Long Non-Coding RNA Modulation of VEGF-A during Hypoxia
<p>The expression of both vascular endothelial growth factor A (VEGF-A) promoter associated antisense long non-coding RNAs (lncRNAs) is upregulated in hypoxia. (<b>A</b>) A schematic is shown depicting the location of <span class="html-italic">RP1-261G23.7</span> (<span class="html-italic">VEGF-AS1</span>) and <span class="html-italic">EST AV731492</span> (<span class="html-italic">VEGF-AS2</span>) in the human genome relative to the <span class="html-italic">VEGF-A</span> gene; (<b>B</b>) fold change in <span class="html-italic">VEGF-AS1</span>, <span class="html-italic">VEGF-AS2</span> and spliced <span class="html-italic">VEGF-A</span> expression levels in EA.hy926 cells ± hypoxia as determined by quantitative reverse transcription -polymerase chain reaction (qRT-PCR) and standardized to β-2-microglobulin (<span class="html-italic">B2M</span>). The data are presented as mean ± standard deviation (SD) (<span class="html-italic">n</span> = 3 independent experiments). Significance was measured by two-way analysis of variance (ANOVA). ** <span class="html-italic">p</span> < 0.01, **** <span class="html-italic">p</span> < 0.0001; (<b>C</b>) qRT-PCR analysis of <span class="html-italic">VEGF-AS1</span>, <span class="html-italic">VEGF-AS2</span> and nuclear paraspeckle assembly transcript 1 (<span class="html-italic">NEAT1</span>) expression in subcellular fractions from EA.hy926 cells ± hypoxia, plotted as percentages in association with nucleus and cytoplasm. The data are presented as mean ± SD (<span class="html-italic">n</span> = 3 independent experiments). Significance was measured by two-way ANOVA. * <span class="html-italic">p</span> < 0.5, *** <span class="html-italic">p</span> < 0.001; (<b>D</b>) RT-PCR analysis of <span class="html-italic">VEGF-AS1</span> expression in polyA depleted and polyA positive fractions in EA.hy926 cells. The data are representative of two independent experiments; (<b>E</b>) RT-PCR analysis of <span class="html-italic">VEGF-AS2</span> expression in polyA depleted and polyA positive fractions in EA.hy926 cells. The data are representative of two independent experiments; (<b>F</b>) Over-expression of <span class="html-italic">VEGF-AS1</span> and <span class="html-italic">VEGF-AS2</span> in EA.hy926 cells 72 h after transfection relative to the pcDNA3.1-GFP control; (<b>G</b>) Fold change in <span class="html-italic">VEGF-A</span> expression in normoxic and hypoxic EA.hy926 cells 72 h after <span class="html-italic">VEGF-AS1</span> and <span class="html-italic">VEGF-AS2</span> transfections relative to the control pcDNA3.1-GFP as determined by qRT-PCR and standardized to <span class="html-italic">B2M</span>. The data are presented as mean ± SD (<span class="html-italic">n</span> = 3 independent experiments). Significance was measured by two-way ANOVA. * <span class="html-italic">p</span> < 0.05. NS, non-significant; RT, reverse transcription; NTC, no template control; Mw, molecular weight; GFP, green fluorescent protein.</p> "> Figure 2
<p>Repression of <span class="html-italic">VEGF-A</span> promoter associated antisense lncRNAs results in the downregulation of <span class="html-italic">VEGF-A</span> expression. (<b>A</b>) Fold change in <span class="html-italic">VEGF-AS1</span> and spliced or unspliced <span class="html-italic">VEGF-A</span> expression levels in normoxic EA.hy926 cells 48 h after antisense phosphorothioate oligonucleotides (PTO) transfections as determined by qRT-PCR and standardized to <span class="html-italic">B2M</span>. The data are presented as mean ± SD (<span class="html-italic">n</span> = 3 independent experiments). Significance was measured by two-way ANOVA. ** <span class="html-italic">p</span> < 0.01; (<b>B</b>) fold change in <span class="html-italic">VEGF-AS1</span> and spliced or unspliced <span class="html-italic">VEGF-A</span> expression levels in hypoxic EA.hy926 cells 48 h after antisense PTO transfections as determined by qRT-PCR and standardized to <span class="html-italic">B2M</span>. The data are presented as mean ± SD (<span class="html-italic">n</span> = 3 independent experiments). Significance was measured by two-way ANOVA. * <span class="html-italic">p</span> < 0.05, *** <span class="html-italic">p</span> < 0.001, **** <span class="html-italic">p</span> < 0.0001; (<b>C</b>) Fold change in <span class="html-italic">VEGF-AS2</span> and spliced or unspliced <span class="html-italic">VEGF-A</span> expression levels in normoxic EA.hy926 cells 48 h after antisense PTO transfections as determined by qRT-PCR and standardized to <span class="html-italic">B2M</span>. The data are presented as mean ± SD (<span class="html-italic">n</span> = 3 independent