Novel circular RNAs expressed in brain microvascular endothelial cells after oxygen-glucose deprivation/recovery

Abstract

Circular RNAs (circRNAs) are generated by head-to-tail splicing and are ubiquitously expressed in all multicellular organisms. Their important biological functions are increasingly recognized. Cerebral ischemia reperfusion injury-induced brain microvascular endothelial cell dysfunction is an initial stage of blood-brain barrier disruption. The expression profile and potential function of circRNAs in brain microvascular endothelial cells is unknown. Rat brain microvascular endothelial cells were extracted and cultured in glucose-free medium for 4 hours with 5% CO₂ and 95% N₂, and the medium was then replaced with complete growth medium for 6 hours. The RNA in these cells was then extracted. The circRNA was identified by Find_circ and CIRI2 software. Functional and pathway enrichment analysis of genes that were common to differentially expressed mRNAs and circRNA host genes was performed by the Database for Annotation, Visualization and Integrated Discovery Functional Annotation Tool. Miranda software was used to predict microRNAs that were potentially sponged by circRNAs. Furthermore, cytoscape depicted the circR-NA-microRNA interaction network. The results showed that there were 1288 circRNAs in normal and oxygen-glucose deprived/recovered primary brain microvascular endothelial cells. There are 211 upregulated and 326 downregulated differentially expressed circRNAs. The host genes of these differentially expressed circRNAs overlapped with those of differentially expressed mRNAs. The shared genes were further studied by functional enrichment analyses, which revealed that circRNAs may contribute to calcium ion function and the cyclic guanosine 3′,5′-monophosphate (CAMP) dependent protein kinase (PKα) signaling pathway. Next, quantitative reverse transcription polymerase chain reaction assays were performed to detect circRNA levels transcribed from the overlapping host genes. Eight out of the ten circRNAs with the highest fold-change identified by sequencing were successfully verified. Subsequently, the circRNA-microRNA interaction networks of these eight circRNAs were explored by bioinformatic analysis. These results demonstrate that altered circRNAs may be important in the pathogenesis of cerebral ischemia reperfusion injury and consequently may also be potential therapeutic targets for cerebral ischemia diseases. All animal experiments were approved by the Chongqing Medical University Committee on Animal Research, China (approval No. CQMU20180086) on March 22, 2018.

Chinese Library Classification No. R446; R743; R741

Introduction

The blood-brain barrier consists of brain microvascular endothelial cells (BMECs), astrocytes, microglial cells, and pericytes and the microvasculature in the brain delivers oxygen and nutrients to neurocytes. Under physiological con-ditions, the blood-brain barrier selectively allows molecules to pass through the barrier to protect the central nervous system. In particular, BMECs can express special transport proteins to carry glucose, amino acids, and other factors. BMECs also secrete neurotrophins and enzymes to nourish neural cells and degrade harmful molecules. Thus, BMECs contribute to revascularization and neurological recovery after ischemic injury (Yu et al., 2015; Toth and Nielsen, 2018).

Circular RNAs (circRNAs) have recently been described as novel regulatory noncoding RNAs (Barrett et al., 2015). circRNAs are transcribed from the exons and introns of genes and form covalently closed head-to-tail (or backspliced) circularized transcripts (Hansen et al., 2013; Barrett et al., 2015). circRNAs can act as competing endogenous RNA or microRNA (miRNA) sponges. circRNAs play important roles in stroke and endothelial function. For example, circRNA-ZNF609 adsorbs miR-615-5p, which withstands oxidative stress and promotes vascular endothelial cell mi-gration (Liu et al., 2017b). Furthermore, circRNA Hectd1 acts as a sponge that inhibits miR-142 and contributes to ischemic stroke via astrocyte activation (Han et al., 2018). Therefore, we suggest that circRNAs are worthy of further exploration.

BMECs contribute significantly to integrity and function of the brain vasculature. Oxygen and nutrient deprivation may induce BMEC dysfunction and increased blood-brain barrier permeability (Yu et al., 2015). However, how oxy-gen-glucose shortages affect circRNAs in BMECs is unknown. Here, we used RNA sequencing to measure the global changes of circRNAs in BMECs subjected to oxygen-glucose deprivation (OGD)/recovery (OGD/R) treatment. Differentially expressed (DE) circRNAs as well as potential mechanisms were explored.

Materials and Methods

Isolation and cultivation of primary BMECs

All animal methods were approved by the Chongqing Medical University Committee on Animal Research, China (approval No. CQMU20180086) on March 22, 2018. Adult male Sprague-Dawley rats aged 6 weeks and weighing 160–200 g were housed in a specific-pathogen-free animal room of the Animal Breeding Center of Chongqing Medical University, China [license No. SYXK (Yu) 2017-0023]. Eight rats were anesthetized by inhalation of isoflurane (2% in oxygen) (RWD Life Science, Shenzhen, China), euthanized, and their brains collected. Tissue was homogenized and then centrifuged at 720 × g for 5 minutes at 4°C. The supernatant was discarded, and the pellet resuspended in phosphate-buffered saline. This was then layered over 15 mL 18% dextran and centrifuged at 4500 × g for 20 minutes at 4°C. The pellet was resuspended in 10 mL phosphate-buffered saline (containing 0.1% bovine serum albumin). Ten milliliters of the suspension were added to 100 µL collagenase (100 mg/mL), 40 µL DNase I (10 mg/mL) and 100 µL N-alpha-tosyl-L-lysine chloromethyl ketone hydrochloride (14.7 µg/mL) and digested for 1 hour at 37°C. After centrifugation at 1000 × g for 5 minutes, the pellet was resuspended in 2 mL phosphate-buffered saline containing 0.1% bovine serum albumin and 100 µL biotin-labeled anti-CD31 antibody (DSB-X Biotin Protein Labeling Kit, Thermo Fisher, Waltham, MA, USA). After incubation for 10 minutes at 4°C, BMECs were isolated with a Dynabeads FlowComp Flexi Kit (Thermo Fisher). Finally, bead-free cells were cultured in ordinary medium. The medium comprised DMEM basic medium (Thermo Fisher), 10% fetal bovine serum, 20 mg/L endothelial cell growth supplement (ECGS), 2 mM L-glutamine and 100 mg/L heparin sodium (Plácido et al., 2017; Sawaguchi et al., 2017). The endothelial cell markers, factor VIII and CD31 (Thermo Fisher), were used to identify BMECs by immunofluorescence (Bachetti and Morbidelli, 2000).

OGD/R treatment

Cells at 90–100% confluency were digested with trypsin and re-seeded in new dishes. The passage dilution was 1:4. Passage 6 BMECs were subjected to OGD/R treatment. At 48 hours after seeding, the medium was replaced by glu-cose-free medium, which was prebubbled with 95% N2 and 5% CO₂ for 1 hour. BMECs were then cultured in an incubator flushed with 5% CO₂ and 95% N₂. The chamber was sealed and kept at 37°C for 4 hours with an oxygen concentration of < 0.2%. Upon OGD termination, the cells were placed back into a normal incubator (5% CO₂, 95% air at 37°C), and the medium was replaced with complete growth medium. Control BMECs were not exposed to OGD. The cells were harvested for further analyses after 6 hours.

RNA extraction and sequencing

A total of 5 µg of RNA per sample was prepared using TRIzol reagent according to the manufacturer’s recommenda-tions (Thermo Fisher). A Nanodrop 2000 (Thermo Fisher), an Agilent Bioanalyzer 2100 (Agilent, USA) and agarose gel electrophoresis were used to determine RNA quality. Samples with an optical density (OD)_260/280 between 1.7–2.0, OD_260/230 > 2, 28S/18S > 1.8 and RNA integrity number > 9 were considered reliable. Five samples of equal mass in each group were mixed. An Epicentre Ribozero ribosomal RNA Removal Kit (Epicentre, Madison, WI, USA) was used to remove ribosomal RNA, and RNase R (Epicentre) was used to digest linear RNA. Subsequently, sequencing libraries were generated using the NEBNext Ultra Directional RNA Library Prep Kit for Illumina (NEB, Ipswich, MA, USA) following the manufacturer’s recommendations. After cluster generation, the libraries were sequenced on an Illumina Hiseq 4000 platform, and 150-bp paired-end reads were generated.

For mRNA sequencing, ribosomal RNA was removed from 3 µg of total RNA. Sequencing libraries were generated directly without linear RNA digestion. Sequencing and DE circRNA/mRNA analysis were completed by Novogene, Beijing, China.

Bioinformatic analysis

Find_circ (GitHub, San Francisco, CA, USA) and CIRI2 (GitHub) software were used to identify circRNAs (Memczak et al., 2013; Gao et al., 2018). Raw data were normalized by standardized TPM (transcripts per million clean tags). The DESeq R package (1.10.1) (Bioconductor, Riverside, CA, USA) calculated differences in the expression of circRNAs.

For mRNA analysis, bowtie2 (v2.2.8) (Johns Hopkins University, Baltimore, MD, USA) and HISAT2 (v2.0.4) (Johns Hopkins University) mapped data to the reference genome. Cuffdiff (v2.1.1) (GenePattern, CA, USA) was used to calculate FPKMs (expected number of Fragments Per Kilobase of transcript sequence per Million base pairs sequenced) of mRNA. Next, the FPKM value was used to calculate mRNA differential expression.

The intersecting genes of DE circRNA host genes and DE mRNAs were subjected to functional and pathway enrichment analysis by the Database for Annotation, Visualization and Integrated Discovery (DAVID) Functional Annotation Tool (DAVID Bioinformatic Team, Frederick, USA) (Huang da et al., 2009). Miranda software (cBio-MSKCC, New York, USA) was used to predict the miRNAs that were potentially sponged by circRNAs (John et al., 2004). Cytoscape (NIGMS, Bethesda, MD, USA) depicted the circRNA-miRNA interaction network (Shannon et al., 2003).

