Laminar blood flow has an anti-inflammatory effect on endothelial cells (ECs) and prevents local atherosclerotic lesion formation. Long non-coding RNAs (lncRNAs) have been described to play a role in many biological processes, including epigenetic regulation of gene expression. We previously showed that the lncRNA MALAT1 is highly expressed in ECs and plays a role in angiogenesis. Since we showed that MALAT1 is induced by anti-inflammatory laminar shear stress (3.4+0.6 fold), we assessed whether MALAT1 regulates the inflammatory response of ECs. Therefore, we used several siRNAs and LNA GapmeRs to specifically silence MALAT1. We noticed that targeting different regions of MALAT1 showed opposite effects on TNFα-induced inflammatory response. Whereas 3 different siRNAs and GapmeRs directed against the 5’end enhanced the TNFα-induced expression of E-selectin, VCAM1 and ICAM1, a GapmeR directed against the 3’end reduced the inflammatory response of ECs (Fig.1). To determine whether these effects are due to targeting of different transcript variants, we designed different PCR primers and identified a novel 1.3 kb 5’ variant of MALAT1, which was confirmed by deep sequencing. Interestingly, the two transcript variants are inversely regulated in human atherosclerotic plaques. Whereas the anti-inflammatory 1.3 kb 5’ variant was down-regulated in advanced plaques in AHA classification type 3 vs. 4 (42%+9%), the long pro-inflammatory variant was increased (158+28%). We further characterized MALAT1 variants and showed that both are transcribed by RNA Polymerase II and are nuclear localized. RNA-IP experiments showed that the short MALAT1 variant binds to Histone H3 whereas the long MALAT1 predominantly binds to H3K27me3 suggesting that the variants exhibit different epigenetic effects. In summary, our data identify for the first time a new 5′ variant of MALAT1, which reduced EC activation by TNFα, whereas the long MALAT1 augments the inflammatory activation of ECs.
Circular RNAs (circRNAs) are non-coding RNAs generated by back-splicing. Back-splicing has been considered as a rare event, but circRNAs were recently found to be abundantly expressed among a variety of human cells and tissues. Nevertheless, the expressional regulation, processing and biological functions of circRNAs are largely unknown. Cytoplasmic circRNAs can bind and trap microRNAs, whereas nuclear circRNAs may affect host gene expression. However, the expression, regulation and functions of circRNAs in endothelial cells have not been determined so far. In this study, basal expression and regulation of circRNAs by hypoxia in human umbilical endothelial cells (HUVEC) were analyzed using deep sequencing. Among the identified 7,388 circRNAs, 2,875 had not been annotated before. We further validated the expression of 40 selected circRNAs by RT-PCR and found that the majority is resistant to RNase R digestion, lacks polyadenylation and is localized to the cytoplasm. Cloning and subsequent sequencing validated the newly generated back splice sites for selected circRNAs. Furthermore, analysis of RNA-seq data revealed that circRNAs, particularly the cytoplasmatic circular RNA cZNF292, are significantly regulated by hypoxia in HUVECs. The siRNA-mediated knockdown of HIF-1α had no effect on cZNF292 induction under hypoxia, suggesting a HIF-1α independent regulation. Most importantly, siRNA-mediated knockdown of cZNF292 significantly reduced spheroid sprouting and network formation of endothelial cells. Furthermore, knockdown of cZNF292 had no effect on its host gene expression. Exon array analysis after cZNF292 knockdown revealed a significant expressional upregulation of 167 as well as a significant expressional downregulation of 123 genes of which most were associated with metabolic processes according to GO annotation. Analysis of Ago-HITS-CLIP data revealed no putative miR-binding sites, suggesting that cZNF292 does not act as a miR-sponge. Taken together, we show for the first time the expression, regulation and function of circRNAs in endothelial cells. The circRNA cZNF292 is regulated by hypoxia and has an important angiogenic function in endothelial cells.
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Introduction: Adenosine-to-Inosine (A-to-I) RNA editing is a post-transcriptional modification process regulating RNA stability and alternative splicing. A-to-I RNA editing is conducted by the enzymes ADAR1 and ADAR2 and mainly targets Alu elements, primate-specific elements which have been associated with the formation of circular RNA (circRNA). Although differential expression of circRNAs has been studied in heart failure (HF), the extent of A-to-I RNA editing and consequences in the human heart remain largely unknown. Methods and Results: We analyzed RNA editing in human heart samples of HF (n=20) patients and controls (n=10) using RNA sequencing. We found a reduction of A-to-I RNA editing in intronic Alu elements of protein-coding genes in HF patients compared to controls. The majority (96%) of regulated circRNAs were upregulated. The predicted back-splice sites (BSS) of 20 circRNAs were validated by qPCR. The circRNA candidates correlated with RNA editing (R=0.47, P=0.02). Among the upregulated circRNAs, we identified two circular transcripts (circAKAP13) derived from the AKAP13 gene, which showed reduced A-to-I RNA editing in HF (-70.7%, n=20). In HF, ADAR2 was reduced (-68.2%) and ADAR1 was increased (7.41±0.13 -fold) on protein level (n=3-6). The knockdown of ADAR1 did not alter circRNA levels, whereas the knockdown of ADAR2 led to significantly upregulated levels of circAKAP13 (1.88±0.42 -fold, n=6). Consistently, ADAR2 overexpression reduced circAKAP13 expression (-41%, n=3). Using two mini-genes containing exons 15-19 of the AKAP13 gene and flanking Alu elements, we found convergent Alu elements enhancing circAKAP13 expression. Conclusion: In conclusion, these data describe the A-to-I RNA editome in the human heart for the first time. Reduced A-to-I RNA editing in HF patients is associated with elevated circRNA levels. We propose a primate-specific splicing mechanism mediated by A-to-I RNA editing in the human heart. These findings contribute to a better mechanistic understanding of A-to-I RNA editing in cardiac diseases.
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