Sprague-Dawley rats, (age 10 days after birth; 18-20 g, without sex selection) were obtained from the Medical Animal Center of Nanjing Medical University (Nanjing, China). All rats were housed for ≥7 days prior to the study in a temperature (22-25°C) and humidity-controlled (50%) animal facility under a 12 h light/dark cycle. Animals had free access to food and water; however, food was withheld overnight prior to surgery. All animal procedures were approved by the Zhongda Hospital Committee on Animal Research and all the experiments were carried out in accordance with the approved guidelines.

HIBD was produced in rats according to previously described methods (16). Briefly, following anesthetization using ether, rats were subjected to ischemia, with the right common carotid artery permanently double ligated and sliced from the middle. Following a recovery time of 1 h, the animals were exposed to hypoxia (8% O₂, 92% N₂) for 2.5 h and then returned to their normal living environment. Sham control rats were subjected to isolation and tying ligatures around vessels without occlusion and subsequent ischemia. A total of 24 h following HI insult, the rats were sacrificed using sodium pentobarbital intraperitoneally (40 mg/kg) and their ipsilateral hemispheres, including the cortex and hippocampus, were harvested. The tissues were stored in liquid nitrogen (−196°C) until the following experiment was carried out.

Total RNA was extracted from the hippocampus using TRIzol Reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer's protocol. The concentration and quality of RNA was assessed using NanoDrop ND-1000 spectrophotometry (NanoDrop, Wilmington, DE, USA) and RNA integrity was determined using 1% denaturing agarose gel electrophoresis.

RNA-seq analysis was performed at the Decode Genomics Biotechnology Co., Ltd. (Nanjing, China). The sequencing library was prepared following the removal of ribosomal RNA, according to Illumina^® TruSeq^® RNA Sample Preparation Guide (Illumina, Inc., San Diego, CA, USA). Ligation of the indexed adaptor was performed following the synthesis of double strand cDNA. Following size selection using Agencourt AMPure XP (Beckman Coulter, Inc., Brea, CA, USA), the libraries were quantified and quality assessed using a Qubit^® 2.0 Fluorometer with the Qubit^® HSDNA BR assay kit (Invitrogen™; Thermo Fisher Scientific, Inc.) and Agilent bioanalyzer (Agilent Technologies, Inc., Santa Clara, CA, USA), respectively. Sequencing was performed using an Illumina HiSeq X-10 (Illumina, Inc.) with 2×150 bp pair-end technology.

Quality control of the sequencing was performed using FastQC version 0.11.2 (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Raw data of poor quality (>50% bases having a low-quality threshold <20) and adapter sequences were removed using SOAPnuke, to improve results. The clean data was mapped to rat genomes, provided by Illumina iGenomes (downloaded from http://cole-trapnell-lab.github.io/cufflinks/cuffdiff/index.html) with Tophat2 (version 2.0.7) calling Bowtie2 (version 2.1.0) using default settings. The read numbers mapped to each circRNA were counted. RPM Reads Per Million mapped reads, including TopHat mapping and TopHat-Fusion mapping, were used to calculate the expression level of individual circRNA. Differential expression between circRNAs was assessed using DEGseq algorithm (17). The statistical criteria for selecting aberrant-expressed circRNA was defined using a q-value [the false discovery rate adjusted P-value <0.05 with a fold-change >2.0 or <0.5 (18,19)]. CircRNA annotation was based on the Gencode rat genome (v10) (20). The circRNA donator/acceptor site was intersected to annotate gene regions, including coding RNA, non-coding RNA, intron, antisense and intergenic regions, with the BEDTools suite (v2.16.2) (21). The number of reads aligning to the circRNA-specific head-to-tail junctions was used as a measurement of circRNAs expression.

GO analysis provides a controlled vocabulary to describe genes and gene product attributes in any organism (http://www.geneontology.org). This ontology includes three domains: Biological processes, cellular components and molecular functions. Fisher's exact test was used to detect overlapping data between the differentially expressed list and GO annotation list, except for that expected by chance. Pathway analysis was used to map genes to the KEGG pathways.

To investigate the regulation between circRNAs and mRNAs in the neonatal rats following HIBD, interaction networks were studied among the 6 differentially expressed circRNAs, whose host gene was located on the exon and was identified along with their corresponding target miRNAs. CircRNAs-targeted miRNAs were predicted using miRNA target prediction software based on TargetScan (http://www.targetscan.org/vert_71/) and miRanda (http://www.microrna.org/microrna/home.do). The co-expression network was illustrated using Cytoscape (v2.8.1, https://cytoscape.org/). The associated analyses and drawing were performed by Decode Genomics Biotechnology Co., Ltd. (Nanjing, China).

