Genome-wide differential expression profiling of lncRNAs and mRNAs associated with early diabetic cardiomyopathy

Abstract

Diabetic cardiomyopathy is one of the main causes of heart failure and death in patients with diabetes. There are no effective approaches to preventing its development in the clinic. Long noncoding RNAs (lncRNA) are increasingly recognized as important molecular players in cardiovascular disease. Herein we investigated the profiling of cardiac lncRNA and mRNA expression in type 2 diabetic db/db mice with and without early diabetic cardiomyopathy. We found that db/db mice developed cardiac hypertrophy with normal cardiac function at 6 weeks of age but with a decreased diastolic function at 20 weeks of age. LncRNA and mRNA transcripts were remarkably different in 20-week-old db/db mouse hearts compared with both nondiabetic and diabetic controls. Overall 1479 lncRNA transcripts and 1109 mRNA transcripts were aberrantly expressed in 6- and 20-week-old db/db hearts compared with nondiabetic controls. The lncRNA-mRNA co-expression network analysis revealed that 5 deregulated lncRNAs having maximum connections with differentially expressed mRNAs were BC038927, G730013B05Rik, 2700054A10Rik, AK089884, and Daw1. Bioinformatics analysis revealed that these 5 lncRNAs are closely associated with membrane depolarization, action potential conduction, contraction of cardiac myocytes, and actin filament-based movement of cardiac cells. This study profiles differently expressed lncRNAs in type 2 mice with and without early diabetic cardiomyopathy and identifies BC038927, G730013B05Rik, 2700054A10Rik, AK089884, and Daw1 as the core lncRNA with high significance in diabetic cardiomyopathy.

Figure 1

Body weight, blood glucose, and insulin levels of C57BL/6J and db/db mice at 6 and 20 weeks of age. (A) body weight; (B) blood glucose; and (C) plasma insulin levels. Data are presented as means ± SEM. Kruskal-Wallis test followed by Dunn’s test was used to analyze multiple group comparisons. *P < 0.05 versus C57BL/6J group at 6 weeks old; ^#P < 0.05 versus C57BL/6J group at 20 weeks old (n = 12–13 mice/group).

Figure 2

Volcano plots showing differently expressed lncRNAs in 6 and 20-week-old db/db mice, respectively compared with controls. The db/db mice developed early diabetic cardiomyopathy at 20 weeks old. The red and green points represented up- and down-regulated lncRNAs, respectively. The horizontal green line depicts *P ≤ 0.05, whereas the vertical green line shows a twofold change of up and down.

Figure 3

Overlapping deregulated lncRNAs in db/db mice between 6 and 20 weeks of age. Colors of red and green represented up- and down-regulated lncRNAs with changes larger than twofold, respectively.

Figure 4

The profile of lncRNA expressions in db/db mouse hearts with and without early diabetic cardiomyopathy compared with controls. (A,B) hierarchical clustering analysis presenting differentially expressed lncRNAs between 6 and 20-week-old db/db and control mice, respectively. The db/db mice developed early diabetic cardiomyopathy at 20 weeks old. Colors of red and green represented up- and down-regulated lncRNAs with changes larger than twofold, respectively. (C,D) chromosomal distribution of deregulated lncRNAs in 6 and 20-week-old db/db mice, respectively. Colors of green and orange represented up- and down-regulated lncRNAs.

Figure 5

Classification of differently expressed lncRNAs in db/db mouse hearts with and without early diabetic cardiomyopathy. (A) percentage of lncRNA categories in 6-week-old db/db mouse hearts; (B) percentage of lncRNA categories in 20-week-old db/db mouse hearts.

Figure 6

Volcano plots representing differently expressed mRNAs in 6 and 20-week-old db/db mice, respectively compared with age-matched controls. Db/db mice had early diabetic cardiomyopathy at 20 weeks old. The red and green points represented up- and down-regulated mRNAs, respectively. The horizontal green line depicts *P ≤ 0.05, whereas the vertical green line shows a twofold change of up and down.

Figure 7

Profile of cardiac mRNA expression in db/db mouse hearts with and without early diabetic cardiomyopathy compared with controls. (A,B) hierarchical clustering analysis showing differently expressed mRNAs between 6 and 20-week-old db/db mice and controls, respectively. Colors of red and green represented up- and down-regulated lncRNAs with changes larger than twofold, respectively (C,D) chromosomal distribution of deregulated mRNAs in 6 and 20-week-old db/db mice, respectively. Colors of green and orange represented up- and down-regulated lncRNAs.

Figure 8

Significantly enriched gene ontology (GO) terms of top 30 up-regulated mRNAs in db/db mice at 6 and 20 weeks of age, respectively compared with controls.

Figure 9

Significantly enriched GO terms of top 30 down-regulated mRNAs in db/db mice at 6 and 20 weeks of age, respectively compared with controls.

Figure 10

KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis of differentially expressed mRNAs in db/db mouse hearts with and without early diabetic cardiomyopathy. (A) the top 4 KEGG pathways of significantly up-regulated mRNAs between 6-week-old db/db and control mice; (B) the top 5 KEGG pathways of significantly up-regulated mRNAs between 20-week-old db/db and control mice; (C) the top 10 KEGG pathways of significantly down-regulated mRNAs between 6-week-old db/db and control mice; (D) the top 10 KEGG pathways of significantly down-regulated mRNAs between 20-week-old db/db and control mice. EGFR, epidermal growth factor receptors; MAPK: mitogen-activated protein kinase; NF-kB, nuclear factor kappa-light-chain-enhancer of activated B cells.

Figure 11

Correlation between significantly differentially expressed lncRNAs and mRNA transcripts. (A) the network of lncRNA-mRNA co-expression in 6-week-old db/db mice; (B) the network of lncRNA-mRNA co-expression in 20-week-old db/db mice. The network represents the co-expression correlations between the significantly differentially expressed lncRNAs and mRNA transcripts. Five lncRNAs having maximum connections with mRNAs were taken to construct the co-expression network (Pearson correlation >0.995 or <−0.995 and P < 0.05). Circles, squares, and V shapes indicate lncRNA transcripts, transcription factors, and mRNA transcripts, respectively. Solid arrows and dashed lines indicate positive and negative correlation, respectively, whereas red and green colors represent up- and down-regulated transcripts. The width of the line is based on Person’s value (stronger correlation corresponds to more width) and color of the line depicts the significance. Nodes indicated lncRNAs or mRNAs.

Figure 12

Gene ontology (GO) analysis of the 5 deregulated lncRNAs that had maximum connections with mRNAs. (A) The network represents the GO pathway terms specific for mRNA genes having co-expression relationship with the significantly differentially expressed top 5 lncRNAs having maximum connections in the Co-expression network (Pearson correlation >0.995 or <−0.995 and P < 0.05) at 6 weeks of age; (B) the network represents the GO pathway terms specific for mRNA genes having co-expression relationship with the significantly differentially expressed top 5 lncRNAs having maximum connections in the Co-expression network (Pearson correlation >0.995 or <−0.995 and P < 0.05) at 20 weeks of age. Functionally grouped networks with GO terms as node are grouped on the basis of kappa score level (>0.03), the most significant groups are shown. Each node represents a GO biological process, and the colors represent the GO group. The node size represents the term enrichment significance. Different GO clusters are shown in different color. The edges reflect the relationships between the terms based on the similarity of their associated genes.