Identification and Characterization of Hypoxia-Regulated Endothelial Circular RNANovelty and Significance
Rationale: Circular RNAs (circRNAs) are noncoding RNAs generated by back splicing. Back splicing has been considered a rare event, but recent studies suggest that circRNAs are widely expressed. However, the expression, regulation, and function of circRNAs in vascular cells is still unknown.
Objective: Here, we characterize the expression, regulation, and function of circRNAs in endothelial cells.
Methods and Results: Endothelial circRNAs were identified by computational analysis of ribo-minus RNA generated from human umbilical venous endothelial cells cultured under normoxic or hypoxic conditions. Selected circRNAs were biochemically characterized, and we found that the majority of them lacks polyadenylation, is resistant to RNase R digestion and localized to the cytoplasm. We further validated the hypoxia-induced circRNAs cZNF292, cAFF1, and cDENND4C, as well as the downregulated cTHSD1 by reverse transcription polymerase chain reaction in cultured endothelial cells. Cloning of cZNF292 validated the predicted back splicing of exon 4 to a new alternative exon 1A. Silencing of cZNF292 inhibited cZNF292 expression and reduced tube formation and spheroid sprouting of endothelial cells in vitro. The expression of pre-mRNA or mRNA of the host gene was not affected by silencing of cZNF292. No validated microRNA-binding sites for cZNF292 were detected in Argonaute high-throughput sequencing of RNA isolated by cross-linking and immunoprecipitation data sets, suggesting that cZNF292 does not act as a microRNA sponge.
Conclusions: We show that the majority of the selected endothelial circRNAs fulfill all criteria of bona fide circRNAs. The circRNA cZNF292 exhibits proangiogenic activities in vitro. These data suggest that endothelial circRNAs are regulated by hypoxia and have biological functions.
Next generation sequencing revealed that the human genome comprises ≈20 000 protein-coding genes, which is much less compared with more primitive organisms, such as plants. However, >70% of the human genome is transcribed giving rise to several classes of noncoding RNAs, which have been shown to control tissue homeostasis and pathophysiological processes.1,2 Alternative RNA splicing represents an additional way to increase the complexity of the transcriptome by modulating the sequential arrangement of exons; in which, particular exons of a gene may be included or excluded from the processed messenger RNA. Thereby, the functional properties of a given coding or noncoding gene might be altered. In the case of coding genes, this leads to the generation of many protein isoforms with different biological properties.3 In addition to linear splicing reactions, the sequence of a primary transcript can be processed by back-splicing reactions leading to the formation of circular RNAs (circRNAs). CircRNAs can be generated from back-spliced exons3–5 or intron-derived RNA.6 Back-splicing has been considered a rare event, although mammalian circRNAs were reported already in the 1990s.7 Recently, computational analysis of deep-sequencing data provided evidence that the number of expressed circRNAs is higher than initially thought, and thousands of circRNAs were identified in different cells and tissues. For example, 1950 circRNAs were detected in HEK293 cells and human leukocytes,4 whereas >25,000 circular transcripts were reported in fibroblasts,3 and meanwhile 7122 human circRNAs were annotated using data sets generated by the ENCODE project.8 circRNAs are also formed from the well-studied transcript ANRIL, a noncoding transcript of the 9q21 locus that is associated with the risk for atherosclerosis.9 However, little is known about the expressional regulation and the biological function of circRNAs. Recent studies suggest that cytoplasmic circRNAs may act by binding and trapping microRNAs,4,10 whereas nuclear circRNAs may regulate expression and splicing of host genes.6 Here, we characterized the expression and hypoxia-induced regulation of circRNAs in endothelial cells and showed that the circRNA cZNF292 is regulated by hypoxia in endothelial cells and controls angiogenic sprouting.
Endothelial cells were purchased from Lonza and cultured as previously described11 under hypoxic (0.2% O2) or normoxic conditions. RNA was isolated using Qiazol and miRNeasy Kits, including additional DNase I digestion. For next generation sequencing, 0.5-μg ribosomal RNA-depleted RNA was fragmented and primed. The sequencing libraries were constructed using Illumina TruSeq RNA Sample Preparation Kits and were sequenced by Illumina HiSeq 2000 flowcell. RNA-seq data were analyzed using circBase.4,12 To annotate the identified circRNAs, HOMER was used.