Quantitative reverse transcription polymerase chain reaction (qRT-PCR)

As described previously (Shang et al., 2016), RNA was extracted using TRIzol reagent (Thermo Fisher). Two mi-crograms of RNA were reverse-transcribed to cDNA using a RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher). qRT-PCR was performed with 20 µL SYBR Green reaction mix containing specific primers (Sangon Biotech, Shanghai, China) using a standard protocol The primers are listed in Additional Table 1. Each group had five experimental replicates; and each experimental replicate was performed three times. The Ct value of target circRNAs was normalized to the geometrical average of glyceraldehyde-3-phosphate dehydrogenase, β-actin and U6.

Additional Table 1.

The list of CircRNAs primers

ID	Primers sequence	Length (bp)
novel_circ_0000173	F: 5’-AAG GAC GTG CTG TCG GTG GAG AGT-3’ R: 5’-GCA GCA AGC GGC AAT TAA CAG AGA CC-3’	103
novel_circ_0004586	F: 5’-CTC CTG CCC TGC TGT GTC CCA TAT CT-3’ R: 5’-CCA CCC ACC ACT GAG AAC AAG CCA AA-3’	268
novel_circ_0003342	F: 5’-TGC TGA GGT GTG GGT GCT GTC A-3’ R: 5’-ACA GTG GTG TGA AGG CTC CAG C-3’	157
novel_circ_0004860	F: 5’-TGG TCT TCT TTG TTC AGG GCC TTG TT-3’ R: 5’-TGG TGG TCA CCC AGT TTG GGT TAT AC-3’	185
novel_circ_0002085	F: 5’-GGT CTT CCT CCT GAT GTG AGC TGA AC-3’ R: 5’-ACA ACA AAG GCA GAG ACT GGC GTT T-3’	216
novel_circ_0003760	F: 5’-ACC TAA AGG AAG CAA GGA CCC GAC AA-3’ R: 5’-CCT TCT TCA GGA CCT TGA GGC AGT AC-3’	105
novel_circ_0002086	F: 5’-GGT TTA TTT CTG TTG GCT TCA GTG CAG T-3’ R: 5’-CCC AGG ATA CAT TGA CCC GGA TTC TTT G-3’	116
novel_circ_0000456	F: 5’-GCT GAC AGG ACC CGA CGA TGG CTT T-3’ R: 5’-CAA CGC ATT CTG CTG ATG CAC CTC CA-3’	108
novel_circ_0001606	F: 5’-TGG TGG TCG TCC GCA TGG TGT T-3’ R: 5’-AAA GAT CTG CTG CCC GGT GAA CCC-3’	109
novel_circ_0002023	F: 5’-CCG TGT GGC TCC GCA AGT GAA TCT T-3’ R: 5’-CAG CAA CTC CAG TGA CCG TGC CAA G-3’	116

F: Forward primer; R: reversed primer.

Statistical analysis

For sequencing, a model based on negative binomial distribution was used. The resulting P values were adjusted to q values using Benjamini and Hochberg’s approach for controlling the false discovery rate. In DAVID enrichment anal-ysis, Fisher’s exact test was used to determine whether the proportions of those falling into each category differed by group. P value was adopted to measure the gene enrichment in annotation terms. In the bubble chart, the number of genes that fall into each category divided by the total number of this category was the RichFactor. Bubble chart analysis was performed using OmicShare tools (Genedenovo, Guangzhou, China). qRT-PCR analysis was performed using Student’s t-test with a two-tailed P value (GraphPad Software Company, San Diego, CA, USA). All summary statistics of the results are presented as the mean ± standard error of the mean (SEM). P < 0.05 was considered statistically significant.

Results

Identification of differentially expressed circRNAs

At least 10G of clean sequencing data were obtained from each sample for further analysis. The intersection of the Find_circ and CIRI2 software results identified 1195 circRNAs in the control group and 1109 circRNAs in the OGD/R group (Memczak et al., 2013; Gao et al., 2018), with 1016 circRNAs found in both groups (Figure 1A). The detailed information of all 1288 identified circRNAs is shown in Additional Table 2 ^{(2MB, pdf)} , including chromosome, length, strand, host genes and expression level. Most circRNAs were transcribed from exons and circRNAs were uniformly distributed across chromosomes. We selected 10 representative chromosomes to show the distribution of circRNAs (Figure 1B).

Figure 1.

Profile of circular RNAs (circRNAs) in brain microvascular endothelial cells in control (Ctrl) and oxygen glucose deprivation/recovery (OGD/R) groups.

(A) RNA sequencing identified 1195 circRNAs in the Ctrl group and 1109 circRNAs in the OGD/R group. Among these circRNAs, 1016 circRNAs existed in both groups. (B) The circRNAs were uniformly distributed across chromosomes. Ten representative chromosomes are shown. (C) Volcano plot showing the global change in circRNAs. A q value (ordinate) < 0.01 and fold change (abscissa) > 2 defined the differentially expressed circRNAs. The blue dots represent the unchanged circRNAs. (D) Heatmaps show 211 upregulated and 326 downregulated circRNAs in primary brain microvascular endothelial cells after OGD/R treatment. FC: Fold change.

Next, we performed a global analysis of the DE circRNAs in the OGD/R and control BMECs. Standardized TPM values were applied to compare gene expression between two groups (Zhou et al., 2010). The fold change (FC) in the expression of each circRNA was calculated as the log2 ratio using normalized TPM values (Audic and Claverie, 1997). Subsequently, the resulting q values for all genes were corrected for multiple tests using a DEGseq adjustment (Wang et al., 2010). A Volcano Plot was used to show the filter and distribution of DE circRNAs (Figure 1C). Finally, the circRNAs with FC > 2 and q values < 0.01 were identified as DE circRNAs. As shown in Figure 1D, there were 211 upregulated and 326 downregulated circRNAs in OGD/R-treated cells. The detailed information is shown in Additional Table 3. These results demonstrate that OGD/R dramatically altered circRNA expression profiles in vascular endothelial cells. Ischemia-reperfusion-induced endothelial dysfunction has been attributed to angiogenesis, oxidative stress, and inflammation (Guo et al., 2018; Pang et al., 2018; Zhu et al., 2018). Whether the OGD/R-induced circRNA alterations participate in endothelial dysfunction by affecting these responses remains to be determined in future studies.

Additional Table 3.