experiments). Significance was measured by two-way ANOVA. ** <span class="html-italic">p</span> < 0.01, *** <span class="html-italic">p</span> < 0.001; (<b>D</b>) Fold change in <span class="html-italic">VEGF-AS2</span> and spliced or unspliced <span class="html-italic">VEGF-A</span> expression levels in hypoxic EA.hy926 cells 48h after antisense PTO transfections as determined by qRT-PCR and standardized to <span class="html-italic">B2M</span>. The data are presented as mean ± SD (<span class="html-italic">n</span> = 3 independent experiments). Significance was measured by two-way ANOVA. ** <span class="html-italic">p</span> < 0.01, **** <span class="html-italic">p</span> < 0.0001; (<b>E</b>) fold change in <span class="html-italic">VEGF-AS2</span> and spliced or unspliced <span class="html-italic">VEGF-A</span> expression levels in normal and knockout (KO) EA.hy926 cells as determined by qRT-PCR and standardized to <span class="html-italic">B2M</span>. The data are presented as mean ± SD (<span class="html-italic">n</span> = 2 independent experiments). Significance was measured by two-way ANOVA. * <span class="html-italic">p</span> < 0.05, ** <span class="html-italic">p</span> < 0.01, *** <span class="html-italic">p</span> < 0.001; (<b>F</b>) Fold change in <span class="html-italic">VEGF-AS2</span> and spliced or unspliced <span class="html-italic">VEGF-A</span> expression levels in KO EA.hy926 cells 72 h after <span class="html-italic">VEGF-AS2</span> transfections relative to the control pcDNA3.1-GFP as determined by qRT-PCR and standardized to <span class="html-italic">B2M</span>. The data are presented as mean ± SD (<span class="html-italic">n</span> = 2 independent experiments). Significance was measured by two-way ANOVA. **** <span class="html-italic">p</span> < 0.0001; (<b>G</b>) fold change in <span class="html-italic">VEGF-AS2</span> and spliced <span class="html-italic">VEGF-A</span> expression levels in EA.hy926 cells 48 h after transfections as determined by qRT-PCR and standardized to <span class="html-italic">B2M</span>. The data are presented as mean ± SD (<span class="html-italic">n</span> = 2 independent experiments). Significance was measured by two-way ANOVA. ** <span class="html-italic">p</span> < 0.01, *** <span class="html-italic">p</span> < 0.001; (<b>H</b>) Fold change in <span class="html-italic">VEGF-S2</span> expression levels in normoxic and hypoxic EA.hy926 cells as determined by qRT-PCR and standardized to <span class="html-italic">B2M</span>. The data are presented as mean ± SD (<span class="html-italic">n</span> = 3 independent experiments). Significance was measured by two-tailed unpaired t test. ** <span class="html-italic">p</span> < 0.01; (<b>I</b>) Fold change in <span class="html-italic">VEGF-S2</span> and unspliced <span class="html-italic">VEGF-A</span> expression levels in hypoxic EA.hy926 cells 48 h after antisense PTO transfections as determined by qRT-PCR and standardized to <span class="html-italic">B2M</span>. The data are presented as mean ± SD (<span class="html-italic">n</span> = 3 independent experiments). Significance was measured by two-way ANOVA. * <span class="html-italic">p</span> < 0.05, **** <span class="html-italic">p</span> < 0.0001.</p> "> Figure 3
<p>Both <span class="html-italic">VEGF-AS1</span> and <span class="html-italic">VEGF-AS2</span> localize to the <span class="html-italic">VEGF-A</span> promoter. (<b>A</b>) Fold change in <span class="html-italic">VEGF-AS1</span> target locus enrichment at the <span class="html-italic">VEGF-A</span> promoter in normoxic and hypoxic EA.hy926 cells as determined by quantitative polymerase chain reaction (qPCR) after pulldown with antisense oligonucleotides with 3′-Biotin modifications. Beads only control is set to be 1. The data are presented as mean ± SD and standardized to inputs (<span class="html-italic">n</span> = 3 independent experiments). Significance was measured by two-way ANOVA. * <span class="html-italic">p</span> < 0.05; (<b>B</b>) fold change in <span class="html-italic">VEGF-AS2</span> target locus enrichment at the <span class="html-italic">VEGF-A</span> promoter in normoxic and hypoxic EA.hy926 cells as determined by qPCR after pulldown with antisense oligonucleotides with 3′-Biotin modifications. Beads only control is set to be 1. The data are presented as mean ± SD and standardized to inputs (<span class="html-italic">n</span> = 3 independent experiments). Significance was measured by two-way ANOVA. * <span class="html-italic">p</span> < 0.05; (<b>C</b>) primer walking at the <span class="html-italic">VEGF-A</span> promoter. A qPCR analysis of <span class="html-italic">VEGF-AS1</span> localization at the <span class="html-italic">VEGF-A</span> promoter in