenosine (m6A) RNA methylation, as a reversible epigenetic modification of mammalian mRNA, holds a critical role in multiple biological processes. m6A modification in Long non-coding RNAs (lncRNAs) has increasingly attracted more attention in recent years, especially in diabetics, with or without metabolic syndrome. We investigated via m6A-sequencing and RNA-sequencing the differentially expressed m6A modification lncRNAs by high glucose and TNF-α induced endothelial cell dysfunction in human umbilical vein endothelial cells. Additionally, gene ontology and kyoto encyclopedia of genes and genomes analyses were performed to analyze the biological functions and pathways for the target of mRNAs. Lastly, a competing endogenous RNA network was established to further reveal a regulatory relationship between lncRNAs, miRNAs and mRNAs. A total of 754 differentially m6A-methylated lncRNAs were identified, including 168 up-regulated lncRNAs and 266 down-regulated lncRNAs. Then, 119 significantly different lncRNAs were screened out, of which 60 hypermethylated lncRNAs and 59 hypomethylated lncRNAs. Moreover, 122 differentially expressed lncRNAs were filtered, containing 14 up-regulated mRNAs and 18 down-regulated lncRNAs. Gene ontology and kyoto encyclopedia of genes and genomes analyses analyses revealed these targets were mainly associated with metabolic process, HIF-1 signaling pathway, and other biological processes. The competing endogenous RNA network revealed the regulatory relationship between lncRNAs, miRNAs and mRNAs, providing potential targets for the treatment and prevention of diabetic endothelial cell dysfunction. This comprehensive analysis for lncRNAs m6A modification in high glucose and TNF-α-induced human umbilical vein endothelial cells not only demonstrated the understanding of characteristics of endothelial cell dysfunction, but also provided the new targets for the clinical treatment of diabetes. Private information from individuals will not be published. This systematic review also does not involve endangering participant rights. Ethical approval will not be required. The results may be published in a peer-reviewed journal or disseminated at relevant conferences.

Figure 1.

Overview of m6A methylation within lncRNAs in HUVECs. (A) Venn diagram showing the numbers of m6A peaks in the two groups. (B) Venn diagram showing the numbers of lncRNAs in the two groups. (C) Volcano plots displaying the differentially expressed m6A peaks between the two groups. (D) Volcano plots displaying the differentially expressed m6A methylated lncRNAs between the two groups. HUVECs = human umbilical vein endothelial cells, lncRNAs = long non-coding RNAs, m6A = N6-methyladenosine.

Figure 2.

Distribution of differentially m6A methylated lncRNAs. (A) Distribution of m6A modification lncRNAs on chromosomes. (B) Distribution of differentially methylated lncRNAs on chromosomes. (C) and (D) Distribution sites of differentially methylated lncRNAs on chromosomes. CDS = coding sequences, lncRNAs = long non-coding RNAs, m6A = N6-methyladenosine.

Figure 3.

Abundance of m6A peaks and the conserved m6A modified motif in lncRNAs (A) Proportions of lncRNAs harboring different numbers of m6A peaks in the two groups. (B) The sequence motifs of the m6A-containing peak regions in the two groups. lncRNAs = long non-coding RNAs, m6A = N6-methyladenosine.

Figure 4.

The features of differentially expressed lncRNAs. (A) Hierarchical cluster analysis of differentially expressed lncRNAs in two groups (CK: control; GT: model). (B) General numbers of differentially expressed lncRNAs. lncRNAs = long non-coding RNAs.

Figure 5.

The association between lncRNAs m6A methylation and expression. (A) Venn diagram showing the relationship between m6A modification and expression. (B) Four-quadrant diagram of the relationship between lncRNAs methylation and expression. lncRNAs = long non-coding RNAs, m6A = N6-methyladenosine.

Figure 6.

Functional analysis of mRNAs located near differentially methylated lncRNAs. (A) GO enrichment analysis of genes near m6A methylated lncRNAs. (B) KEGG enrichment analysis of genes near m6A methylated lncRNAs. GO = gene ontology, KEGG = kyoto encyclopedia of genes and genomes, lncRNAs = long non-coding RNAs, m6A = N6-methyladenosine.

Figure 7.

The networks of lncRNA-miRNA-mRNA regulation. Green represent the lncRNAs, genes in red are coding miRNA and mRNAs. lncRNAs = long non-coding RNAs.