The list of differentially expressed circRNAs

ID	OGD/R	Ctrl	log2.Fold_c	p.value	q.value	Expression change
novel_circ_0000007	979.7346	474.0868	1.0472	1.63E-44	5.22E-45	up
novel_circ_0000012	1110.366	474.0868	1.2278	1.55E-63	7.84E-64	up
novel_circ_0000062	2612.625	0	12.351	0	0	up
novel_circ_0000083	1045.05	355.5651	1.5554	9.69E-84	1.22E-83	up
novel_circ_0000104	457.2095	118.5217	1.9477	2.70E-50	9.10E-51	up
novel_circ_0000123	522.5251	177.7826	1.5554	9.16E-43	2.85E-43	up
novel_circ_0000147	326.5782	0	9.3513	6.42E-61	2.91E-61	up
novel_circ_0000153	326.5782	0	9.3513	6.42E-61	2.91E-61	up
novel_circ_0000167	391.8938	0	9.6143	3.13E-70	2.21E-70	up
novel_circ_0000170	522.5251	177.7826	1.5554	9.16E-43	2.85E-43	up
novel_circ_0000211	391.8938	0	9.6143	3.13E-70	2.21E-70	up
novel_circ_0000217	457.2095	177.7826	1.3627	1.98E-31	5.23E-32	up
novel_circ_0000228	457.2095	0	9.8367	3.80E-79	3.69E-79	up
novel_circ_0000238	326.5782	0	9.3513	6.42E-61	2.91E-61	up
novel_circ_0000243	2286.047	0	12.159	0	0	up
novel_circ_0000257	326.5782	0	9.3513	6.42E-61	2.91E-61	up
novel_circ_0000261	979.7346	177.7826	2.4623	0	0	up
novel_circ_0000301	391.8938	118.5217	1.7253	3.16E-37	9.25E-38	up
novel_circ_0000327	914.4189	414.826	1.1403	2.97E-47	9.81E-48	up
novel_circ_0000328	391.8938	0	9.6143	3.13E-70	2.21E-70	up
novel_circ_0000335	457.2095	0	9.8367	3.80E-79	3.69E-79	up
novel_circ_0000353	326.5782	0	9.3513	6.42E-61	2.91E-61	up
novel_circ_0000360	391.8938	177.7826	1.1403	3.37E-21	7.14E-22	up
novel_circ_0000368	326.5782	118.5217	1.4623	3.32E-25	7.63E-26	up
novel_circ_0000371	326.5782	0	9.3513	6.42E-61	2.91E-61	up
novel_circ_0000377	522.5251	0	10.029	9.88E-88	1.52E-87	up
novel_circ_0000379	457.2095	118.5217	1.9477	2.70E-50	9.10E-51	up
novel_circ_0000396	587.8407	0	10.199	4.93E-96	9.65E-96	up
novel_circ_0000407	457.2095	0	9.8367	3.80E-79	3.69E-79	up
novel_circ_0000431	522.5251	0	10.029	9.88E-88	1.52E-87	up
novel_circ_0000446	457.2095	177.7826	1.3627	1.98E-31	5.23E-32	up
novel_circ_0000447	522.5251	177.7826	1.5554	9.16E-43	2.85E-43	up
novel_circ_0000456	522.5251	118.5217	2.1403	3.83E-64	2.40E-64	up
novel_circ_0000486	783.7876	296.3043	1.4034	7.08E-55	2.46E-55	up
novel_circ_0000533	326.5782	0	9.3513	6.42E-61	2.91E-61	up
novel_circ_0000619	391.8938	118.5217	1.7253	3.16E-37	9.25E-38	up
novel_circ_0000665	457.2095	0	9.8367	3.80E-79	3.69E-79	up
novel_circ_0000667	587.8407	0	10.199	4.93E-96	9.65E-96	up
novel_circ_0000691	1632.891	0	11.673	0	0	up
novel_circ_0000714	326.5782	118.5217	1.4623	3.32E-25	7.63E-26	up
novel_circ_0000766	718.472	296.3043	1.2779	3.42E-44	1.09E-44	up
novel_circ_0000776	979.7346	474.0868	1.0472	1.63E-44	5.22E-45	up
novel_circ_0000784	783.7876	0	10.614	0	0	up
novel_circ_0000791	1045.05	355.5651	1.5554	9.69E-84	1.22E-83	up
novel_circ_0000814	457.2095	177.7826	1.3627	1.98E-31	5.23E-32	up
novel_circ_0000863	391.8938	177.7826	1.1403	3.37E-21	7.14E-22	up
novel_circ_0000883	522.5251	237.0434	1.1403	1.00E-27	2.48E-28	up
novel_circ_0000908	326.5782	0	9.3513	6.42E-61	2.91E-61	up
novel_circ_0000988	457.2095	0	9.8367	3.80E-79	3.69E-79	up
novel_circ_0001002	326.5782	118.5217	1.4623	3.32E-25	7.63E-26	up
novel_circ_0001012	326.5782	118.5217	1.4623	3.32E-25	7.63E-26	up
novel_circ_0001025	391.8938	177.7826	1.1403	3.37E-21	7.14E-22	up
novel_circ_0001031	653.1564	0	10.351	0	0	up
novel_circ_0001041	457.2095	177.7826	1.3627	1.98E-31	5.23E-32	up
novel_circ_0001051	2090.1	1007.434	1.0529	1.01E-93	1.86E-93	up
novel_circ_0001053	783.7876	0	10.614	0	0	up
novel_circ_0001073	391.8938	0	9.6143	3.13E-70	2.21E-70	up
novel_circ_0001090	718.472	355.5651	1.0148	1.35E-31	3.64E-32	up
novel_circ_0001092	3004.519	0	12.553	0	0	up
novel_circ_0001121	391.8938	0	9.6143	3.13E-70	2.21E-70	up
novel_circ_0001156	326.5782	0	9.3513	6.42E-61	2.91E-61	up
novel_circ_0001158	326.5782	118.5217	1.4623	3.32E-25	7.63E-26	up
novel_circ_0001167	457.2095	0	9.8367	3.80E-79	3.69E-79	up
novel_circ_0001221	391.8938	0	9.6143	3.13E-70	2.21E-70	up
novel_circ_0001225	391.8938	177.7826	1.1403	3.37E-21	7.14E-22	up
novel_circ_0001235	391.8938	0	9.6143	3.13E-70	2.21E-70	up
novel_circ_0001237	1306.313	533.3477	1.2924	3.40E-80	4.09E-80	up
novel_circ_0001275	457.2095	0	9.8367	3.80E-79	3.69E-79	up
novel_circ_0001300	522.5251	237.0434	1.1403	1.00E-27	2.48E-28	up
novel_circ_0001327	587.8407	237.0434	1.3103	8.75E-38	2.61E-38	up
novel_circ_0001339	391.8938	118.5217	1.7253	3.16E-37	9.25E-38	up
novel_circ_0001425	457.2095	118.5217	1.9477	2.70E-50	9.10E-51	up
novel_circ_0001428	457.2095	0	9.8367	3.80E-79	3.69E-79	up
novel_circ_0001498	326.5782	0	9.3513	6.42E-61	2.91E-61	up
novel_circ_0001503	979.7346	414.826	1.2399	4.47E-57	1.98E-57	up
novel_circ_0001517	391.8938	118.5217	1.7253	3.16E-37	9.25E-38	up
novel_circ_0001523	522.5251	177.7826	1.5554	9.16E-43	2.85E-43	up
novel_circ_0001541	326.5782	0	9.3513	6.42E-61	2.91E-61	up
novel_circ_0001551	1371.628	651.8694	1.0732	6.21E-64	3.54E-64	up
novel_circ_0001589	326.5782	118.5217	1.4623	3.32E-25	7.63E-26	up
novel_circ_0001606	522.5251	118.5217	2.1403	3.83E-64	2.40E-64	up
novel_circ_0001624	1045.05	474.0868	1.1403	9.39E-54	3.26E-54	up
novel_circ_0001670	391.8938	0	9.6143	3.13E-70	2.21E-70	up
novel_circ_0001676	391.8938	0	9.6143	3.13E-70	2.21E-70	up
novel_circ_0001687	457.2095	177.7826	1.3627	1.98E-31	5.23E-32	up
novel_circ_0001688	326.5782	0	9.3513	6.42E-61	2.91E-61	up
novel_circ_0001696	457.2095	118.5217	1.9477	2.70E-50	9.10E-51	up
novel_circ_0001736	1110.366	0	11.117	0	0	up
novel_circ_0001737	326.5782	0	9.3513	6.42E-61	2.91E-61	up
novel_circ_0001739	522.5251	0	10.029	9.88E-88	1.52E-87	up
novel_circ_0001744	457.2095	0	9.8367	3.80E-79	3.69E-79	up
novel_circ_0001764	783.7876	355.5651	1.1403	9.47E-41	2.91E-41	up
novel_circ_0001811	326.5782	118.5217	1.4623	3.32E-25	7.63E-26	up
novel_circ_0001814	391.8938	0	9.6143	3.13E-70	2.21E-70	up
novel_circ_0001841	1567.575	770.3911	1.0249	6.51E-68	4.43E-68	up
novel_circ_0001850	326.5782	0	9.3513	6.42E-61	2.91E-61	up
novel_circ_0001894	522.5251	0	10.029	9.88E-88	1.52E-87	up
novel_circ_0001898	391.8938	177.7826	1.1403	3.37E-21	7.14E-22	up
novel_circ_0001910	326.5782	118.5217	1.4623	3.32E-25	7.63E-26	up
novel_circ_0001943	326.5782	118.5217	1.4623	3.32E-25	7.63E-26	up
novel_circ_0001962	326.5782	118.5217	1.4623	3.32E-25	7.63E-26	up
novel_circ_0001996	326.5782	0	9.3513	6.42E-61	2.91E-61	up
novel_circ_0002023	522.5251	118.5217	2.1403	3.83E-64	2.40E-64	up
novel_circ_0002025	522.5251	0	10.029	9.88E-88	1.52E-87	up
novel_circ_0002053	326.5782	118.5217	1.4623	3.32E-25	7.63E-26	up
novel_circ_0002067	653.1564	177.7826	1.8773	7.23E-68	4.86E-68	up
novel_circ_0002086	587.8407	118.5217	2.3103	1.37E-78	1.32E-78	up
novel_circ_0002090	522.5251	0	10.029	9.88E-88	1.52E-87	up
novel_circ_0002105	522.5251	177.7826	1.5554	9.16E-43	2.85E-43	up
novel_circ_0002193	457.2095	177.7826	1.3627	1.98E-31	5.23E-32	up
novel_circ_0002208	718.472	177.7826	2.0148	2.44E-81	3.04E-81	up
novel_circ_0002217	391.8938	0	9.6143	3.13E-70	2.21E-70	up
novel_circ_0002226	1175.681	296.3043	1.9883	0	0	up
novel_circ_0002230	522.5251	118.5217	2.1403	3.83E-64	2.40E-64	up
novel_circ_0002240	2743.257	0	12.422	0	0	up
novel_circ_0002241	522.5251	0	10.029	9.88E-88	1.52E-87	up
novel_circ_0002242	1306.313	0	11.351	0	0	up
novel_circ_0002255	522.5251	177.7826	1.5554	9.16E-43	2.85E-43	up
novel_circ_0002289	326.5782	0	9.3513	6.42E-61	2.91E-61	up
novel_circ_0002327	326.5782	0	9.3513	6.42E-61	2.91E-61	up
novel_circ_0002336	653.1564	237.0434	1.4623	1.00E-48	3.37E-49	up
novel_circ_0002346	391.8938	177.7826	1.1403	3.37E-21	7.14E-22	up
novel_circ_0002397	457.2095	0	9.8367	3.80E-79	3.69E-79	up
novel_circ_0002409	522.5251	237.0434	1.1403	1.00E-27	2.48E-28	up
novel_circ_0002433	849.1033	0	10.73	0	0	up
novel_circ_0002493	718.472	296.3043	1.2779	3.42E-44	1.09E-44	up
novel_circ_0002513	1045.05	296.3043	1.8184	0	0	up
novel_circ_0002520	1306.313	355.5651	1.8773	0	0	up
novel_circ_0002540	326.5782	0	9.3513	6.42E-61	2.91E-61	up
novel_circ_0002559	326.5782	0	9.3513	6.42E-61	2.91E-61	up
novel_circ_0002570	1567.575	770.3911	1.0249	6.51E-68	4.43E-68	up
novel_circ_0002599	849.1033	0	10.73	0	0	up
novel_circ_0002643	391.8938	0	9.6143	3.13E-70	2.21E-70	up
novel_circ_0002649	457.2095	177.7826	1.3627	1.98E-31	5.23E-32	up
novel_circ_0002667	391.8938	0	9.6143	3.13E-70	2.21E-70	up
novel_circ_0002679	326.5782	0	9.3513	6.42E-61	2.91E-61	up
novel_circ_0002715	849.1033	237.0434	1.8408	1.69E-85	2.19E-85	up
novel_circ_0002740	326.5782	0	9.3513	6.42E-61	2.91E-61	up
novel_circ_0002743	522.5251	118.5217	2.1403	3.83E-64	2.40E-64	up
novel_circ_0002812	2155.416	1007.434	1.0973	0	0	up
novel_circ_0002853	522.5251	0	10.029	9.88E-88	1.52E-87	up
novel_circ_0002859	979.7346	237.0434	2.0472	0	0	up
novel_circ_0002877	326.5782	0	9.3513	6.42E-61	2.91E-61	up
novel_circ_0002881	587.8407	0	10.199	4.93E-96	9.65E-96	up
novel_circ_0002894	849.1033	355.5651	1.2558	1.26E-50	4.30E-51	up
novel_circ_0002903	522.5251	177.7826	1.5554	9.16E-43	2.85E-43	up
novel_circ_0002958	326.5782	118.5217	1.4623	3.32E-25	7.63E-26	up
novel_circ_0002986	979.7346	414.826	1.2399	4.47E-57	1.98E-57	up
novel_circ_0002991	326.5782	0	9.3513	6.42E-61	2.91E-61	up
novel_circ_0002999	326.5782	118.5217	1.4623	3.32E-25	7.63E-26	up
novel_circ_0003074	457.2095	177.7826	1.3627	1.98E-31	5.23E-32	up
novel_circ_0003096	1110.366	533.3477	1.0579	5.53E-51	1.89E-51	up
novel_circ_0003103	326.5782	0	9.3513	6.42E-61	2.91E-61	up
novel_circ_0003115	653.1564	0	10.351	0	0	up
novel_circ_0003129	391.8938	0	9.6143	3.13E-70	2.21E-70	up
novel_circ_0003154	326.5782	0	9.3513	6.42E-61	2.91E-61	up
