Long Non-Coding RNA Modulation of VEGF-A during Hypoxia

Abstract

The role and function of long non-coding RNAs (lncRNAs) in modulating gene expression is becoming apparent. Vascular endothelial growth factor A (VEGF-A) is a key regulator of blood vessel formation and maintenance making it a promising therapeutic target for activation in ischemic diseases. In this study, we uncover a functional role for two antisense VEGF-A lncRNAs, RP1-261G23.7 and EST AV731492, in transcriptional regulation of VEGF-A during hypoxia. We find here that both lncRNAs are polyadenylated, concordantly upregulated with VEGF-A, localize to the VEGF-A promoter and upstream elements in a hypoxia dependent manner either as a single-stranded RNA or DNA bound RNA, and are associated with enhancer marks H3K27ac and H3K9ac. Collectively, these data suggest that VEGF-A antisense lncRNAs, RP1-261G23.7 and EST AV731492, function as VEGF-A promoter enhancer-like elements, possibly by acting as a local scaffolding for proteins and also small RNAs to tether.

Keywords: antisense RNA; long non-coding RNA (lncRNA); transcriptional activation; vascular endothelial growth factor A (VEGF-A).

Figure 1

The expression of both vascular endothelial growth factor A (VEGF-A) promoter associated antisense long non-coding RNAs (lncRNAs) is upregulated in hypoxia. (A) A schematic is shown depicting the location of RP1-261G23.7 (VEGF-AS1) and EST AV731492 (VEGF-AS2) in the human genome relative to the VEGF-A gene; (B) fold change in VEGF-AS1, VEGF-AS2 and spliced VEGF-A expression levels in EA.hy926 cells ± hypoxia as determined by quantitative reverse transcription -polymerase chain reaction (qRT-PCR) and standardized to β-2-microglobulin (B2M). The data are presented as mean ± standard deviation (SD) (n = 3 independent experiments). Significance was measured by two-way analysis of variance (ANOVA). ** p < 0.01, **** p < 0.0001; (C) qRT-PCR analysis of VEGF-AS1, VEGF-AS2 and nuclear paraspeckle assembly transcript 1 (NEAT1) 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 (n = 3 independent experiments). Significance was measured by two-way ANOVA. * p < 0.5, *** p < 0.001; (D) RT-PCR analysis of VEGF-AS1 expression in polyA depleted and polyA positive fractions in EA.hy926 cells. The data are representative of two independent experiments; (E) RT-PCR analysis of VEGF-AS2 expression in polyA depleted and polyA positive fractions in EA.hy926 cells. The data are representative of two independent experiments; (F) Over-expression of VEGF-AS1 and VEGF-AS2 in EA.hy926 cells 72 h after transfection relative to the pcDNA3.1-GFP control; (G) Fold change in VEGF-A expression in normoxic and hypoxic EA.hy926 cells 72 h after VEGF-AS1 and VEGF-AS2 transfections relative to the control pcDNA3.1-GFP as determined by qRT-PCR and standardized to B2M. The data are presented as mean ± SD (n = 3 independent experiments). Significance was measured by two-way ANOVA. * p < 0.05. NS, non-significant; RT, reverse transcription; NTC, no template control; Mw, molecular weight; GFP, green fluorescent protein.

