Transcriptome-wide analysis reveals the molecular mechanisms of cannabinoid type II receptor agonists in cardiac injury induced by chronic psychological stress

Abstract

Background: Growing evidence has supported that chronic psychological stress would cause heart damage, However the mechanisms involved are not clear and effective interventions are insufficient. Cannabinoid type 2 receptor (CB2R) can be a potential treatment for cardiac injury. This study is aimed to investigate the protective mechanism of CB2R agonist against chronic psychological stress-induced cardiac injury. Methods: A mouse chronic psychological stress model was constructed based on a chronic unpredictable stress pattern. Mice were performed a three-week psychological stress procedure, and cardiac tissues of them were collected for whole-transcriptome sequencing. Overlap analysis was performed on differentially expressed mRNAs (DE-mRNAs) and ER stress-related genes (ERSRGs), and bioinformatic methods were used to predict the ceRNA networks and conduct pathway analysis. The expressions of the DE-ERSRGs were validated by RT-qPCR. Results: In the comparison of DE mRNA in Case group, Control group and Treatment group, three groups of ceRNA networks and ceRNA (circ) networks were constructed. The DE-mRNAs were mainly enriched in chromatid-relevant terms and Hematopoietic cell lineage pathway. Additionally, 13 DE-ERSRGs were obtained by the overlap analysis, which were utilized to establish a ceRNA network with 15 nodes and 14 edges and a ceRNA (circ) network with 23 nodes and 28 edges. Furthermore, four DE-ERSRGs (Cdkn1a, Atf3, Fkbp5, Gabarapl1) in the networks were key, which were mainly enriched in response to extracellular stimulus, response to nutrient levels, cellular response to external stimulus, and FoxO signaling pathway. Finally, the RT-qPCR results showed almost consistent expression patterns of 13 DE-ERSRGs between the transcriptome and tissue samples. Conclusion: The findings of this study provide novel insights into the molecular mechanisms of chronic psychological stress-induced cardiac diseases and reveal novel targets for the cardioprotective effects of CB2R agonists.

Keywords: CB2R; cardiac injuries; ceRNA network; chronic psychological stress; endoplasmic reticulum stress; functional enrichment analysis.

FIGURE 1

Differential expression analysis. Volcano plot for differentially expressed mRNAs (DE-mRNAs), lncRNAs (DE-lncRNAs), miRNAs (DE-miRNAs) and circRNAs (DE-circRNAs) in the three comparison groups. (A) Control vs. Case; (B) Case vs. Treatment; (C) Control vs. Treatment. Blue: downregulation, red: upregulation.

FIGURE 2

Functional Enrichment Analysis. (A) Bar plots of KEGG pathway and GO enrichment analyses in the control and case groups. (B) Bar plots of KEGG pathway and GO enrichment analyses in the case and treatment groups. (C) Bar plots of KEGG pathway and GO enrichment analyses in the control and case groups. Each GO map presents the top 10 enriched terms in BP, CC, and MF. Each KEGG map shows the top 15 enriched KEGG pathways.

FIGURE 3

ceRNA network construction. (A) mRNA-miRNA-lncRNA network in control and case groups. (B) mRNA-miRNA-circRNA network in control and case groups. In the mRNA-miRNA-lncRNA (circRNA) network, the hexagon represents miRNA, the rhombus represents lncRNA (circRNA), the oval represents mRNA, and the color represents logFC value. The redder the color, the more upregulated the expression of the gene, and the bluer the color, the more downregulated the expression of the gene.

FIGURE 4

GO enrichment and KEGG pathway analyses of mRNAs enrolled in ceRNA networks. (A) KEGG pathway and GO enrichment analysis of mRNAs in the ceRNA (lncRNA) network in control and case groups. (B) KEGG pathway and GO enrichment analysis of mRNAs in the ceRNA (lncRNA) network in the case and treatment groups. (C) KEGG pathway and GO enrichment analysis of mRNAs in the ceRNA (lncRNA) network in the control and treatment groups. Each GO map shows the top 10 enriched phrases in BP, CC, and MF. On (A, B), the top 9 enriched KEGG pathways are shown, while (C) shows the top 15 enriched KEGG pathways.

FIGURE 5

Identification of DE-ERSRGs and functional enrichement analysis. (A) Venn diagram of 13 DE-ERSRGs by overlapping DEGs in control vs. case group, DEGs in case group vs. treatment, with 826 ER stress-related genes. (B) Heat map of expression of 13 DE-ERSRGs in control and case groups. (C) Heat map of expression of 13 DE-ERSRGs in the case and treatment groups. (D) KEGG pathway analysis of 13 DE-ERSRGs. (E) GO enrichment analysis of 13 DE-ERSRGs.

FIGURE 6

Construction of ceRNA networks for 13 DE-ERSRGs. (A) mRNA-miRNA-lncRNA regulatory network. (B) Sankey diagram of the mRNA-miRNA-lncRNA regulatory. (C) mRNA-miRNA-circRNA regulatory network. (D) Sankey diagram of mRNA-miRNA-circRNA regulatory. In the mRNA-miRNA-lncRNA (circRNA) network, the hexagon represents miRNA, the rhombus represents lncRNA (circRNA), the oval represents mRNA, and the color represents logFC value. The closer the color is to red, the more upregulated the expression is, and the closer the color is to blue, the downregulated the expression is.

FIGURE 7

Validation of the expression of 13 DE-ERSRGs by RT-qPCR. Validation of the expression levels of 13 DE-ERSRGs in mice tissue samples by quantitative RT-PCR. Error bars show mean ± SD. Data are representative of three independent experiments. *p < .05, **p < .01, ***p < .001.