novel_circ_0003162	783.7876	237.0434	1.7253	1.10E-72	1.01E-72	up
novel_circ_0003222	391.8938	118.5217	1.7253	3.16E-37	9.25E-38	up
novel_circ_0003226	1828.838	651.8694	1.4883	0	0	up
novel_circ_0003243	653.1564	177.7826	1.8773	7.23E-68	4.86E-68	up
novel_circ_0003259	391.8938	118.5217	1.7253	3.16E-37	9.25E-38	up
novel_circ_0003270	783.7876	237.0434	1.7253	1.10E-72	1.01E-72	up
novel_circ_0003274	391.8938	0	9.6143	3.13E-70	2.21E-70	up
novel_circ_0003280	522.5251	177.7826	1.5554	9.16E-43	2.85E-43	up
novel_circ_0003377	1045.05	355.5651	1.5554	9.69E-84	1.22E-83	up
novel_circ_0003426	1436.944	0	11.489	0	0	up
novel_circ_0003437	1110.366	414.826	1.4205	2.57E-78	2.46E-78	up
novel_circ_0003459	326.5782	0	9.3513	6.42E-61	2.91E-61	up
novel_circ_0003471	1502.26	592.6085	1.342	4.26E-97	9.01E-97	up
novel_circ_0003487	522.5251	0	10.029	9.88E-88	1.52E-87	up
novel_circ_0003492	457.2095	0	9.8367	3.80E-79	3.69E-79	up
novel_circ_0003520	457.2095	177.7826	1.3627	1.98E-31	5.23E-32	up
novel_circ_0003528	522.5251	177.7826	1.5554	9.16E-43	2.85E-43	up
novel_circ_0003530	326.5782	0	9.3513	6.42E-61	2.91E-61	up
novel_circ_0003535	326.5782	0	9.3513	6.42E-61	2.91E-61	up
novel_circ_0003591	979.7346	0	10.936	0	0	up
novel_circ_0003621	326.5782	118.5217	1.4623	3.32E-25	7.63E-26	up
novel_circ_0003630	457.2095	0	9.8367	3.80E-79	3.69E-79	up
novel_circ_0003686	522.5251	118.5217	2.1403	3.83E-64	2.40E-64	up
novel_circ_0003730	391.8938	0	9.6143	3.13E-70	2.21E-70	up
novel_circ_0003754	326.5782	0	9.3513	6.42E-61	2.91E-61	up
novel_circ_0003760	718.472	118.5217	2.5998	0	0	up
novel_circ_0003787	457.2095	177.7826	1.3627	1.98E-31	5.23E-32	up
novel_circ_0003829	522.5251	0	10.029	9.88E-88	1.52E-87	up
novel_circ_0003844	391.8938	0	9.6143	3.13E-70	2.21E-70	up
novel_circ_0003856	783.7876	296.3043	1.4034	7.08E-55	2.46E-55	up
novel_circ_0003868	522.5251	0	10.029	9.88E-88	1.52E-87	up
novel_circ_0004003	979.7346	474.0868	1.0472	1.63E-44	5.22E-45	up
novel_circ_0004015	783.7876	237.0434	1.7253	1.10E-72	1.01E-72	up
novel_circ_0004057	391.8938	0	9.6143	3.13E-70	2.21E-70	up
novel_circ_0004100	457.2095	118.5217	1.9477	2.70E-50	9.10E-51	up
novel_circ_0004120	326.5782	0	9.3513	6.42E-61	2.91E-61	up
novel_circ_0004238	1502.26	592.6085	1.342	4.26E-97	9.01E-97	up
novel_circ_0004333	326.5782	118.5217	1.4623	3.32E-25	7.63E-26	up
novel_circ_0004399	718.472	355.5651	1.0148	1.35E-31	3.64E-32	up
novel_circ_0004468	2547.31	1125.956	1.1778	0	0	up
novel_circ_0004527	391.8938	0	9.6143	3.13E-70	2.21E-70	up
novel_circ_0004605	1045.05	414.826	1.333	1.89E-67	1.26E-67	up
novel_circ_0004616	522.5251	0	10.029	9.88E-88	1.52E-87	up
novel_circ_0004652	1045.05	177.7826	2.5554	0	0	up
novel_circ_0004711	522.5251	237.0434	1.1403	1.00E-27	2.48E-28	up
novel_circ_0004761	391.8938	0	9.6143	3.13E-70	2.21E-70	up
novel_circ_0004762	391.8938	118.5217	1.7253	3.16E-37	9.25E-38	up
novel_circ_0004783	914.4189	296.3043	1.6258	4.70E-78	4.47E-78	up
novel_circ_0004796	1306.313	651.8694	1.0028	4.73E-55	1.66E-55	up
novel_circ_0004890	326.5782	118.5217	1.4623	3.32E-25	7.63E-26	up
novel_circ_0004913	587.8407	0	10.199	4.93E-96	9.65E-96	up
novel_circ_0004933	718.472	237.0434	1.5998	2.14E-60	9.66E-61	up
novel_circ_0004934	457.2095	177.7826	1.3627	1.98E-31	5.23E-32	up
novel_circ_0004961	326.5782	118.5217	1.4623	3.32E-25	7.63E-26	up
novel_circ_0005050	391.8938	177.7826	1.1403	3.37E-21	7.14E-22	up
novel_circ_0000010	0	414.826	-9.6964	4.73E-72	3.76E-72	down
novel_circ_0000032	0	296.3043	-9.2109	2.33E-55	8.23E-56	down
novel_circ_0000045	130.6313	296.3043	-1.1816	5.87E-15	1.05E-15	down
novel_circ_0000068	0	296.3043	-9.2109	2.33E-55	8.23E-56	down
novel_circ_0000070	0	533.3477	-10.059	1.54E-87	2.07E-87	down
novel_circ_0000077	0	296.3043	-9.2109	2.33E-55	8.23E-56	down
novel_circ_0000101	0	355.5651	-9.474	6.97E-64	3.54E-64	down
novel_circ_0000113	0	355.5651	-9.474	6.97E-64	3.54E-64	down
novel_circ_0000141	0	414.826	-9.6964	4.73E-72	3.76E-72	down
novel_circ_0000158	0	414.826	-9.6964	4.73E-72	3.76E-72	down
novel_circ_0000173	130.6313	948.1736	-2.8597	0	0	down
novel_circ_0000178	130.6313	474.0868	-1.8597	1.11E-44	3.61E-45	down
novel_circ_0000182	0	414.826	-9.6964	4.73E-72	3.76E-72	down
novel_circ_0000204	0	296.3043	-9.2109	2.33E-55	8.23E-56	down
novel_circ_0000210	130.6313	414.826	-1.667	8.02E-34	2.21E-34	down
novel_circ_0000224	261.2625	592.6085	-1.1816	2.42E-28	6.10E-29	down
novel_circ_0000245	326.5782	829.6519	-1.3451	7.94E-48	2.63E-48	down
novel_circ_0000282	0	296.3043	-9.2109	2.33E-55	8.23E-56	down
novel_circ_0000297	130.6313	296.3043	-1.1816	5.87E-15	1.05E-15	down
novel_circ_0000305	457.2095	1066.695	-1.2222	1.97E-52	6.79E-53	down
novel_circ_0000306	130.6313	592.6085	-2.1816	2.83E-68	1.96E-68	down
novel_circ_0000333	0	355.5651	-9.474	6.97E-64	3.54E-64	down
novel_circ_0000342	195.9469	533.3477	-1.4446	6.55E-35	1.87E-35	down
novel_circ_0000404	130.6313	414.826	-1.667	8.02E-34	2.21E-34	down
novel_circ_0000406	0	296.3043	-9.2109	2.33E-55	8.23E-56	down
novel_circ_0000408	0	355.5651	-9.474	6.97E-64	3.54E-64	down
novel_circ_0000414	522.5251	1066.695	-1.0296	1.22E-39	3.72E-40	down
novel_circ_0000428	261.2625	651.8694	-1.3191	7.92E-37	2.30E-37	down
novel_circ_0000434	0	533.3477	-10.059	1.54E-87	2.07E-87	down
novel_circ_0000442	0	296.3043	-9.2109	2.33E-55	8.23E-56	down
novel_circ_0000470	522.5251	1185.217	-1.1816	5.50E-55	1.92E-55	down
novel_circ_0000471	0	1837.086	-11.843	0	0	down
novel_circ_0000475	130.6313	533.3477	-2.0296	3.29E-56	1.42E-56	down
novel_circ_0000480	0	355.5651	-9.474	6.97E-64	3.54E-64	down
novel_circ_0000482	0	592.6085	-10.211	6.25E-95	1.17E-94	down
novel_circ_0000494	457.2095	1422.26	-1.6373	0	0	down
novel_circ_0000501	0	296.3043	-9.2109	2.33E-55	8.23E-56	down
novel_circ_0000507	0	355.5651	-9.474	6.97E-64	3.54E-64	down
novel_circ_0000525	0	296.3043	-9.2109	2.33E-55	8.23E-56	down
novel_circ_0000601	130.6313	296.3043	-1.1816	5.87E-15	1.05E-15	down
novel_circ_0000604	130.6313	355.5651	-1.4446	7.99E-24	1.76E-24	down
novel_circ_0000638	1045.05	2133.391	-1.0296	1.54E-77	1.45E-77	down
novel_circ_0000640	0	414.826	-9.6964	4.73E-72	3.76E-72	down
novel_circ_0000642	195.9469	474.0868	-1.2747	7.44E-26	1.78E-26	down
novel_circ_0000643	1306.313	2666.738	-1.0296	1.76E-96	3.66E-96	down
novel_circ_0000673	0	355.5651	-9.474	6.97E-64	3.54E-64	down
novel_circ_0000686	587.8407	1363	-1.2133	1.01E-65	6.50E-66	down
novel_circ_0000707	130.6313	651.8694	-2.3191	8.94E-81	1.08E-80	down
novel_circ_0000719	849.1033	2785.26	-1.7138	0	0	down
novel_circ_0000721	130.6313	296.3043	-1.1816	5.87E-15	1.05E-15	down
novel_circ_0000757	0	296.3043	-9.2109	2.33E-55	8.23E-56	down
novel_circ_0000781	0	533.3477	-10.059	1.54E-87	2.07E-87	down
novel_circ_0000812	0	296.3043	-9.2109	2.33E-55	8.23E-56	down
novel_circ_0000815	130.6313	355.5651	-1.4446	7.99E-24	1.76E-24	down
novel_circ_0000836	0	355.5651	-9.474	6.97E-64	3.54E-64	down
novel_circ_0000850	0	474.0868	-9.889	6.37E-80	6.85E-80	down
novel_circ_0000871	130.6313	414.826	-1.667	8.02E-34	2.21E-34	down
novel_circ_0000884	130.6313	296.3043	-1.1816	5.87E-15	1.05E-15	down
novel_circ_0000926	0	355.5651	-9.474	6.97E-64	3.54E-64	down
novel_circ_0000936	0	355.5651	-9.474	6.97E-64	3.54E-64	down
novel_circ_0000964	0	355.5651	-9.474	6.97E-64	3.54E-64	down
novel_circ_0000976	0	474.0868	-9.889	6.37E-80	6.85E-80	down
novel_circ_0000991	849.1033	2133.391	-1.3291	0	0	down
novel_circ_0001048	261.2625	829.6519	-1.667	6.75E-66	4.38E-66	down
novel_circ_0001049	457.2095	1363	-1.5759	1.37E-98	2.99E-98	down
novel_circ_0001082	0	474.0868	-9.889	6.37E-80	6.85E-80	down
novel_circ_0001089	0	296.3043	-9.2109	2.33E-55	8.23E-56	down
novel_circ_0001096	0	414.826	-9.6964	4.73E-72	3.76E-72	down
novel_circ_0001099	0	355.5651	-9.474	6.97E-64	3.54E-64	down
novel_circ_0001114	130.6313	296.3043	-1.1816	5.87E-15	1.05E-15	down
novel_circ_0001115	0	592.6085	-10.211	6.25E-95	1.17E-94	down
novel_circ_0001138	0	474.0868	-9.889	6.37E-80	6.85E-80	down
novel_circ_0001163	0	296.3043	-9.2109	2.33E-55	8.23E-56	down
novel_circ_0001170	0	533.3477	-10.059	1.54E-87	2.07E-87	down
novel_circ_0001183	0	414.826	-9.6964	4.73E-72	3.76E-72	down
novel_circ_0001250	261.2625	592.6085	-1.1816	2.42E-28	6.10E-29	down
novel_circ_0001260	326.5782	770.3911	-1.2382	3.85E-39	1.16E-39	down
novel_circ_0001274	0	296.3043	-9.2109	2.33E-55	8.23E-56	down
novel_circ_0001279	0	296.3043	-9.2109	2.33E-55	8.23E-56	down
novel_circ_0001280	0	355.5651	-9.474	6.97E-64	3.54E-64	down
novel_circ_0001283	195.9469	533.3477	-1.4446	6.55E-35	1.87E-35	down
novel_circ_0001290	0	355.5651	-9.474	6.97E-64	3.54E-64	down
novel_circ_0001310	261.2625	948.1736	-1.8597	1.47E-87	2.07E-87	down
novel_circ_0001312	326.5782	948.1736	-1.5377	1.11E-66	7.34E-67	down
novel_circ_0001316	326.5782	829.6519	-1.3451	7.94E-48	2.63E-48	down
novel_circ_0001345	0	1244.478	-11.281	0	0	down
novel_circ_0001348	261.2625	651.8694	-1.3191	7.92E-37	2.30E-37	down
novel_circ_0001367	0	355.5651	-9.474	6.97E-64	3.54E-64	down
novel_circ_0001384	0	355.5651	-9.474	6.97E-64	3.54E-64	down
novel_circ_0001390	0	474.0868	-9.889	6.37E-80	6.85E-80	down
novel_circ_0001412	0	296.3043	-9.2109	2.33E-55	8.23E-56	down
novel_circ_0001413	0	296.3043	-9.2109	2.33E-55	8.23E-56	down
novel_circ_0001429	0	414.826	-9.6964	4.73E-72	3.76E-72	down
novel_circ_0001443	0	296.3043	-9.2109	2.33E-55	8.23E-56	down
novel_circ_0001446	0	414.826	-9.6964	4.73E-72	3.76E-72	down
novel_circ_0001490	130.6313	296.3043	-1.1816	5.87E-15	1.05E-15	down
novel_circ_0001506	0	355.5651	-9.474	6.97E-64	3.54E-64	down
novel_circ_0001524	261.2625	770.3911	-1.5601	1.09E-55	4.70E-56	down
