Long Noncoding RNA MANTIS Facilitates Endothelial Angiogenic Function

Abstract

Background: The angiogenic function of endothelial cells is regulated by numerous mechanisms, but the impact of long noncoding RNAs (lncRNAs) has hardly been studied. We set out to identify novel and functionally important endothelial lncRNAs.

Methods: Epigenetically controlled lncRNAs in human umbilical vein endothelial cells were searched by exon-array analysis after knockdown of the histone demethylase JARID1B. Molecular mechanisms were investigated by RNA pulldown and immunoprecipitation, mass spectrometry, microarray, several knockdown approaches, CRISPR-Cas9, assay for transposase-accessible chromatin sequencing, and chromatin immunoprecipitation in human umbilical vein endothelial cells. Patient samples from lung and tumors were studied for MANTIS expression.

Results: A search for epigenetically controlled endothelial lncRNAs yielded lncRNA n342419, here termed MANTIS, as the most strongly regulated lncRNA. Controlled by the histone demethylase JARID1B, MANTIS was downregulated in patients with idiopathic pulmonary arterial hypertension and in rats treated with monocrotaline, whereas it was upregulated in carotid arteries of Macaca fascicularis subjected to atherosclerosis regression diet, and in endothelial cells isolated from human glioblastoma patients. CRISPR/Cas9-mediated deletion or silencing of MANTIS with small interfering RNAs or GapmeRs inhibited angiogenic sprouting and alignment of endothelial cells in response to shear stress. Mechanistically, the nuclear-localized MANTIS lncRNA interacted with BRG1, the catalytic subunit of the switch/sucrose nonfermentable chromatin-remodeling complex. This interaction was required for nucleosome remodeling by keeping the ATPase function of BRG1 active. Thereby, the transcription of key endothelial genes such as SOX18, SMAD6, and COUP-TFII was regulated by ensuring efficient RNA polymerase II machinery binding.

Conclusion: MANTIS is a differentially regulated novel lncRNA facilitating endothelial angiogenic function.

Keywords: RNA, long noncoding; epigenomics; glioblastoma; hypertension, pulmonary; neovascularization, physiologic.

Figure 1.

Endothelial angiogenic capacity is dependent on lncRNA MANTIS. A, Affymetrix Exon-array heatmap comparing siJARID1B-1/siScr, siJARID1B-2/siScr, siJARID1B-1/siGFP, siJARID1B-2/siGFP, and siScr/siGFP levels of HUVEC batches 1 to 3. Scale bar shows color code from –2.7 (blue) to 2.4 (yellow) log2 fold change. lncRNAs marked by an asterisk revealed >1 noncode accession numbers. See online-only Data Supplement Table III and online-only Data Supplement Figure IA for all lncRNA names. B, qRT-PCR of MANTIS and JARID1B in lungs from control donors (CTL) or patients with IPAH. n=12. Median with interquartile range is shown, and Mann-Whitney test was used. C, qRT-PCR of MANTIS and JARID1B in monkey vessels treated either with a normal diet (CTL), a high-fat diet (Ath), or a high-fat diet and a subsequent recovery phase (Reg). n=3. One-way ANOVA, Bonferroni. D, qRT-PCR of MANTIS and JARID1B from endothelial cells isolated from glioblastoma (GBM) or adjacent healthy control (CTL) tissue. n=5. Paired t test. E, RNA in situ hybridization of endothelium of healthy brain or glioblastoma with RNAscope. Scale bar indicates 50 µm. Arrows point to dots indicating MANTIS RNA. F, qRT-PCR measurements relative to β-Actin of MANTIS and JARID1B after laminar flow exposure (shr, 20 dyn/cm²) for 48 hours or 72 hours in HUVECs as indicated. Static (stc) samples served as control. n=4. Unpaired t test. G, CRISPR/Cas9 MANTIS guide RNAs (MTS gRNA) and control cells (CTL) after in vivo matrigel angiogenesis assay in mice. HUVECs were embedded in matrigel, stained with Vybrant dil (red), and injected. Isolectin GS-IB4 Alexa 647 conjugated stained vessels (green). Images were taken by light sheet microscopy 26 days after injection. Representative pictures are shown. Scale bar indicates 200 µm. H, Quantification of cells per plug as shown in G 26 days after injection. n=3. Unpaired t test. I, Tube formation assay performed with MTS gRNA and CTL. Numbers indicate number of tubes ± SEM. n=3. Scale bar indicates 200 µm. J, Spheroid outgrowth assay with MANTIS gRNA and CTL. Cells treated with or without VEGF-A are shown. Scale bar indicates 50 µm. K, Spheroid outgrowth assay after siMTS. Scr served as negative control. Cells treated ± VEGF-A are shown. Scale bar indicates 50 µm. L, Quantification of sprout numbers from the spheroid outgrowth assays seen in K or online-only Data Supplement Figure IIE. n=10. One-way ANOVA, Bonferroni. M, Tube formation assay after siMTS. Scr served as negative control. Numbers indicate number of tubes ± SEM. n=3. Scale bar indicates 200 µm. N, Images of cells treated with scrambled or siMTS after 72 h of laminar flow (LSS, 20 dyn/cm²). Numbers indicate number of cells orientated in direction of the flow ± SEM. n=4. Scale bar indicates 100 µm. All qRT-PCR data are relative to β-actin. Error bars are defined as mean ±SEM. *P<0.05. ANOVA indicates analysis of variance; HUVEC, human umbilical vein endothelial cell; IPAH, idiopathic pulmonary arterial hypertension; lncRNA, long noncoding RNA; qRT-PCR, quantitative real-time polymerase chain reaction; Scr, scrambled; SEM, standard error of the mean; siMTS, MANTIS siRNA; and VEGF-A, vascular endothelial growth factor A.

