Intracerebral hemorrhage alters circular RNA expression profiles in the rat brain

Abstract

Circular RNAs (circRNAs), formed from pre-messenger RNAs by back-splicing, are a novel class of evolutionarily-conserved endogenous non-coding RNAs. While circRNAs are involved in various diseases, the role of circRNAs in intracerebral hemorrhage (ICH) remains unknown. In the present study, we performed high-throughput sequencing to profile the expression of circRNAs in the rat brain at 24 and 48 hours after ICH onset, and utilized bioinformatics methods to make predictions about the function of dysregulated circRNAs. Compared with the sham group, 346 and 389 circRNAs changed significantly (|log2 (fold change)| > 1 and P < 0.05) at 24 and 48 hours after ICH, respectively. Bioinformatics analyses indicated that parent genes of dysregulated circRNAs were involved in biological processes, cellular component, and molecular function following ICH, and that they were enriched in the dopaminergic synapses, glutamatergic synapses, endocytosis, regulation of actin cytoskeleton, the mitogen-activated protein kinase signaling pathway, and the retrograde endocannabinoid signaling pathway. Enrichment analyses of target mRNAs showed that these mRNAs were enriched in synaptic plasticity, ion channel activity, and pathways including the phospholipase D signaling and the cGMP-PKG signaling. Our study indicates that the expression profile of circRNAs changes significantly after ICH in rat brains, and suggests that circRNAs may be crucial for the pathophysiological process following ICH.

Keywords: Intracerebral hemorrhage; circular RNAs; high-throughput sequencing; rat.

Figure 1

The expression profile of circRNAs in the perihematomal brain tissue after ICH. The hierarchical clustering analyses of circRNAs differentially expressed at 24 hours (A) and 48 hours (C) after ICH compared with the sham group, “Blue” represents low intensity, and “Red” represents strong intensity. Volcano plots show all the detected circRNAs (sham vs. 24 hours (B) and sham vs. 48 hours after ICH (D)), the red points represent the differentially upregulated circRNAs, while the green points represent the differentially downregulated circRNAs (|log2 (fold change)| > 1, P-value < 0.05 (-log10 scaled)).

Figure 2

Features of differentially expressed circRNAs. Distribution of dysregulated circRNAs in rat chromosomes (A). Most circRNAs after ICH are from the exonic region, and few are from the splicing region (B).

Figure 3

GO enrichment analyses for the parent genes of altered circRNAs. Major enriched GO terms for biological process, cellular component, and molecular function at 24 hours and 48 hours after ICH in rats (A, B).

Figure 4

KEGG pathway analyses for the parent genes of altered circRNAs at 24 hours (A, B) and 48 hours (C, D) after ICH in rats. The top 10 (A, C) and top 30 (B, D) enriched pathways.

Figure 5

qRT-PCR validation results of four randomly selected circRNAs (rno_circ:chr9:31604268-31660864, rno_circ:chr18:17224244-17264715, rno_circ:chr13:35769419-35784163, and rno_circ:chr1:281093685-281095506). Data are shown as mean ± SD. *P < 0.05, **P < 0.01, compared with the sham group.

Figure 6

The circRNA-miRNA network. The network consists of 40 differentially expressed circRNAs and their top five miRNAs. Red squares, green squares, and orange spheres indicate downregulated circRNAs, upregulated circRNAs, and miRNAs, respectively.

Figure 7

The circRNA-miRNA-mRNA interaction network. The network includes two validated circRNAs, 10 miRNAs, and 72 mRNAs. Green arrows, red rhombuses, and blue spheres indicate circRNAs, miRNAs, and mRNAs, respectively.

Figure 8

GO and KEGG pathway analyses for mRNAs targeted by the circRNA (confirmed by qRT-PCR)-miRNA regulatory network. Major enriched GO terms for biological process, cellular component, and molecular function (A) and the 30 major enriched pathways (B) at 48 hours after ICH in rats.