Figure 2

Repression of VEGF-A promoter associated antisense lncRNAs results in the downregulation of VEGF-A expression. (A) Fold change in VEGF-AS1 and spliced or unspliced VEGF-A expression levels in normoxic EA.hy926 cells 48 h after antisense phosphorothioate oligonucleotides (PTO) transfections as determined by qRT-PCR and standardized to B2M. The data are presented as mean ± SD (n = 3 independent experiments). Significance was measured by two-way ANOVA. ** p < 0.01; (B) fold change in VEGF-AS1 and spliced or unspliced VEGF-A expression levels in hypoxic EA.hy926 cells 48 h after antisense PTO transfections as determined by qRT-PCR and standardized to B2M. The data are presented as mean ± SD (n = 3 independent experiments). Significance was measured by two-way ANOVA. * p < 0.05, *** p < 0.001, **** p < 0.0001; (C) Fold change in VEGF-AS2 and spliced or unspliced VEGF-A expression levels in normoxic EA.hy926 cells 48 h after antisense PTO transfections as determined by qRT-PCR and standardized to B2M. The data are presented as mean ± SD (n = 3 independent experiments). Significance was measured by two-way ANOVA. ** p < 0.01, *** p < 0.001; (D) Fold change in VEGF-AS2 and spliced or unspliced VEGF-A expression levels in hypoxic EA.hy926 cells 48h after antisense PTO transfections as determined by qRT-PCR and standardized to B2M. The data are presented as mean ± SD (n = 3 independent experiments). Significance was measured by two-way ANOVA. ** p < 0.01, **** p < 0.0001; (E) fold change in VEGF-AS2 and spliced or unspliced VEGF-A expression levels in normal and knockout (KO) EA.hy926 cells as determined by qRT-PCR and standardized to B2M. The data are presented as mean ± SD (n = 2 independent experiments). Significance was measured by two-way ANOVA. * p < 0.05, ** p < 0.01, *** p < 0.001; (F) Fold change in VEGF-AS2 and spliced or unspliced VEGF-A expression levels in KO EA.hy926 cells 72 h after VEGF-AS2 transfections relative to the control pcDNA3.1-GFP as determined by qRT-PCR and standardized to B2M. The data are presented as mean ± SD (n = 2 independent experiments). Significance was measured by two-way ANOVA. **** p < 0.0001; (G) fold change in VEGF-AS2 and spliced VEGF-A expression levels in EA.hy926 cells 48 h after transfections as determined by qRT-PCR and standardized to B2M. The data are presented as mean ± SD (n = 2 independent experiments). Significance was measured by two-way ANOVA. ** p < 0.01, *** p < 0.001; (H) Fold change in VEGF-S2 expression levels in normoxic and hypoxic EA.hy926 cells as determined by qRT-PCR and standardized to B2M. The data are presented as mean ± SD (n = 3 independent experiments). Significance was measured by two-tailed unpaired t test. ** p < 0.01; (I) Fold change in VEGF-S2 and unspliced VEGF-A expression levels in hypoxic EA.hy926 cells 48 h after antisense PTO transfections as determined by qRT-PCR and standardized to B2M. The data are presented as mean ± SD (n = 3 independent experiments). Significance was measured by two-way ANOVA. * p < 0.05, **** p < 0.0001.

Figure 3

Both VEGF-AS1 and VEGF-AS2 localize to the VEGF-A promoter. (A) Fold change in VEGF-AS1 target locus enrichment at the VEGF-A 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 (n = 3 independent experiments). Significance was measured by two-way ANOVA. * p < 0.05; (B) fold change in VEGF-AS2 target locus enrichment at the VEGF-A 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 (n = 3 independent experiments). Significance was measured by two-way ANOVA. * p < 0.05; (C) primer walking at the VEGF-A promoter. A qPCR analysis of VEGF-AS1 localization at the VEGF-A 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 (n = 2 independent experiments); (D) primer walking at the VEGF-A promoter. A qPCR analysis of VEGF-AS2 localization at the VEGF-A 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 (n = 2 independent experiments); (E) RT-PCR analysis of VEGF-AS1 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; (F) RT-PCR analysis of VEGF-AS2 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.

Figure 4

Strong enhancer marks are associated with VEGF-AS1 and VEGF-AS2; (A) A qPCR analysis of H3K27ac and H3K9ac enrichment at the VEGF-AS1, VEGF-AS2 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 (n = 3 independent experiments). Significance was measured by two-way ANOVA. **** p < 0.0001; (B) A qRT-PCR analysis of H3K27ac and H3K9ac association with VEGF-AS1 in normoxic and hypoxic EA.hy926 cells. IgG is used as a control. The data are presented as mean ± SD and standardized to inputs (n = 3 independent experiments). Significance was measured by two-way ANOVA. * p < 0.05; (C) A qRT-PCR analysis of H3K27ac and H3K9ac association with VEGF-AS2 in normoxic and hypoxic EA.hy926 cells. IgG is used as a control. The data are presented as mean ± SD and standardized to inputs (n = 2–3 independent experiments); (D) the prediction of physical contacts between the VEGF-AS2 locus and the VEGF-A promoter in human umbilical vein endothelial cells (HUVECs). The figure was generated by using three-dimensional (3D) Genome Browser (http://biorxiv.org/content/early/2017/02/27/112268). The track around the VEGF-A gene loci was selected in 5 kilobase resolution in HUVECs with genome version HG19.