CircRNAs were validated by semiquantitative reverse transcription polymerase chain reaction (RT-PCR) using the KOD Xtreme HotStart Polymerase Kit. For RNase R digestion, 6-µg RNA was incubated with 6 U RNase R in 1× RNase R buffer in a 20-µL reaction volume at 37°C for 10 minutes followed by heat inactivation at 95°C for 3 minutes.
Angiogenesis and proliferation assays were performed after transfection with GeneTransII or RNAiMAX as described.11 An extended method section is available in the Online Data Supplement.
Identification and Characterization of Endothelial circRNAs
Endothelial circRNAs were identified in ribosomal RNA-depleted RNA of human umbilical vein endothelial cells (HUVEC) by using PYTHON scripts provided by circBase.12 Bowtie2,13 a short read aligner which is used to align reads from a deep-sequencing experiment to a reference genome was used to select and analyze splice junction reads followed by filtering out low-quality candidates (Figure 1A). The highest expressed transcripts are shown in Figure 1B. Among the 7388 identified circRNAs of our study, we identified several new circRNAs, which are not deposited in circBase (eg, cSIRT1, cFN1, and cTGFB1; Online Table I). The majority of the circRNAs (95.6%) derive from the gene body, whereas 1.6% are of intergenic origin and 0.5% derive from noncoding sequences (Online Table II). Interestingly, circRNA expression did not correlate with the expression of the host genes suggesting an independent regulation of transcription versus circRNA formation (Figure 1C).
On the basis of basal expression levels estimated using RNA-seq data and the number of reads covering the back-splice sites, we selected 8 circRNAs for further characterization. Reads of 5 selected circRNAs were derived from exon–exon back-splicing events, whereas 2 circRNAs showed back-splicing of exons to intronic sequences or intron–intron head to tail joining (Online Table III). To confirm the expression of these circular transcripts, we performed semiquantitative reverse transcription PCR (RT-PCR) experiments using primers encompassing the circRNA-specific back-splice sites (Online Figure I) and showed that all 8 selected candidates are expressed in HUVEC (Figure 2A) as well as in microvascular and aortic ECs (Online Figure II). Next, we determined whether the selected transcripts are indeed circRNAs by testing their enrichment in poly-A–negative fractions (as circRNAs are expected to have no poly-A tail8) and resistance to 3′-5′ exoribonuclease RNase R digestion (as circRNAs are expected to be resistant to exonuclease RNase R14). We demonstrate that 7 of 8 circRNAs are predominantly detected in the poly-A–negative fraction (Figure 2B and 2C), whereas the protein-coding mRNA for endothelial NO synthase was enriched in the poly-A–positive fraction (Figure 2B and 2C). Only cMED13L was also detected in the poly-A-positive fraction (Figure 2B). Furthermore, we show that all 8 transcripts are resistant to RNase R, whereas linear vascular endothelial growth factor-A mRNA is degraded (Figure 2D and 2E). Notably, all circRNAs except cMED13L were enriched in the cytoplasmic fraction, whereas the nuclear long noncoding RNA MALAT1 is detected in the nuclear fraction (Figure 2F). In summary, 7 of the 8 selected circRNAs fulfilled all criteria of bona fide cytoplasmic circRNAs.
Identification of Hypoxia-Regulated circRNAs
Because hypoxia is a key stimulus for angiogenesis, we next exposed HUVECs to hypoxia and determined the expression of circRNAs. Interestingly, hypoxia induced a significant regulation of circRNAs as depicted by the heat map (Figure 3A) and the bar graph of selected circRNAs (Figure 3B). The regulation of selected circRNAs by hypoxia and the hypoxia mimic CoCl2 was subsequently validated by reverse transcription PCR (Figure 3C and 3D; Online Figure IIIA). In this context, cZNF292, cAFF1, and cDENND4C are upregulated, whereas cTHSD1 is reduced by hypoxia. cSRSF4 is only upregulated by 1.2-fold (Figure 3C and 3D). PCR against cFOXJ3 yielded multiple bands excluding its analysis. With the exception of cAFF1, the regulation of circRNAs by hypoxia was even more pronounced when cells were cultured under dense conditions (Online Figure IIIB). To determine whether the observed regulation of circRNAs is because of transcriptional regulation of host genes, host gene expression was analyzed from RNA-seq data (Figure 3E) and validated by quantitative reverse transcription PCR (Figure 3F). A correlation of host gene expression and circRNA formation under hypoxia was observed for cZNF292, cDENNDA4C, and cTHSD1, whereas only the upregulation of cAFF1 was found to be independent of hypoxia-induced transcriptional changes (Figure 3E and 3F).
