Biliverdin administration regulates the microRNA-mRNA expressional network associated with neuroprotection in cerebral ischemia reperfusion injury in rats

Abstract

Inflammatory response has an important role in the outcome of cerebral ischemia reperfusion injury (CIR). Biliverdin (BV) administration can relieve CIR in rats, but the mechanism remains unknown. The aim of the present study was to explore the expressional network of microRNA (miRNA)‑mRNA in CIR rats following BV administration. A rat middle cerebral artery occlusion model with BV treatment was established. After neurobehavior was evaluated by neurological severity scores (NSS), miRNA and mRNA expressional profiles were analyzed by microarray technology from the cerebral cortex subjected to ischemia and BV administration. Then, bioinformatics prediction was used to screen the correlation between miRNA and mRNA, and 20 candidate miRNAs and 33 candidate mRNAs were verified by reverse transcription‑quantitative polymerase chain reaction. Furthermore, the regulation relationship between ETS proto‑oncogene 1 (Ets1) and miRNA204‑5p was examined by luciferase assay. A total of 86 miRNAs were differentially expressed in the BV group compared with the other groups. A total of 10 miRNAs and 26 candidate genes were identified as a core 'microRNA‑mRNA' regulatory network that was linked with the functional improvement of BV administration in CIR rats. Lastly, the luciferase assay results confirmed that miRNA204‑5p directly targeted Ets1. The present findings suggest that BV administration may regulate multiple miRNAs and mRNAs to improve neurobehavior in CIR rats, by influencing cell proliferation, apoptosis, maintaining ATP homeostasis, and angiogenesis.

Figure 1

TTC stain and NSS test in the experimental groups. (A) Representative images of TTC-stained coronal slices at 48 h post-reperfusion from the three groups. Non-ischemic region is red, and infarct region appears as white color. (B) The infarct volume % of cerebral hemisphere in the three groups. (C) NSS test results in the three groups. Data are presented as mean ± standard deviation (n=3). ^**P<0.01 vs. group S; ^#P<0.05 vs. group C. TTC, 2,3,5-triphenyltetrazolium chloride; NSS, neurological severity score; BV, biliverdin; dpr, days post reperfusion.

Figure 2

Heatmap of miRNA profiling following different treatments. Heatmap of hierarchical clustering showing 86 significantly regulated miRNAs in S, C, and BV groups. (A) A total of 54 miRNAs were downregulated in the C group compared with eth S group, and upregulated in the BV group compared with the C group. (B) The other 32 miRNAs displayed an opposite trend to panel A. The color code in the heat maps is linear with green as the lowest and red as the highest expression. BV, biliverdin.

Figure 3

qPCR validation of miRNA array. (A) Normalizing ratios of candidate miRNAs detected on the microarrays. (B) qPCR results of selected miRNAs. Results are presented as mean ± standard deviation (n=6). ^&P<0.05 group BV vs. group S; ^#P<0.01 group C vs. group S; ^*P<0.01 group BV vs. group C. qPCR, quantitative polymerase chain reaction; BV, biliverdin.

Figure 4

GO and KEGG analysis of candidate miRNA target genes. (A) The top 10 significantly GO and KEGG terms of 'Up/Down' miRNA group target genes. (B) The top 10 GO and KEGG terms of 'Down/Up' miRNA group target genes. The shared GO terms of the two microRNA target gene groups are associated with receptor signaling protein serine/threonine kinase activity (molecular function), ubiquitin ligase complex, and chromatin (cellular component). Blue, biological process; purple, molecular function; green, cellular component complete; yellow, KEGG. GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes.

Figure 4

GO and KEGG analysis of candidate miRNA target genes. (A) The top 10 significantly GO and KEGG terms of 'Up/Down' miRNA group target genes. (B) The top 10 GO and KEGG terms of 'Down/Up' miRNA group target genes. The shared GO terms of the two microRNA target gene groups are associated with receptor signaling protein serine/threonine kinase activity (molecular function), ubiquitin ligase complex, and chromatin (cellular component). Blue, biological process; purple, molecular function; green, cellular component complete; yellow, KEGG. GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes.

Figure 5

Relationships between miRNAs and their predicted gene targets. (A) Relationships between 11 miRNAs and their 126 overlapped predicted target genes. (B) Relationships between 10 miRNAs and their 33 candidate genes verified through qPCR testing. Orange, qPCR-verified miRNAs; blue, overlapped predicted target genes of 10 microRNAs verified through qPCR; purple, qPCR-verified miRNAs; green, the candidate genes of the core 'miRNA-mRNA' regulatory network after qPCR testing; yellow, the candidate genes not consistent with the 'microRNA-mRNA' regulatory mechanism after qPCR testing. qPCR, quantitative polymerase chain reaction.

Figure 6

qPCR validation of target mRNAs. Data are presented as mean ± standard deviation (n=6). ^&P<0.05 group BV vs. group S; ^#P<0.01 group C vs. group S; ^*P<0.01 group BV vs. group C. qPCR, quantitative polymerase chain reaction; BV, biliverdin.

Figure 7

Determination of Ets1 as a direct target of mo-miR-204-5p. (A) The miR-204-5p binding site in the 3'UTR region of Ets1 was highly conserved among several miRNAs. (B) 293T cells were transfected with the luciferase construct containing the 3'UTR of 6 targeted genes of miR-204-5p together with the miR-204-5p mimics or NC miRNA. (C-H) Quantification of luciferase activity. ^*P<0.05 compared with the NC plasmid. UTR, untranslated region; NC, negative control.

Figure 8

Experimental flow diagram. The blue portion explains the animal experimental groups. The white portion of the diagram describes the miRNA-gene integration and analysis. Significantly regulated miRNAs were selected and separated into two groups based on expression pattern. miRNAs that were succesfully validated by qPCR were selected for further analysis. TargetScanv6.2, miRmap, miRDB analyses were used to identify gene targets of candidate miRNAs; only those genes that overlapped in all three analyses were included for GO and KEGG analysis. A total of 49 genes from the mRNA array overlapped with miRNA target genes, and 33 of them were selected for qPCR testing. As a result, 26 candidate genes and 10 miRNAs were identified as a core 'microRNA-mRNA' regulatory network. qPCR, quantitative polymerase chain reaction; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; MCAO, middle cerebral artery occlusion.