novel_circ_0001588	130.6313	296.3043	-1.1816	5.87E-15	1.05E-15	down
novel_circ_0001621	0	296.3043	-9.2109	2.33E-55	8.23E-56	down
novel_circ_0001626	0	296.3043	-9.2109	2.33E-55	8.23E-56	down
novel_circ_0001630	0	296.3043	-9.2109	2.33E-55	8.23E-56	down
novel_circ_0001643	130.6313	414.826	-1.667	8.02E-34	2.21E-34	down
novel_circ_0001653	130.6313	414.826	-1.667	8.02E-34	2.21E-34	down
novel_circ_0001675	0	296.3043	-9.2109	2.33E-55	8.23E-56	down
novel_circ_0001690	195.9469	414.826	-1.082	9.99E-18	1.94E-18	down
novel_circ_0001726	0	355.5651	-9.474	6.97E-64	3.54E-64	down
novel_circ_0001732	0	3200.086	-12.644	0	0	down
novel_circ_0001768	195.9469	533.3477	-1.4446	6.55E-35	1.87E-35	down
novel_circ_0001777	130.6313	355.5651	-1.4446	7.99E-24	1.76E-24	down
novel_circ_0001779	195.9469	474.0868	-1.2747	7.44E-26	1.78E-26	down
novel_circ_0001790	0	1422.26	-11.474	0	0	down
novel_circ_0001804	195.9469	592.6085	-1.5966	1.01E-44	3.30E-45	down
novel_circ_0001866	130.6313	474.0868	-1.8597	1.11E-44	3.61E-45	down
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novel_circ_0001902	195.9469	711.1302	-1.8597	3.96E-66	2.60E-66	down
novel_circ_0001904	326.5782	829.6519	-1.3451	7.94E-48	2.63E-48	down
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novel_circ_0001963	0	355.5651	-9.474	6.97E-64	3.54E-64	down
novel_circ_0001969	0	296.3043	-9.2109	2.33E-55	8.23E-56	down
novel_circ_0001972	0	296.3043	-9.2109	2.33E-55	8.23E-56	down
novel_circ_0001986	261.2625	592.6085	-1.1816	2.42E-28	6.10E-29	down
novel_circ_0002027	0	474.0868	-9.889	6.37E-80	6.85E-80	down
novel_circ_0002037	195.9469	414.826	-1.082	9.99E-18	1.94E-18	down
novel_circ_0002039	522.5251	1303.739	-1.3191	6.65E-72	5.25E-72	down
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novel_circ_0002085	130.6313	711.1302	-2.4446	1.24E-93	2.25E-93	down
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novel_circ_0002127	130.6313	296.3043	-1.1816	5.87E-15	1.05E-15	down
novel_circ_0002141	130.6313	296.3043	-1.1816	5.87E-15	1.05E-15	down
novel_circ_0002145	0	296.3043	-9.2109	2.33E-55	8.23E-56	down
novel_circ_0002155	0	296.3043	-9.2109	2.33E-55	8.23E-56	down
novel_circ_0002177	0	474.0868	-9.889	6.37E-80	6.85E-80	down
novel_circ_0002182	195.9469	414.826	-1.082	9.99E-18	1.94E-18	down
novel_circ_0002185	326.5782	711.1302	-1.1227	4.86E-31	1.27E-31	down
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novel_circ_0002385	0	414.826	-9.6964	4.73E-72	3.76E-72	down
novel_circ_0002418	261.2625	651.8694	-1.3191	7.92E-37	2.30E-37	down
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novel_circ_0002672	130.6313	296.3043	-1.1816	5.87E-15	1.05E-15	down
novel_circ_0002692	0	355.5651	-9.474	6.97E-64	3.54E-64	down
novel_circ_0002741	326.5782	770.3911	-1.2382	3.85E-39	1.16E-39	down
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novel_circ_0002772	195.9469	711.1302	-1.8597	3.96E-66	2.60E-66	down
novel_circ_0002778	0	355.5651	-9.474	6.97E-64	3.54E-64	down
novel_circ_0002781	195.9469	474.0868	-1.2747	7.44E-26	1.78E-26	down
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novel_circ_0002824	130.6313	414.826	-1.667	8.02E-34	2.21E-34	down
novel_circ_0002837	130.6313	296.3043	-1.1816	5.87E-15	1.05E-15	down
novel_circ_0002847	457.2095	1066.695	-1.2222	1.97E-52	6.79E-53	down
novel_circ_0002849	195.9469	533.3477	-1.4446	6.55E-35	1.87E-35	down
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novel_circ_0002928	0	355.5651	-9.474	6.97E-64	3.54E-64	down
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novel_circ_0002939	0	414.826	-9.6964	4.73E-72	3.76E-72	down
novel_circ_0002952	326.5782	770.3911	-1.2382	3.85E-39	1.16E-39	down
novel_circ_0003011	0	355.5651	-9.474	6.97E-64	3.54E-64	down
novel_circ_0003044	0	296.3043	-9.2109	2.33E-55	8.23E-56	down
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novel_circ_0003086	0	592.6085	-10.211	6.25E-95	1.17E-94	down
novel_circ_0003088	195.9469	474.0868	-1.2747	7.44E-26	1.78E-26	down
novel_circ_0003118	130.6313	296.3043	-1.1816	5.87E-15	1.05E-15	down
novel_circ_0003119	0	414.826	-9.6964	4.73E-72	3.76E-72	down
novel_circ_0003126	195.9469	414.826	-1.082	9.99E-18	1.94E-18	down
novel_circ_0003130	0	296.3043	-9.2109	2.33E-55	8.23E-56	down
novel_circ_0003138	0	296.3043	-9.2109	2.33E-55	8.23E-56	down
novel_circ_0003153	0	296.3043	-9.2109	2.33E-55	8.23E-56	down
novel_circ_0003160	130.6313	296.3043	-1.1816	5.87E-15	1.05E-15	down
novel_circ_0003173	0	355.5651	-9.474	6.97E-64	3.54E-64	down
novel_circ_0003201	0	296.3043	-9.2109	2.33E-55	8.23E-56	down
novel_circ_0003206	130.6313	296.3043	-1.1816	5.87E-15	1.05E-15	down
novel_circ_0003211	0	296.3043	-9.2109	2.33E-55	8.23E-56	down
novel_circ_0003214	0	355.5651	-9.474	6.97E-64	3.54E-64	down
novel_circ_0003229	0	296.3043	-9.2109	2.33E-55	8.23E-56	down
novel_circ_0003254	0	355.5651	-9.474	6.97E-64	3.54E-64	down
novel_circ_0003256	130.6313	296.3043	-1.1816	5.87E-15	1.05E-15	down
novel_circ_0003312	0	296.3043	-9.2109	2.33E-55	8.23E-56	down
novel_circ_0003316	0	296.3043	-9.2109	2.33E-55	8.23E-56	down
novel_circ_0003319	0	355.5651	-9.474	6.97E-64	3.54E-64	down
novel_circ_0003327	0	296.3043	-9.2109	2.33E-55	8.23E-56	down
novel_circ_0003342	130.6313	770.3911	-2.5601	0	0	down
novel_circ_0003355	195.9469	651.8694	-1.7341	3.67E-55	1.29E-55	down
novel_circ_0003364	0	296.3043	-9.2109	2.33E-55	8.23E-56	down
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novel_circ_0003438	130.6313	533.3477	-2.0296	3.29E-56	1.42E-56	down
novel_circ_0003441	0	296.3043	-9.2109	2.33E-55	8.23E-56	down
novel_circ_0003453	0	355.5651	-9.474	6.97E-64	3.54E-64	down
novel_circ_0003462	0	533.3477	-10.059	1.54E-87	2.07E-87	down
novel_circ_0003468	130.6313	355.5651	-1.4446	7.99E-24	1.76E-24	down
novel_circ_0003499	195.9469	414.826	-1.082	9.99E-18	1.94E-18	down
novel_circ_0003501	130.6313	651.8694	-2.3191	8.94E-81	1.08E-80	down
novel_circ_0003509	0	296.3043	-9.2109	2.33E-55	8.23E-56	down
novel_circ_0003532	261.2625	829.6519	-1.667	6.75E-66	4.38E-66	down
novel_circ_0003553	0	296.3043	-9.2109	2.33E-55	8.23E-56	down
novel_circ_0003563	1306.313	2963.043	-1.1816	0	0	down
novel_circ_0003564	0	296.3043	-9.2109	2.33E-55	8.23E-56	down
novel_circ_0003586	261.2625	533.3477	-1.0296	1.23E-20	2.56E-21	down
novel_circ_0003587	261.2625	533.3477	-1.0296	1.23E-20	2.56E-21	down
novel_circ_0003608	1240.997	3081.564	-1.3122	0	0	down
novel_circ_0003620	130.6313	533.3477	-2.0296	3.29E-56	1.42E-56	down
novel_circ_0003632	0	3081.564	-12.589	0	0	down
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novel_circ_0003649	0	296.3043	-9.2109	2.33E-55	8.23E-56	down
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novel_circ_0003661	195.9469	829.6519	-2.082	2.40E-89	4.32E-89	down
novel_circ_0003680	0	296.3043	-9.2109	2.33E-55	8.23E-56	down
novel_circ_0003695	130.6313	474.0868	-1.8597	1.11E-44	3.61E-45	down
novel_circ_0003702	0	296.3043	-9.2109	2.33E-55	8.23E-56	down
novel_circ_0003746	0	474.0868	-9.889	6.37E-80	6.85E-80	down
novel_circ_0003748	0	414.826	-9.6964	4.73E-72	3.76E-72	down
novel_circ_0003750	130.6313	355.5651	-1.4446	7.99E-24	1.76E-24	down
novel_circ_0003764	326.5782	1066.695	-1.7076	3.60E-87	4.72E-87	down
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novel_circ_0003806	261.2625	533.3477	-1.0296	1.23E-20	2.56E-21	down
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novel_circ_0003825	130.6313	355.5651	-1.4446	7.99E-24	1.76E-24	down
novel_circ_0003832	130.6313	296.3043	-1.1816	5.87E-15	1.05E-15	down
novel_circ_0003849	0	355.5651	-9.474	6.97E-64	3.54E-64	down
novel_circ_0003862	326.5782	888.9128	-1.4446	5.01E-57	2.21E-57	down
novel_circ_0003869	130.6313	414.826	-1.667	8.02E-34	2.21E-34	down
novel_circ_0003907	130.6313	296.3043	-1.1816	5.87E-15	1.05E-15	down
novel_circ_0003940	0	296.3043	-9.2109	2.33E-55	8.23E-56	down
novel_circ_0003947	130.6313	296.3043	-1.1816	5.87E-15	1.05E-15	down
novel_circ_0003952	0	533.3477	-10.059	1.54E-87	2.07E-87	down
novel_circ_0003970	130.6313	355.5651	-1.4446	7.99E-24	1.76E-24	down
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novel_circ_0004011	0	296.3043	-9.2109	2.33E-55	8.23E-56	down
novel_circ_0004013	130.6313	355.5651	-1.4446	7.99E-24	1.76E-24	down
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novel_circ_0004105	0	355.5651	-9.474	6.97E-64	3.54E-64	down
novel_circ_0004118	0	355.5651	-9.474	6.97E-64	3.54E-64	down
novel_circ_0004121	130.6313	296.3043	-1.1816	5.87E-15	1.05E-15	down
novel_circ_0004129	0	355.5651	-9.474	6.97E-64	3.54E-64	down
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novel_circ_0004193	326.5782	1066.695	-1.7076	3.60E-87	4.72E-87	down
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novel_circ_0004522	0	474.0868	-9.889	6.37E-80	6.85E-80	down
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novel_circ_0004935	130.6313	296.3043	-1.1816	5.87E-15	1.05E-15	down
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novel_circ_0004943	130.6313	355.5651	-1.4446	7.99E-24	1.76E-24	down
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novel_circ_0004969	0	296.3043	-9.2109	2.33E-55	8.23E-56	down
novel_circ_0004989	130.6313	533.3477	-2.0296	3.29E-56	1.42E-56	down
novel_circ_0004997	0	355.5651	-9.474	6.97E-64	3.54E-64	down
novel_circ_0005014	0	296.3043	-9.2109	2.33E-55	8.23E-56	down
novel_circ_0005016	391.8938	829.6519	-1.082	7.57E-34	2.14E-34	down
novel_circ_0005018	130.6313	651.8694	-2.3191	8.94E-81	1.08E-80	down
novel_circ_0005035	0	533.3477	-10.059	1.54E-87	2.07E-87	down
novel_circ_0005045	130.6313	296.3043	-1.1816	5.87E-15	1.05E-15	down
novel_circ_0005052	0	474.0868	-9.889	6.37E-80	6.85E-80	down

