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. 2023 Apr 1;20(5):627-638.
doi: 10.7150/ijms.82503. eCollection 2023.

Transcriptomic Profiling of circRNAs in rat Hippocampus after Deep Hypothermic Circulatory Arrest

Tianlong Wang  1 , Weidong Yan  1 , Shengqiang Pei  2 , Mingru Zhang  3 , Qiaoni Zhang  1 , Yuan Teng  1 , Gang Liu  1 , Jian Wang  1 , Shujie Yan  1 , Bingyang Ji  1
Affiliations

Transcriptomic Profiling of circRNAs in rat Hippocampus after Deep Hypothermic Circulatory Arrest

Tianlong Wang et al. Int J Med Sci. .

Abstract

Neurologic abnormalities occurring after deep hypothermic circulatory arrest (DHCA) remain a significant concern. However, molecular mechanisms leading to DHCA-related cerebral injury are still ill-defined. Circular RNAs (circRNAs) are a class of covalently closed non-coding RNAs and can play important roles in different types of cerebral injury. This study aimed to investigate circRNAs expression profiles in rat hippocampus after DHCA and explore the potential functions of circRNAs in DHCA-related cerebral injury. Hence, the DHCA procedure in rats was established and a transcriptomic profiling of circRNAs in rat hippocampus was done. As a result, a total of 35192 circRNAs were identified. Among them, 339 circRNAs were dysregulated, including 194 down-regulated and 145 up-regulated between DHCA and sham group. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses were performed based on the host genes of all dysregulated circRNAs. Also, 4 circRNAs were validated by RT-qPCR (rno_circ_0028462, rno_circ_0037165, rno_circ_0045161 and rno_circ_0019047). Then a circRNA-microRNA (miRNA) interaction network involving 4 candidate circRNAs was constructed. Furthermore, functional enrichment analysis of the miRNA-targeting mRNAs of every candidate circRNA was conducted to gain insight into each of the 4 circRNAs. Our study provided a better understanding of circRNAs in the mechanisms of DHCA-related cerebral injury and some potential targets for neuroprotection.

Keywords: Cerebral injury; Circular RNAs; Deep hypothermic circulatory arrest; Hippocampus; Rat.

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Conflict of interest statement

Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

Figure 1
Figure 1
Workflow for the whole study. DHCA, Deep hypothermic circulatory arrest; RT-qPCR, reverse transcription-quantitative polymerase chain reaction.
Figure 2
Figure 2
Physiological parameters for the rats throughout the experimental period. Temperature (A), heart rate (HR, B) and mean arterial pressure (MAP, C) were measured over time in the sham and DHCA groups (values: mean ± SEM). bpm, beat per minute. P > 0.05 was not shown.
Figure 3
Figure 3
Expression profiling data of circRNAs in the rat hippocampal after DHCA. (A) Length distribution of the identified circRNAs in all samples. (B) Genomic location distribution of the identified circRNAs in all samples. (C) Chromosome distribution of the identified circRNA in all samples. (D) The boxplot of normalized expression values in all samples. (E) Volcano plot analysis for DECs (P < 0.05 and |log2FC| ≥ 1.5 are set as the cut-off criteria). Red, blue, and green points represent circRNAs that are down-regulated, up-regulated, and not significantly different. (F) Heatmap presents the expression of all DECs. Red and blue indicates increased and decreased expression, respectively. DHCA, deep hypothermic circulatory arrest; TPM, transcripts per million; DECs, differentially expressed circRNAs; log2FC, log2 fold-change.
Figure 4
Figure 4
GO and KEGG enrichment analyses on DECs in the hippocampal after DHCA. (A) Top 10 GO terms in the molecular function, cellular component and biological process categories of DECs in the rat hippocampal after DHCA. (B) Top 15 KEGG pathways of DECs in the rat hippocampal after DHCA. GO: Gene Ontology; KEGG: Kyoto Encyclopedia of Genes and Genomes; DECs, differentially expressed circRNAs; DHCA, deep hypothermic circulatory arrest.
Figure 5
Figure 5
Validation of differentially expressed circRNAs. (A-B) The high expression of rno_circRNA_0028462 and rno_circRNA_0037165 and the low expression of rno_circRNA_0045161 and rno_circRNA_0019047 were confirmed with RT-qPCR method (n = 4, each group). (C) The RT-qPCR data of 4 verified circRNAs displayed the tendency in accordance with our expression profiling results. *p < 0.05, **p < 0.001.
Figure 6
Figure 6
CircRNA-miRNA interaction network. The 4 DECs (rno_circRNA_0028462, rno_circRNA_0037165, rno_circRNA_0045161 and rno_circRNA_0019047) verified by RT‑qPCR were selected to construct a representative circRNA-miRNA network. Red represents up-regulated DECs. Green represents down-regulated DECs. Blue represents the targeted miRNAs. DECs, differentially expressed circRNAs.
Figure 7
Figure 7
GO and KEGG enrichment analyses of the miRNA-targeted mRNAs of 2 confirmed up-regulated DECs. (A, C) Top 10 GO terms in the molecular function, cellular component and biological process categories of the miRNA-targeted mRNAs of rno_circRNA_0028462 and rno_circRNA_0037165, respectively. (B, D) Top 15 KEGG pathways of the miRNA-targeted mRNAs of rno_circRNA_0028462 and rno_circRNA_0037165, respectively. GO: Gene Ontology; KEGG: Kyoto Encyclopedia of Genes and Genomes; DECs, differentially expressed circRNAs.
Figure 8
Figure 8
GO and KEGG enrichment analyses of the miRNA-targeted mRNAs of 2 confirmed down-regulated DECs. (A, C) Top 10 GO terms in the molecular function, cellular component and biological process categories of the miRNA-targeted mRNAs of rno_circRNA_0045161 and rno_circRNA_0019047, respectively. (B, D) Top 15 KEGG pathways of the miRNA-targeted mRNAs of rno_circRNA_0045161 and rno_circRNA_0019047, respectively. GO: Gene Ontology; KEGG: Kyoto Encyclopedia of Genes and Genomes; DECs, differentially expressed circRNAs.
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