Figure 2.

The nuclear localized MANTIS lncRNA interacts with the SWI/SNF complex member BRG1. A, RNA fluorescence in situ hybridization (FISH) of HUVECs with TYE-665–modified probes against ACTINB and MANTIS. DAPI was used to stain nuclei. MANTIS overexpression (OE) samples were treated with an additional overexpression of pcDNA3.1+MANTIS for 48 hours before FISH. Scale bar indicates 20 µm. B, Scheme of RNA pulldown assay with subsequent preparation for mass spectrometric measurements. b-RNA indicates biotinylated RNA. C, qRT-PCR after RNA pulldown assay by measuring the amount of MANTIS RNA (MTS) in the eluates relative to the negative control RNA (CTL). n=4. Paired t test. D, Volcano plot of log2 ratio of MANTIS versus control interaction partner proteins after RNA pulldown assay and ESI-MS/MS measurements. n=3. Noncorrected –log₁₀ Student t test. LFQ indicates label-free quantification. E, Proteins enriched after RNA pulldown assay, their score, ratio MANTIS/CONTROL (MTS/CTL), and t value. F, MANTIS lncRNA (Left), U12 snRNA (Middle), and MEG3 lncRNA (Right) binding to complexes RNA immunoprecipitated with IgG, anti-BRG1, anti-SMARCA5, and anti-H3 were measured with qRT-PCR. The binding was analyzed relative to the input. Log2 values are shown. n=6. One-way ANOVA, Bonferroni. G, MANTIS lncRNA (Left), U12 snRNA (Middle), and MEG3 lncRNA (Right) binding to complexes RNA-chromatin immunoprecipitated with IgG, anti-BRG1, anti-SMARCA5, and anti-H3 were measured with qRT-PCR. The binding was analyzed relative to the input. Log10 values are shown. n=6. One-way ANOVA, Bonferroni. Error bars are defined as mean ± SEM. *P<0.05. ANOVA indicates analysis of variance; DAPI, 4′,6-diamidino-2-phenylindole; ESI-MS/MS, electrospray ionization-tandem mass spectrometry; HUVEC, human umbilical vein endothelial cell; lncRNA, long noncoding RNA; qRT-PCR, quantitative real-time polymerase chain reaction; SEM, standard error of the mean; and SWI/SNF, switch/sucrose nonfermentable.

Figure 3.