OGD/R,oxygen-glucose deprivation/recovery; Ctrl, control

Functional enrichment of the overlapping host genes for DE circRNAs and mRNAs

circRNAs are synthesized via backsplicing and are generated from mRNA precursors. Several circRNAs can also reg-ulate the expression of their host genes (Barrett et al., 2015). Therefore, characterizing the function of these mRNAs may enhance our understanding of the features of circRNAs. We analyzed the genes that colocalized on chromosomes with these DE circRNAs and found that the exonic and intronic circRNAs originated from 416 host genes (Figure 2B).

Figure 2.

Functional annotation clustering of differentially expressed (DE) circular RNAs (circRNAs) combined with DE mRNAs.

(A) Heatmaps show mRNAs with FC > 2 in the control (Ctrl) and oxygen glucose deprivation/recovery (OGD/R) groups. There were 448 upregulated and 439 downregulated mRNAs. (B) The intersection of DE mRNA host genes and DE circRNA host genes. A total of 36 genes were identified in the intersection. (C, D) The functional (C) and signaling (D) pathway enrichment analyses of these genes, respectively. The P value indicates significance for enrichment. P < 0.05 was considered significant. The RichFactor is calculated as the number of genes that fall into each category divided by the total number in that category. The size of the circle reflects the absolute number of genes that fall into each category. FC: Fold change.