cZNF292 Regulates Angiogenic Sprouting
With respect to circRNAs, one of the key questions is whether these transcripts have biological functions or are mere byproducts of mRNA processing. To address this question, we focused on cZNF292, which was among the highest expressed and significantly hypoxia-regulated circRNAs, and confirmed its expression by Northern blot (Online Figure IV). Using RNase R-treated RNA, we cloned cZNF292 by using primers directly flanking the predicted back-splice site and confirmed the circularization of exon 4 to intron 1 (Figure 4A; Online Figure V). A detailed analysis uncovered an alternative exon 1A located in intron 1 of which the 5′ end functions as splice acceptor site during back-splicing. Next, we silenced cZNF292 by using 2 siRNAs directed against exon 1A (Figure 4B) since the sequence of the back-splice site was not suitable for the generation of specific siRNAs (because of potential off target effects and high GC [guanine and cytosine base pairs] content). We confirmed that both siRNAs silenced cZNF292 but did not affect pre-mRNA levels or ZNF292 protein expression (Online Figure VI). siRNA-mediated silencing of cZNF292 significantly inhibited spheroid sprouting (Figure 4C), tube formation in matrigel assays (Online Figure VII), and reduced endothelial cell proliferation (Figure 4D). Transfection of a plasmid that was designed to generate the circular transcript of ZNF292 enhanced proliferation, whereas overexpression of linear exon 1A to 4 did not affect cell-cycle progression (Online Figure VIII).
Having demonstrated that cZNF292 regulates angiogenic sprouting, we aimed to obtain insights into its mechanism. Although nuclear circRNAs may interfere with the generation of their host gene,6 siRNA-mediated silencing of cZNF292 did not affect ZNF292 mRNA expression (Figure 4E). Because circRNAs were shown to act as sponges for microRNAs (miRNAs), we determined whether this mechanism may also account for cZNF292. By merging our identified circRNAs with the published high-throughput sequencing of RNA isolated by cross-linking and immunoprecipitation (HITS-CLIP) data from HUVEC15 (experimentally determined binding sites of Ago proteins), we bioinformatically characterized putative miRNA-binding sites within endothelial circRNAs (Figure 4F; Online Table IV). However, no HITS-CLIP peaks were detected for cZNF292 indicating that cZNF292 does not associate with Argonaute, making a potential function as miRNA sponge unlikely.
This study confirms that circRNAs are abundantly expressed in endothelial cells. Many of the identified circRNAs have already been described and annotated by others.3–5,10 Among the highest expressed circRNAs, cEphB4 was recently identified and characterized in other cell types.16 Besides identifying back-splicing events based on the usage of constitutive splice sites, we additionally demonstrate the usage of alternative 3′ splice sites (eg, cZNF292). Furthermore, we characterized hypoxia-controlled endothelial circRNAs and showed that one of the highly expressed and significantly regulated circRNAs, cZNF292, exhibits biological functions in endothelial cells. Selective depletion of cZNF292 by 2 different siRNAs inhibits angiogenic sprouting of endothelial cells, suggesting that cZNF292 exhibits a proangiogenic function. The proproliferative effect of cZNF292 was further confirmed by overexpression of cZNF292.