LncRNA MANTIS is required for SMAD6, COUP-TFII, and SOX18 expression. A, Illumina Bead-Chip Array heat map comparing gene expression after MANTIS LNA-GapmeR versus Control LNA-GapmeR treatments for 48 hours. Scale bar shows color code from –2.8 (green) to 2.4 (red) log2 fold change. Only representative genes were shown. B, qRT-PCR after LNA-GapmeR knockdown of MANTIS lncRNA. Expression levels of MANTIS, ANXA4, GCML1, snRNP27, and AAK1 are shown. CTL LNA served as negative control and was set to 1. n=4, Paired t test. C, qRT-PCR after LNA-GapmeR based knockdown of MANTIS lncRNA. CTL served as negative control and was set to 1. Expression levels of MANTIS, SOX18, SMAD6, COUP-TFII, and SOD2 are shown. n=6. Paired t test. D, qRT-PCR after knockdown of MANTIS lncRNA with siRNA-1. Scrambled siRNA (Scr) and MANTIS siRNA-specific control siRNA (CTL) served as negative controls. Expression levels of MANTIS, SOX18, SMAD6, COUP-TFII, and SOD2 are shown. Scr was set to 1. n=7. One-way ANOVA, Bonferroni. E, Gene ontology (GO) analyses made with Gorilla using all significantly downregulated genes after MANTIS knockdown found in the array. F, Representative Western blot of HUVECs treated either with control or MANTIS LNA-GapmeRs. SMAD6, SOX18, and COUP-TFII antibodies were used. GAPDH served as control. G, Quantification of blots shown in F. CTL was set to 1. n=3, Paired t test. H, Representative Western blot of HUVECs treated either with control or MANTIS siRNA-1. SMAD6, SOX18, COUP-TFII, and ANXA4 antibodies were used. GAPDH served as control. I, Quantification of blots shown in H. Scr was set to 1. n=4. Paired t test. J through L, qRT-PCR of SMAD6 (J), COUP-TFII (K), and SOX18 (L) in lungs from control donors (CTL) or patients with IPAH. n=12. Unpaired t test. All qRT-PCRs are relative to ß-Actin. Error bars are defined as mean ± SEM. *P<0.05. CTL indicates control; HUVEC, human umbilical vein endothelial cell; IPAH, idiopathic pulmonary arterial hypertension; lncRNA, long noncoding RNA; MTS, MANTIS; qRT-PCR, quantitative real-time polymerase chain reaction; Scr, scrambled; SEM, standard error of the mean; and SOD2, superoxide dismutase 2.

Figure 4.

SMAD6, SOX18, or COUP-TFII knockdown resemble MANTIS-deficient phenotype. A, qRT-PCR measurements after siRNA-based knockdown for 48 hours of MANTIS lncRNA (MTS), SMAD6, COUP-TFII, or SOX18. Scrambled siRNA (Scr) served as negative control and was set to 1. Expression levels of MANTIS (MTS), SMAD6, COUP-TFII, and SOX18 relative to β-ACTIN are shown. n=9. Paired t test. B, Spheroid outgrowth assay after MANTIS, SMAD6, COUP-TFII, or SOX18 siRNA-based knockdown for 48 hours. Scrambled siRNA (Scr) served as negative control. Cells treated with or without VEGF-A (±VEGF-A) are shown. Scale bar indicates 50 µm. C, D, Quantifications of sprout numbers (C) and cumulative sprout length (D) from the spheroid outgrowth assays shown in B. n=4. One-way ANOVA, Bonferroni. E, qRT-PCR measurements of MANTIS-mutant (MTS-mut) after overexpression (OE) of HUVEC with CTL or MTS-mut. n=6. Paired t test. F, Spheroid outgrowth assay after Scrambled (Scr) or MANTIS siRNA knockdown (siMTS) with subsequent overexpression of either CTL or MTS-mut for 24 hours. Cells treated with or without VEGF-A (±VEGF-A) are shown. Scale bar indicates 50 µm. G, H, Quantification of sprout numbers (G) and cumulative sprout length (H) from the spheroid outgrowth assays shown in F. n=5. One-way ANOVA, Bonferroni. I, qRT-PCR of MANTIS, SOX18, SMAD6, and COUP-TFII in PECAM-positive lung endothelial cells isolated from rats treated with saline (CTL) or monocrotaline (MCT). n=3. rn indicates rattus norvegicus. Unpaired t test. Error bars are defined as mean ± SEM. *P<0.05. ANOVA indicates analysis of variance; HUVEC, human umbilical vein endothelial cell; PECAM, platelet endothelial cell adhesion molecule; qRT-PCR, quantitative real-time polymerase chain reaction; SEM, standard error of the mean; and VEGF-A, vascular endothelial growth factor A.

Figure 5.