Next, RNA sequencing was used to explore the mRNA profile of BMECs. After quality trimming of raw reads, 13G high-quality data remained. We mapped the clean reads to the Ensembl human genome database. The proportion of total reads in the OGD/R and control transcriptome libraries that mapped to the genome ranged from 92.9% to 93.2%. Cuffdiff calculated the FPKM of each transcript and the FC of DE mRNAs. We set the threshold as FC > 2.0 and P < 0.05 and identified 808 DE mRNAs, including 448 upregulated mRNAs and 439 downregulated mRNAs (Figure 2A and B).

To predict the potential function of the DE circRNAs, software was used to identify the host genes of the DE circRNAs and DE mRNAs. Figure 2B shows the intersection of host genes for DE circRNAs and DE mRNA. A total of 36 genes were identified to transcribe both DE mRNA and circRNA and were then subjected to functional and pathway enrichment analysis. In Figure 2C and D, the color of the bubble distinguishes the P value, and the size represents the number of genes that are enriched. More detailed information is shown in Additional Table 4. We identified calcium ion export, cellular calcium ion homeostasis and calcium ion transmembrane transport to be significantly enriched functional terms and the calcium signaling pathway as a top term in pathway enrichment analysis.

Additional Table 4.

The list of functional and signal pathway enrichment

Term	Genes
Functional enrichment list
GO:1901660~calcium ion export	Slc8a1, Atp2b1
GO:0010468~regulation of gene expression	ATPBS, Thoc2, Rc3h2, Bcl11b
GO:0006874~cellular calcium ion homeostasis	ATPBS, Slc8a1, Atp2b1
GO:0001666~response to hypoxia	ATPBS, Slc8a1, Kcnma1, Flt1
GO:0009791~post-embryonic development	Slc8a1, Rc3h2, Bcl11b
GO:0070588~calcium ion transmembrane transport	Slc8a1, Atp2b1, Cacna1d
GO:0007568~aging	Kcnma1, Atp2b1, Flt1, Cacna1d
GO:0030007~cellular potassium ion homeostasis	ATPBS, Kcnma1
GO:0045475~locomotor rhythm	Mapk10, Kcnma1
GO:0007165~signal transduction	Ptprm, Srgap2, Magi3, Bcl11b, Cap2
GO:0006883~cellular sodium ion homeostasis	ATPBS, Slc8a1
GO:0070509~calcium ion import	Slc8a1, Cacna1d
GO:0002028~regulation of sodium ion transport	Slc8a1, Wnk1
GO:0001782~B cell homeostasis	Sos2, Rc3h2
GO:0001824~blastocyst development	ATPBS, Thoc2
GO:0050769~positive regulation of neurogenesis	Spen, Man2a1
GO:0001569~patterning of blood vessels	Flt1, Sema5a
GO:0051924~regulation of calcium ion transport	Slc8a1, Cacna1d
GO:0006468~protein phosphorylation	Mapk10, Flt1, Nek4, Wnk1
GO:0045909~positive regulation of vasodilation	Ptprm, Kcnma1
GO:0007628~adult walking behavior	Kcnma1, Oxr1
GO:0060048~cardiac muscle contraction	ATPBS, Slc8a1
GO:0048286~lung alveolus development	Man2a1, Rc3h2
GO:0010923~negative regulation of phosphatase activity	Tmem132d, Wnk1
Pathway enrichment list
rno04022:cGMP-PKG signaling pathway	ATPBS, Ppp3cc, Slc8a1, Kcnma1, Atp2b1, Cacna1d
rno04020:Calcium signaling pathway	Ppp3cc, Slc8a1, Atp2b1, Cacna1d
rno04024:cAMP signaling pathway	ATPBS, Mapk10, Atp2b1, Cacna1d
rno04924:Renin secretion	Ppp3cc, Kcnma1, Cacna1d
rno04970:Salivary secretion	ATPBS, Kcnma1, Atp2b1
rno04911:Insulin secretion	ATPBS, Kcnma1, Cacna1d
rno04912:GnRH signaling pathway	Mapk10, Sos2, Cacna1d
rno04010:MAPK signaling pathway	Mapk10, Sos2, Ppp3cc, Cacna1d
rno04972:Pancreatic secretion	ATPBS, Kcnma1, Atp2b1
rno04360:Axon guidance	Srgap2, Ppp3cc, Sema5a
rno04728:Dopaminergic synapse	Mapk10, Ppp3cc, Cacna1d
rno04261:Adrenergic signaling in cardiomyocytes	ATPBS, Atp2b1, Cacna1d
rno04510:Focal adhesion	Mapk10, Sos2, Flt1
rno04973:Carbohydrate digestion and absorption	ATPBS, Cacna1d
rno04014:Ras signaling pathway	Mapk10, Sos2, Flt1

Validation of the DE circRNAs

Next, we continued to investigate the circRNAs transcribed from the overlapping host genes (Figure 2B). We adopted qRT-PCR to verify changes in the expression of circRNAs. According to the FC value, we selected the five most up-regulated or downregulated circRNAs for expression validation. As shown in Figure 3A and B, the quantification of circRNA expression was well correlated with the qRT-PCR results. Nine out of ten circRNA candidates identified in the samples were successfully amplified by qRT-PCR using the RevertAid First Strand cDNA Synthesis Kit and SYBR Green PCR Kits (Thermo Fisher). Additionally, the PCR results of eight circRNAs were consistent with sequencing, demonstrating the high reliability of the high-throughput RNA sequencing of circRNA.

Figure 3.

The microRNA (miRNA)-circular RNA (circRNA) interaction network map.

(A, B) The five most upregulated or downregulated circRNAs, the host genes of which were identified in both the differentially expressed (DE) mRNA and DE circRNA analyses, were selected for verification. One circRNA was not successfully amplified by quantitative reverse transcription-polymerase chain reaction (RT-PCR). The quantification of eight circRNAs was consistent with the RNA sequencing results. (C) The miRNA-circRNA interaction network map of these eight circRNAs created by bioinformatic analysis. ①novel_circ_0003760; ②novel_circ_0002086; ③novel_circ_0001606; ④;novel_circ_0002023; ⑤novel_circ_0000173; ⑥novel_circ_0004586; ⑦nov-el_circ_0003342; ⑧novel_circ_0004860. Red dots indicate miRNAs.

circRNA-miRNA network analysis

Recent studies have shown that some circRNAs act as miRNA sponges (Hansen et al., 2013). To determine the possible miRNA targets of OGD/R-induced DE circRNAs, Miranda software was employed to analyze the binding sites of DE circRNAs and miRNAs. Figure 3C shows the circRNA-miRNA interaction networks of the eight circRNAs verified by qRT-PCR. A detailed list of the predicted circRNA-miRNA interactions is provided in Additional Table 5. In cerebral ischemia, some miRNAs are sponged by circRNAs. For instance, circRNA DLGAP4 functions as an endogenous miR-143 sponge to inhibit miR-143 activity, resulting in the inhibition of endothelial-mesenchymal transition by regulating tight junction protein and mesenchymal cell marker expression (Bai et al., 2018). Our results also show that miR-143 may interact with novel_circ_0003342 (Figure 3C).

Additional Table 5.

The list of the predicted circRNA-miRNA interactions

miRNA	CircRNAs
rno-let-7g-3p	novel_circ_0004860
rno-miR-1188-5p	novel_circ_0003342
rno-miR-122-5p	novel_circ_0004860
rno-miR-124-3p	novel_circ_0004586
rno-miR-128-3p	novel_circ_0003342
rno-miR-129-1-3p	novel_circ_0001606
rno-miR-129-2-3p	novel_circ_0001606
rno-miR-1297	novel_circ_0004586
rno-miR-1306-5p	novel_circ_0001606
rno-miR-134-3p	novel_circ_0004860
rno-miR-1-3p	novel_circ_0004860
rno-miR-143-3p	novel_circ_0003342
rno-miR-143-5p	novel_circ_0004586
rno-miR-148a-5p	novel_circ_0003760
rno-miR-148b-5p	novel_circ_0003760
rno-miR-17-2-3p	novel_circ_0000173
rno-miR-17-2-3p	novel_circ_0002086
rno-miR-18a-5p	novel_circ_0004586
rno-miR-190a-3p	novel_circ_0004860
rno-miR-191a-3p	novel_circ_0000173
rno-miR-191a-3p	novel_circ_0002086
rno-miR-193b-5p	novel_circ_0004860
rno-miR-196a-5p	novel_circ_0003760
rno-miR-196b-3p	novel_circ_0000173
rno-miR-196b-5p	novel_circ_0003760
rno-miR-196c-5p	novel_circ_0003760
rno-miR-1b	novel_circ_0004860
rno-miR-201-5p	novel_circ_0000173
rno-miR-203a-5p	novel_circ_0004586
rno-miR-204-3p	novel_circ_0000173
rno-miR-206-3p	novel_circ_0004860
rno-miR-211-3p	novel_circ_0000173
rno-miR-211-3p	novel_circ_0004860
rno-miR-214-3p	novel_circ_0004586
rno-miR-216a-3p	novel_circ_0003342
rno-miR-218a-1-3p	novel_circ_0000173
rno-miR-23b-5p	novel_circ_0004586
rno-miR-25-3p	novel_circ_0003342
rno-miR-26a-3p	novel_circ_0003342
rno-miR-296-3p	novel_circ_0003760
rno-miR-29a-3p	novel_circ_0002023
rno-miR-29b-3p	novel_circ_0002023
rno-miR-29c-3p	novel_circ_0002023
rno-miR-3065-3p	novel_circ_0002023
rno-miR-3065-3p	novel_circ_0003342
rno-miR-3075	novel_circ_0000173
rno-miR-3084a-5p	novel_circ_0002023
rno-miR-3084b-5p	novel_circ_0002023
rno-miR-3084c-5p	novel_circ_0002023
rno-miR-30b-3p	novel_circ_0000173
rno-miR-30c-1-3p	novel_circ_0001606
rno-miR-30c-2-3p	novel_circ_0001606
rno-miR-323-5p	novel_circ_0004860
rno-miR-32-5p	novel_circ_0003342
rno-miR-327	novel_circ_0003760
rno-miR-336-5p	novel_circ_0000173
rno-miR-344a-5p	novel_circ_0002023
rno-miR-344b-3p	novel_circ_0000173
rno-miR-345-3p	novel_circ_0004860
rno-miR-349	novel_circ_0003760
rno-miR-34a-5p	novel_circ_0003342
rno-miR-34b-5p	novel_circ_0003342
rno-miR-34c-5p	novel_circ_0003342
rno-miR-3559-5p	novel_circ_0004586
rno-miR-3560	novel_circ_0000173
rno-miR-3561-3p	novel_circ_0004586
rno-miR-3564	novel_circ_0004860
rno-miR-3569	novel_circ_0002023
rno-miR-3576	novel_circ_0003760
rno-miR-3584-5p	novel_circ_0000173
rno-miR-3585-5p	novel_circ_0000173
rno-miR-3588	novel_circ_0000173
rno-miR-3590-5p	novel_circ_0001606
rno-miR-3591	novel_circ_0001606
rno-miR-361-3p	novel_circ_0000173
rno-miR-362-5p	novel_circ_0000173
rno-miR-363-3p	novel_circ_0003342
rno-miR-383-3p	novel_circ_0004586
rno-miR-410-5p	novel_circ_0003760
rno-miR-412-3p	novel_circ_0001606
rno-miR-448-5p	novel_circ_0003342
rno-miR-449a-5p	novel_circ_0003342
rno-miR-449c-5p	novel_circ_0003342
rno-miR-450a-3p	novel_circ_0000173
rno-miR-465-5p	novel_circ_0001606
rno-miR-466b-5p	novel_circ_0004860
rno-miR-484	novel_circ_0002023
rno-miR-494-5p	novel_circ_0003760
rno-miR-496-5p	novel_circ_0003760
rno-miR-500-5p	novel_circ_0000173
rno-miR-505-5p	novel_circ_0002023
rno-miR-509-5p	novel_circ_0002023
rno-miR-542-3p	novel_circ_0003342
rno-miR-544-3p	novel_circ_0000173
rno-miR-547-5p	novel_circ_0000173
rno-miR-6315	novel_circ_0004860
rno-miR-6316	novel_circ_0000173
rno-miR-6320	novel_circ_0000173
rno-miR-6329	novel_circ_0003342
rno-miR-6334	novel_circ_0002023
rno-miR-672-3p	novel_circ_0000173
rno-miR-674-5p	novel_circ_0004586
rno-miR-742-3p	novel_circ_0003342
rno-miR-761	novel_circ_0004586
rno-miR-880-3p	novel_circ_0002086
rno-miR-883-3p	novel_circ_0002086
rno-miR-92a-3p	novel_circ_0003342
rno-miR-92b-3p	novel_circ_0003342
rno-miR-935	novel_circ_0003342