Little is known about the function of the zinc finger domain-containing transcription factor ZNF292 in the cardiovascular system: ZNF292 is expressed at a biologically meaningful level (fragments per kilobase of transcript per million mapped reads ≈10) in endothelial cells, but its regulation has only been described in hematopoietic cells.17 Furthermore, the mechanism by which cZNF292 controls angiogenic functions is unclear. Our results argue against a recently described cis-regulatory function of circular intronic transcripts on host gene expression6 because siRNA-mediated silencing of cZNF292 did not affect expression of ZNF292 mRNA. The lack of cis-regulatory activity is also consistent with a predominant cytoplasmic localization of cZNF292 raising the possibility that cZNF292 may act as miRNA sponge rather than regulating host gene expression. However, there was no evidence for Argonaute binding to cZNF292 from HITS-CLIP data, suggesting that a putative function as an miRNA sponge, at least under unstimulated conditions, is unlikely. Moreover, for the top 30 expressed circRNAs, predicted miRNA-binding sites were only verified in Argonaute HITS-CLIP data for cHIPK3 and cCRIM1 (Online Tables V and VI) suggesting that most endothelial-enriched circRNAs do not act as miRNA sponges. A recent study demonstrated that circular viroid RNAs can exhibit coding potential.18 The presence of internal ribosomal entry sites might allow formation of peptides or proteins from circular RNA. Thus, in vitro translation studies are necessary to experimentally address this potential function of cZNF292.
The mechanism by which circularization is affected by hypoxia also deserves further exploration: whereas under basal conditions levels of circRNAs and host gene expression did not significantly correlate (Figure 1C), hypoxia-induced changes of host gene levels were found to be reflected in most of the selected circRNAs. In the case of cZNF292, we demonstrate that hypoxia-induced upregulation is not mediated by the hypoxia-induced transcription factor 1a (Online Figure IX), but further studies are required to elucidate whether this is true for other circRNA transcripts. Because no correlation between circRNA and mRNA expression was observed when all transcripts were included into the analysis (Online Figure X), hypoxia may additionally regulate back-splicing and thereby generation of circRNAs. In this context, it is known that hypoxia regulates kinases that interfere with splicing19 and additionally modulates the expression and activity of JmjC domain-containing proteins, which interact with splicing factors.11
We thank Andrea Knau, Tino Roexe, Katrin Wetekam, and Nicole Konecny for expert technical support. The study is supported by the Deutsche Forschungsgemeinschaft (SFB834 and Exc115), the German Center for Cardiovascular Research (DZHK), the European Research Council, and the Leducq Foundation Network MIRVAD.
J.-N Boeckel, N. Jaé, S. Dimmeler, and A.M. Zeiher applied for a patent.
In August 2015, the average time from submission to first decision for all original research papers submitted to Circulation Research was 14.31 days.
This manuscript was sent to Evangelos D. Michelakis, Consulting Editor, for review by expert referees, editorial decision, and final disposition.
The online-only Data Supplement is available with this article at http://circres.ahajournals.org/lookup/suppl/doi:10.1161/CIRCRESAHA.115.306319/-/DC1.
- Nonstandard Abbreviations and Acronyms
- circular RNA
- polymerase chain reaction
- Received February 24, 2015.
- Revision received September 15, 2015.
- Accepted September 16, 2015.
- © 2015 American Heart Association, Inc.
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Novelty and Significance
What Is Known?
Over 70% of the human genome is transcribed, but merely 3% is translated into proteins.
Several classes of noncoding RNAs have been shown to be involved in the regulation of physiological and pathophysiological processes.
Circular RNAs are widely expressed among cells and tissues and are mainly generated from back-spliced exons of protein coding genes.
What New Information Does This Article Contribute?
Circular RNAs are expressed in endothelial cells and are regulated by hypoxia.
The circular RNA cZNF292 controls proliferation and angiogenic sprouting in endothelial cells.
Next generation sequencing revealed >70% of the human genome is transcribed giving rise to several classes of noncoding RNAs. Of those, circular RNAs are generated from back-spliced exons or introns mainly of protein-coding genes. In our study, we found 7388 circular RNAs expressed in endothelial cells and biochemically validated 7 candidates. Several circRNAs were significantly regulated by hypoxia, which is a key stimulus for angiogenesis. Circular ZNF292 was found to be regulated by hypoxia and involved in the regulation of proliferation and angiogenic sprouting in endothelial cells.