MANTIS facilitates nucleosome remodeling by BRG1. A, Representative Western blot of HUVECs treated either with scrambled (Scr) or with MANTIS siRNA-1 (MTS) for 48 hours. GAPDH served as loading control. B, ATAC-Seq profiles of genomic loci of SOX18, SMAD6, COUP-TFII, and ANXA4 after transfection of HUVECs with scrambled (CTL) or MANTIS siRNA (siMTS) underlined with Refseq and University of California, Santa Cruz annotation. For all, number of reads ranges from 0 to 13. r indicates reads; and a, alignment. Arrows indicate regions of strong differences between CTL and siMTS. C, D, qPCR of indicated genomic loci relative to GAPDH after transfection of HUVEC with scrambled (CTL) or MANTIS siRNA (siMTS) with subsequent FAIRE (C) or Mnase (D). Numbers indicate nucleotide positions upstream of the TSS. CTL was set to 1. n=4, paired t test. E through G, ChIP of HUVECs transfected with scrambled (C) or MANTIS siRNA (M) with H3K27me3 (E, n=6), RNA Pol II (F, n=3), and BRG1 (G, n=4) followed by qPCR for GAPDH promoter, Sox18 promoter regions at the transcription start site (TSS, –39 nt) or 586 nt and 1022 nt, SMAD6 promoter regions at the transcription start site (TSS, –29 nt) or 1491 nt and 3031 nt, and COUP-TFII promoter regions at the transcription start site (TSS, –137 nt) or 534 nt and 3185 nt. Numbers indicate nucleotide positions upstream of the TSS. Unpaired t test. Error bars are defined as mean ± SEM. *P<0.05. ChIP indicates chromatin immunoprecipitation; FAIRE, Formaldehyde-Assisted Isolation of Regulatory Elements; HUVEC, human umbilical vein endothelial cell; MNase, micrococcal nuclease; nt, nucleotide; qPCR, quantitative polymerase chain reaction; RNA Pol II, RNA polymerase II; SEM, standard error of the mean; and TSS, transcriptional start site.

Figure 6.

MANTIS improves ATPase activity of BRG1 by stabilizing its interaction with BAF155. A, Proximity ligation assay (PLA) of HUVECs transfected with scrambled (Scr) or MANTIS siRNA-1 (siMTS) for BRG1 with BAF155, BAF53a, or LIS1. LIS1 served as negative control. Red dots indicate polymerase-amplified interaction signals. Scale bar indicates 20 µm. B, Quantifications of PLA shown in A. n=3, Unpaired t test. Maxima indicate number of dots originating from polymerase-amplified interaction signal. C, Scheme of different MANTIS mutants used in D. Numbers indicate Exon number; A, Alu element. D, Relative increase of BRG1/BAF155 interaction from a PLA after overexpression of MANTIS mutants. n=6, Unpaired t test. E, Representative Western blot of HUVECs treated either with scrambled (Scr) or with MANTIS siRNA-1 (MTS) for 48 hours for BRG1, BAF155, and BAF53a. F, ATPase activity assay (absorbance at 620 nm) after BRG1 immunoprecipitation of HUVECs transfected with scrambled (Scr) or MANTIS siRNA-1 (MTS) for 48 hours. n=5, Paired t test. Error bars are defined as mean ± SEM. *P<0.05. HUVEC indicates human umbilical vein endothelial cell.

Figure 7.

Model of mechanism of action for the lncRNA MANTIS. Upper, MANTIS lncRNA expression is controlled by the histone demethylase JARID1B. Because of the limited expression of MANTIS, BRG1 and BAF155 assembly is decreased, leading to more heterochromatin formation at the TSS of SOX18, SMAD6, and COUP-TFII, limiting RNA Pol II binding and transcription of those genes. Lower, In case of JARID1B knockdown, H3K4me3 levels arise at the TSS of MANTIS, allowing more MANTIS expression. MANTIS interacts with BRG1, allowing increased binding of BAF155, which leads to a higher ATPase activity of BRG1 and euchromatin formation at the TSS of SOX18, SMAD6, and COUP-TFII allowing RNA Pol II binding and thereby transcription of SOX18, SMAD6, and COUP-TFII, which leads to increased angiogenic function. lncRNA indicates long noncoding RNA; RNA Pol II, RNA polymerase II; and TSS, transcriptional start site.