Discussion

Lin et al. (2016) explored the circRNA profile of mouse hippocampal HT22 cells in an OGD/R model and identified several DE circRNAs that may be involved in apoptosis, metabolism and immunoreaction. A further study also identified 1027 DE circRNAs in a middle cerebral artery occlusion model (Liu et al., 2017a). However, none of the circRNAs that we identified in BMECs overlapped with those identified in neural cells or tissues in previous papers. In view of the specificity of circRNA expression, endothelial cells and neural cells likely have different expression profiles in hypoxic-ischemic conditions. These newly identified circRNAs might also contribute to the features and functions of BMECs.

We analyzed the intersection of DE circRNA and DE mRNA host genes. The results indicate the circRNAs are closely related to calcium ions. Calcium signaling plays an important role in the regulation of vascular endothelial cell function. For example, increased calcium binds to calmodulin and interacts with related proteins to release vasodilators, such as nitric oxide and prostacyclin (Yamamoto et al., 2000). Differences in the amplitude and duration of intracellular calcium oscillations contribute to the differential activation of various transcription factors, leading to regulated gene expression (Chen et al., 2019). This is consistent with the high score for “regulation of gene expression” in our results. Additionally, “response to hypoxia” had a high enrichment score. Therefore, we suggest that circRNAs transcribed from the 36 genes identified may play important roles in calcium ion regulation during cerebral ischemia-reperfusion injury.

Surprisingly, the most significant term in the pathway results was cyclic guanosine 3′,5′-monophosphate (cGMP)-cGMP-dependent protein kinase (PKG) signaling. The first mechanism proposed for cGMP-dependent relaxation of smooth muscle was the reduction of free intracellular cytosolic calcium concentration (Johnson and Lincoln, 1985). Several sites of action have been proposed to account for cGMP-dependent regulation of cytosolic calcium, and these have been reviewed (Lincoln et al., 2001). Nitric oxide-cGMP signaling was recognized by the 1998 Nobel Prize in Physiology and Medicine (Arnold et al., 1977). Nitric oxide diffuses across vascular smooth muscle cell membranes and activates the enzyme-soluble guanylate cyclase, which catalyzes the conversion of guanosine tri-phosphate into cGMP (Denninger and Marletta, 1999). cGMP activates PKG, which promotes multiple phosphorylation of targets, lowering cellular calcium concentrations and promoting vascular relaxation (Surks et al., 1999). The cGMP-PKG pathway also decreases calcium release, which inhibits caspase-3 activation and apoptosis. Therefore, identification of this term in our pathway enrichment analysis is consistent with the identification of calcium ion terms in the functional enrichment analysis.

circRNA was first identified in 1976 by Sanger et al. (1976). circRNAs are not susceptible to degradation by RNA exonucleases because of their covalently closed circular structure that lacks accessible ends (Altesha et al., 2019). These characteristics give circRNAs significantly longer half-lives than linear RNAs. BMEC-released circRNAs can be easily detected in plasma; for example circHectd1 is significantly increased in the plasma of model stroke mice (Han et al., 2018). circRNAs are, therefore, potential candidates for diagnostic and prognostic biomarkers of disease.

miRNAs negatively regulate gene expression by partial base pairing with the untranslated region of its target mRNA. But interaction of the miRNA seed region with the mRNA is not unidirectional. Transcribed pseudogenes, long noncoding RNAs and circRNAs compete for the same pool of miRNAs, thereby regulating miRNA activity. This phenomenon of regulating other RNA transcripts by competing for shared miRNAs is performed by RNAs termed competing endogenous RNAs. The binding and holding of miRNAs by circRNAs has been termed the “sponging effect”, which results in the increased expression of miRNA target mRNAs (Altesha et al., 2019). In the present study, we obtained several circRNA-miRNA networks by bioinformatics. Some of the miRNAs have been reported in cerebral ischemia reperfusion injury. For example, miR-26a can promote endothelial lumen formation and cell proliferation in BMECs via the phosphatidylinositol 3′-kinase/Akt and mitogen-activated protein kinase/extracellular signal-regulated kinase pathway (Liang et al., 2018). In addition, overexpression of miR-544 ameliorated the inflammation and apoptotic responses in brain tissue after ischemia reperfusion by down-regulating the expression of interleukin-1 receptor-associated kinase 4 (Fang et al., 2018). Moreover, miR-124, one the most abundant miRNAs in the central nervous system, is a potential partner of novel_circ_0004586. miR-124 plays multiple functions in brain ischemia, such as in inflammation, glycolysis and cell damage (Zhu et al., 2014; Hamzei Taj et al., 2016; Caruso et al., 2017). Future research should explore the regulatory mechanisms of circRNAs and functional miRNAs.

The most significant result of the present study is the association of circRNAs with calcium ion-related pathways. Calcium overload is well known to be induced by brain ischemia and oxygen and glucose deprivation and to induce dysfunctional adenosine triphosphate and cell damage. Targeting calcium ion-related proteins, such as sodium-calcium exchanger, voltage-sensitive calcium channels, transient receptor potential channels, and N-methyl-D-aspartate recep-tors has been confirmed to be an effective treatment (Khananshvili, 2013; Kumar et al., 2014). cGMP and PKGs are widely involved in the physiological processes of the vascular system. This pathway stimulates endothelial cell prolif-eration and inhibits vascular smooth muscle cell proliferation. Dysfunction of the cGMP-PKG signaling pathway at any step of the cascade has been implicated in numerous vascular diseases, ranging from cerebral ischemia to atherosclerosis and angiogenesis (Zhang et al., 2003; Tsai and Kass, 2009). In future studies we will, therefore, explore the mechanism between circRNAs and calcium ion-related pathways.

In the present study, we used rat BMECs to detect DE circRNAs and mRNAs after OGD/R injury. Bioinformatics predicted the functions of circRNAs and indicated downstream pathways. We believe this will be benefit treatment strategies for cerebral ischemia reperfusion injury. However, one contentious question is whether animal samples and in vitro experimental results can be used to predict responses in human. circRNAs are poorly conserved among species (Chen and Yang, 2015) and all biological functions are determined by the genes of the individual. Every species has a unique genetic code for the biological activities associated with that species (Shanks et al., 2009). Currently, nine out of ten experimental drugs fail in clinical studies because we cannot accurately predict how they will behave in people based on laboratory and animal studies (Shanks et al., 2009). Therefore, we will explore the circRNAs from the present study that are homologous with human orthologs.

In summary, a number of previously unrecorded circRNAs have been revealed to be differentially expressed in primary BMECs after OGD/R treatment. Furthermore, calcium ion and cGMP-PKG signaling pathways may be important regulatory targets of circRNAs. Altered circRNAs may be important in the pathogenesis of cerebral ische-mia-reperfusion injury and, consequently, may be potential therapeutic targets for cerebral ischemia diseases.

Additional files:

Additional Table 1: The list of CircRNAs primers.

Additional Table 2 ^{(2MB, pdf)} : Identified 1288 circRNAs by RNA sequencing.

Additional Table 2

Identified 1288 circRNAs by RNA sequencing

Additional Table 3: The list of differentially expressed circRNAs.

Additional Table 4: The list of functional and signal pathway enrichment.

Additional Table 5: The list of the predicted circRNA-miRNA interactions.