RESUMO
Impaired oxygen homeostasis is a frequently encountered pathophysiological factor in multiple complex diseases, including cardiovascular disease and cancer. While the canonical hypoxia response pathway is well characterized, less is known about the role of noncoding RNAs in this process. Here, we investigated the nascent and steady-state noncoding transcriptional responses in endothelial cells and their potential roles in regulating the hypoxic response. Notably, we identify a novel antisense long noncoding RNA that convergently overlaps the majority of the hypoxia inducible factor 1 alpha (HIF1A) locus, which is expressed across several cell types and elevated in atherosclerotic lesions. The antisense (HIF1A-AS) is produced as a stable, unspliced, and polyadenylated nuclear retained transcript. HIF1A-AS is highly induced in hypoxia by both HIF1A and HIF2A and exhibits anticorrelation with the coding HIF1A transcript and protein expression. We further characterized this functional relationship by CRISPR-mediated bimodal perturbation of the HIF1A-AS promoter. We provide evidence that HIF1A-AS represses the expression of HIF1a in cis by repressing transcriptional elongation and deposition of H3K4me3, and that this mechanism is dependent on the act of antisense transcription itself. Overall, our results indicate a critical regulatory role of antisense mediated transcription in regulation of HIF1A expression and cellular response to hypoxia.
Assuntos
Células Endoteliais , RNA Longo não Codificante , Humanos , Hipóxia Celular , Células Endoteliais/metabolismo , Hipóxia/genética , Fator 1 Induzível por Hipóxia/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Regiões Promotoras Genéticas , RNA Longo não Codificante/genética , Células CultivadasRESUMO
[Figure: see text].
Assuntos
Aorta/metabolismo , Doenças da Aorta/genética , Aterosclerose/genética , Perfilação da Expressão Gênica , MicroRNAs/genética , Transcriptoma , Aorta/patologia , Doenças da Aorta/metabolismo , Doenças da Aorta/patologia , Aterosclerose/metabolismo , Aterosclerose/patologia , Células Cultivadas , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/patologia , Humanos , Macrófagos/metabolismo , Macrófagos/patologia , MicroRNAs/metabolismo , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Placa Aterosclerótica , RNA-SeqRESUMO
Vascular endothelial growth factors (VEGFs) are best known as key regulators of angiogenesis and lymphangiogenesis. Although VEGFs have been promising therapeutic targets for various cardiovascular diseases, their regulatory landscape in endothelial cells remains elusive. Several studies have highlighted the involvement of noncoding RNAs (ncRNAs) in the modulation of VEGF expression. In this study, we investigated the role of two classes of ncRNAs, long ncRNAs (lncRNAs) and enhancer RNAs (eRNAs), in the transcriptional regulation of VEGFA and VEGFC. By integrating genome-wide global run-on sequencing (GRO-Seq) and chromosome conformation capture (Hi-C) data, we identified putative lncRNAs and eRNAs associated with VEGFA and VEGFC genes in endothelial cells. A subset of the identified putative enhancers demonstrated regulatory activity in a reporter assay. Importantly, we demonstrate that deletion of enhancers and lncRNAs by CRISPR/Cas9 promoted significant changes in VEGFA and VEGFC expression. Transcriptome sequencing (RNA-Seq) data from lncRNA deletions showed downstream factors implicated in VEGFA- and VEGFC-linked pathways, such as angiogenesis and lymphangiogenesis, suggesting functional roles for these lncRNAs. Our study uncovers novel lncRNAs and eRNAs regulating VEGFA and VEGFC that can be targeted to modulate the expression of these important molecules in endothelial cells.
Assuntos
Células Endoteliais/metabolismo , RNA não Traduzido/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator C de Crescimento do Endotélio Vascular/metabolismo , Elementos Facilitadores Genéticos/genética , Elementos Facilitadores Genéticos/fisiologia , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/fisiologia , Humanos , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , RNA Longo não Codificante/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Fator C de Crescimento do Endotélio Vascular/genéticaRESUMO
AIMS: Oxidized phospholipids and microRNAs (miRNAs) are increasingly recognized to play a role in endothelial dysfunction driving atherosclerosis. NRF2 transcription factor is one of the key mediators of the effects of oxidized phospholipids, but the gene regulatory mechanisms underlying the process remain obscure. Here, we investigated the genome-wide effects of oxidized phospholipids on transcriptional gene regulation in human umbilical vein endothelial cells and aortic endothelial cells with a special focus on miRNAs. METHODS AND RESULTS: We integrated data from HiC, ChIP-seq, ATAC-seq, GRO-seq, miRNA-seq, and RNA-seq to provide deeper understanding of the transcriptional mechanisms driven by NRF2 in response to oxidized phospholipids. We demonstrate that presence of NRF2 motif and its binding is more prominent in the vicinity of up-regulated transcripts and transcriptional initiation represents the most likely mechanism of action. We further identified NRF2 as a novel regulator of over 100 endothelial pri-miRNAs. Among these, we characterize two hub miRNAs miR-21-5p and miR-100-5p and demonstrate their opposing roles on mTOR, VEGFA, HIF1A, and MYC expressions. Finally, we provide evidence that the levels of miR-21-5p and miR-100-5p in exosomes are increased upon senescence and exhibit a trend to correlate with the severity of coronary artery disease. CONCLUSION: Altogether, our analysis provides an integrative view into the regulation of transcription and miRNA function that could mediate the proatherogenic effects of oxidized phospholipids in endothelial cells.