Total RNA was extracted from hippocampus samples using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and converted to cDNA using PrimeScript RT-PCR kit (cat. no. RR037A; Takara Bio, Inc., Otsu, Japan), according to the manufacturer's protocol. In brief, the reaction system was incubated at 25°C for 10 min, and then at 42°C for 60 min. PCR was performed in a total reaction volume of 25 µl, containing 12.5 µl SYBR Premix Ex Taq (2X), 2 µl cDNA, 1 µl forward primer (10 µM), 1 µl reverse primer (10 µM), 0.5 µl ROX Reference Dye II (50X) and 8 µl double-distilled water. The amplification procedures were as follows: 10 min at 95°C to initiate denaturation; 40 cycles of 5 sec at 95°C, 30 sec at 63°C and 30 sec at 72°C; and a final extension of 5 min at 72°C. Amplification efficiency was evaluated using standard curve analysis. All samples were normalized using the 2^−ΔΔCq method against GAPDH and the experiment was repeated in triplicate (22). The primers of the circRNAs and GAPDH are listed in Table I.

Data analyses were performed using GraphPad Prism 5.0 (GraphPad, Inc., La Jolla, CA, USA), Cluster 3.0 (Human Genome Center, University of Tokyo, Tokyo, Japan) and Cytoscape 2.8.1 (https://cytoscape.org/). Data are presented as the with mean ± standard error of the mean and three repeats were performed for each experiment. Statistical differences were determined using Students t-test and one-way analysis of variance. Multiple comparisons between the groups were performed using the Student-Newman-Kuels method. K-means clustering algorithms were performed to classify the samples according to the selected differently expressed genes. All statistical tests were performed as two-sided tests and P<0.05 was considered to indicate a statistically significant difference.

RNA-Seq was used to investigate circRNAs expression profiles from the hippocampus of HIBD neonatal rats. Hierarchical clustering was performed to demonstrate the circRNAs expression pattern between the HIBD and control groups (Fig. 2A). As presented in the box plot, the distribution of the circRNAs expression profiles across all the samples are similar (Fig. 2B). Statistically significant alterations in circRNAs with between the sham and HIBD groups were identified using a volcano plot (Fig. 2C). Compared with the sham group, 66 circRNAs were demonstrated to be differentially expressed [fold change (FC) >2.0 and P<0.05] following HIBD, including 20 upregulated and 46 downregulated circRNAs. The top 30 altered circRNAs are listed in Table II. The differentially expressed circRNAs following HIBD included 70% exonic and 28% intergenic in origin (Fig. 3A). The distributions of altered circRNAs revealed that the upregulated and downregulated circRNAs were transcribed from all chromosomes, with the exceptions of chr12, chr16 and chr20 (Fig. 3B). The downregulated circRNAs were transcribed from all chromosomes with the exceptions of chr12, chr13 chr16 and chr17 and the upregulated circRNAs were transcribed from most chromosomes (Fig. 3B).

To determine the roles of circRNAs in physiological and pathological processes following HIBD, GO and KEGG pathway analyses were conducted for mRNAs transcribed from the parent genes of altered circRNAs. The results indicated that significant enriched and meaningful GO terms in biological process were ‘positive regulation of guanosine monophosphatase (GTPase) activity’, ‘intracellular signal transduction’ and ‘γ-aminobutyric acid (GABA) signaling pathway’ (Fig. 4A). In terms of molecular function, the majority of the altered circRNA associated mRNAs were identified to be ‘protein binding’, ‘Rab GTPase binding’ and ‘adenosine triphosphate binding’ (Fig. 4B). The cellular component (CC) included enriched CC terms, including ‘postsynaptic density’, ‘synapse’ and ‘cytoplasm’ (Fig. 4C). Gene-enriched KEGG pathway analysis suggested pathways that could be involved during the development of HIBD. The majority of these circRNAs were target genes involved in morphine addiction, nicotine addiction, axon guidance, GABAergic synapse and glutamatergic synapse (Figs. 4D and 5). Glutamatergic synapse and axon guidance have been reported to be involved in neuron injury and regeneration following brain injury in rats (23,24).

To verify the RNA-Seq data, 4 differentially expressed circRNAs were selected for qPCR and compared with the sham group, including circRNAs chr13: 31092209|31209056 and chr1: 200899066|201028171, which were upregulated, and chr11: 34065887|34070810 and chr10: 13931236|13935484, which were downregulated significantly following HIBD (P<0.01; Fig. 6). The expression patterns of these circRNAs were consistent with the RNA-Seq data.

circRNAs serve a crucial role in sponging and regulating the expression of miRNA, their interaction with HIBD associated miRNAs suggests that circRNAs could serve an important role in pathophysiological processes of HIBD. Therefore, potential circRNA/microRNA interactions were investigated based on TargetScan and miRanda. The circRNA/miRNA interaction of the differentially expressed circRNAs is presented in Fig. 7. These results are in agreement with previously reported differential expression of miRNA in neonatal rats (25-29).