EA.hy926 cells after pulldown with antisense oligonucleotides with 3′-Biotin modifications. The data are presented as mean ± SD and standardized to inputs (<span class="html-italic">n</span> = 2 independent experiments); (<b>D</b>) primer walking at the <span class="html-italic">VEGF-A</span> promoter. A qPCR analysis of <span class="html-italic">VEGF-AS2</span> localization at the <span class="html-italic">VEGF-A</span> promoter in EA.hy926 cells after pulldown with antisense oligonucleotides with 3′-Biotin modifications. The data are presented as mean ± SD and standardized to inputs (<span class="html-italic">n</span> = 2 independent experiments); (<b>E</b>) RT-PCR analysis of <span class="html-italic">VEGF-AS1</span> expression in hypoxic and normoxic EA.hy926 cells after pulldown with antisense oligonucleotides with 3′-Biotin modifications and treatments with RNase A and RNase H. The data are representative of three independent experiments; (<b>F</b>) RT-PCR analysis of <span class="html-italic">VEGF-AS2</span> expression in hypoxic and normoxic EA.hy926 cells after pulldown with antisense oligonucleotides with 3′-Biotin modifications and treatments with RNase A and RNase H. The data are representative of three independent experiments.</p> "> Figure 4
<p>Strong enhancer marks are associated with <span class="html-italic">VEGF-AS1</span> and <span class="html-italic">VEGF-AS2</span>; (<b>A</b>) A qPCR analysis of H3K27ac and H3K9ac enrichment at the <span class="html-italic">VEGF-AS1</span>, <span class="html-italic">VEGF-AS2</span> and off-target loci in normoxic and hypoxic EA.hy926 cells. IgG is used as a control. The data are presented as mean ± SD and standardized to inputs (<span class="html-italic">n</span> = 3 independent experiments). Significance was measured by two-way ANOVA. **** <span class="html-italic">p</span> < 0.0001; (<b>B</b>) A qRT-PCR analysis of H3K27ac and H3K9ac association with <span class="html-italic">VEGF-AS1</span> in normoxic and hypoxic EA.hy926 cells. IgG is used as a control. The data are presented as mean ± SD and standardized to inputs (<span class="html-italic">n</span> = 3 independent experiments). Significance was measured by two-way ANOVA. * <span class="html-italic">p</span> < 0.05; (<b>C</b>) A qRT-PCR analysis of H3K27ac and H3K9ac association with <span class="html-italic">VEGF-AS2</span> in normoxic and hypoxic EA.hy926 cells. IgG is used as a control. The data are presented as mean ± SD and standardized to inputs (<span class="html-italic">n</span> = 2–3 independent experiments); (<b>D</b>) the prediction of physical contacts between the <span class="html-italic">VEGF-AS2</span> locus and the VEGF-A promoter in human umbilical vein endothelial cells (HUVECs). The figure was generated by using three-dimensional (3D) Genome Browser (<a href="http://biorxiv.org/content/early/2017/02/27/112268" target="_blank">http://biorxiv.org/content/early/2017/02/27/112268</a>). The track around the <span class="html-italic">VEGF-A</span> gene loci was selected in 5 kilobase resolution in HUVECs with genome version HG19.</p> ">
Abstract
:1. Introduction
2. Results
2.1. Antisense Transcript Expression Is Concordantly Upregulated with Vascular Endothelial Growth Factor A in Hypoxia
2.2. Antisense Transcripts Modify Vascular Endothelial Growth Factor A Expression
2.3. Antisense Transcrpits Localize at the Vascular Endothelial Growth Factor A Promoter as a Single-Stranded RNA or Bound to the DNA
2.4. Enhancer Marks Are Associated with Antisense Transcripts
3. Discussion
4. Materials and Methods
4.1. Expression Vectors and Antisense Oligonucleotides
4.2. Cell Culture and Transfections
4.3. Reverse Transcription and Quantitative Polymerase Chain Reaction
4.4. Subcellular Fractionation
4.5. Polyadenylation (PolyA) Study
4.6. Heterozygous VEGF-AS2 Knockout Cell Line
4.7. Chromatin Isolation by RNA Purification (ChIRP)
4.8. Chromatin Immunoprecipitation and RNA Immunoprecipitation
Supplementary Materials
Author Contributions
Funding
Conflicts of Interest
References
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Nieminen, T.; Scott, T.A.; Lin, F.-M.; Chen, Z.; Yla-Herttuala, S.; Morris, K.V. Long Non-Coding RNA Modulation of VEGF-A during Hypoxia. Non-Coding RNA 2018, 4, 34. https://doi.org/10.3390/ncrna4040034
Nieminen T, Scott TA, Lin F-M, Chen Z, Yla-Herttuala S, Morris KV. Long Non-Coding RNA Modulation of VEGF-A during Hypoxia. Non-Coding RNA. 2018; 4(4):34. https://doi.org/10.3390/ncrna4040034
Chicago/Turabian StyleNieminen, Tiina, Tristan A. Scott, Feng-Mao Lin, Zhen Chen, Seppo Yla-Herttuala, and Kevin V. Morris. 2018. "Long Non-Coding RNA Modulation of VEGF-A during Hypoxia" Non-Coding RNA 4, no. 4: 34. https://doi.org/10.3390/ncrna4040034