Long Noncoding RNA MALAT1 Regulates Endothelial Cell Function and Vessel GrowthNovelty and Significance
Rationale: The human genome harbors a large number of sequences encoding for RNAs that are not translated but control cellular functions by distinct mechanisms. The expression and function of the longer transcripts namely the long noncoding RNAs in the vasculature are largely unknown.
Objective: Here, we characterized the expression of long noncoding RNAs in human endothelial cells and elucidated the function of the highly expressed metastasis-associated lung adenocarcinoma transcript 1 (MALAT1).
Methods and Results: Endothelial cells of different origin express relative high levels of the conserved long noncoding RNAs MALAT1, taurine upregulated gene 1 (TUG1), maternally expressed 3 (MEG3), linc00657, and linc00493. MALAT1 was significantly increased by hypoxia and controls a phenotypic switch in endothelial cells. Silencing of MALAT1 by small interfering RNAs or GapmeRs induced a promigratory response and increased basal sprouting and migration, whereas proliferation of endothelial cells was inhibited. When angiogenesis was further stimulated by vascular endothelial growth factor, MALAT1 small interfering RNAs induced discontinuous sprouts indicative of defective proliferation of stalk cells. In vivo studies confirmed that genetic ablation of MALAT1 inhibited proliferation of endothelial cells and reduced neonatal retina vascularization. Pharmacological inhibition of MALAT1 by GapmeRs reduced blood flow recovery and capillary density after hindlimb ischemia. Gene expression profiling followed by confirmatory quantitative reverse transcriptase-polymerase chain reaction demonstrated that silencing of MALAT1 impaired the expression of various cell cycle regulators.
Conclusions: Silencing of MALAT1 tips the balance from a proliferative to a migratory endothelial cell phenotype in vitro, and its genetic deletion or pharmacological inhibition reduces vascular growth in vivo.
With the use of modern molecular biology techniques, such as deep sequencing, it has become evident that the majority of the genome is transcribed, whereas only 2% of the transcribed genome codes for protein. The rest of the transcribed part of the genome is known as noncoding RNA. Noncoding RNAs can be divided in small (<200 nt) noncoding RNAs, which include microRNAs and transfer RNAs, and longer RNAs (>200 nt) include ribosomal RNAs and long noncoding RNAs (lncRNAs).1,2 Although microRNAs are shown to play important roles in the post-transcriptional regulation of gene expression and to control endothelial cell function, vessel growth and remodeling,3,4 little is known about the function of lncRNAs in the endothelium.
Editorial see p 1366
In This Issue, see p 1361
Several subclasses of lncRNAs have been described, such as intragenic natural antisense transcripts (NATs) that are transcribed in the opposite direction to a particular gene.5,6 LncRNAs can also be located between protein-coding genes or in introns.6 LncRNAs are involved in the regulation of gene expression through epigenetic mechanisms that include chromatin remodeling, the regulation of splicing, and by acting as sponges for microRNAs.2,7 For example, genomic imprinting is controlled by lncRNAs, and the lncRNA X-inactive–specific transcript is involved in the initiation of X chromosome inactivation.8 NATs often regulate the associated sense transcript.6 In endothelial cells, a NAT for tyrosine kinase containing immunoglobulin and epidermal growth factor homology domain-1 (tie-1) was shown to bind tie-1 mRNA and reduced tie-1 transcript levels selectively, resulting in specific defects in endothelial cell contact junctions.9
One of the lncRNAs that has been described to control both epigenetic gene regulation and splicing is metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), which was first described to be associated with metastasis of lung tumors.10 MALAT1 was shown to interact with polycomb 2 (CBX4) and thereby regulate histone modifications to control cellular proliferation.11 Furthermore, splicing regulation by MALAT1 was identified in HeLa cells, where MALAT1 interacts with serine-/arginine-rich proteins, thereby regulating subcellular localization of splicing regulating proteins.12
Here, we characterize the expression of noncoding RNAs in endothelial cells and define a functional role of MALAT1 in the regulation of the angiogenic response of endothelial cells in vitro and vascularization in vivo.
Primary Cell Culture and Angiogenesis Assays
Cell culture conditions and methods to determine cell cycle progression, spheroid growth, and migration are described in the online Method section of this article.
For RNA sequencing, 0.5 μg total RNA isolated from human umbilical vein endothelial cells (HUVECs) was used. Poly-A RNA was selected using poly-T oligo-attached magnetic beads. After the elution of the poly-A RNA, the RNA is fragmented and primed for cDNA synthesis. The sequencing libraries were constructed using Illumina TruSeq RNA Sample Preparation Kit, according to the manufacturer’s protocol. Four libraries indexed with different barcodes were pooled and sequenced on one lane of an Illumina HiSeq 2000 flowcell. The reads were mapped using tophat with 2 mismatches allowed. Cuffdiff was used to determine expression of RNA in HUVEC. The lncRNA annotation was based on the NONCODE database (www.noncode.org). The sequence data have been deposited in the NCBI GEO database under accession number GSE54384.
HUVECs were transfected at 60% to 75% confluence with 100 nmol/L synthesized small interfering RNAs (siRNAs; Sigma, St Louis, MO) or 1- to 50-nmol/L locked nucleic acid (LNA) GapmeR (Exiqon, Vedbaek, Denmark) targeting MALAT1 using Lipofectamine RNAiMax (Life Technologies, Carlsbad, CA) according to the manufacturer’s protocol. As controls, the siFirefly luciferase or scrambled LNA GapmeR were transfected. Four hours after transfection, the medium was replaced by EBM (Lonza) supplemented with EGM-SingleQuots (Lonza), and 10% FCS (Invitrogen, Carlsbad, CA).
RNA Isolation and Quantitative Real-Time Polymerase Chain Reaction
Total RNA from cultured cells and mouse tissue was isolated using miRNeasy kits (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. Nuclear and cytoplasmic extracts were prepared using the protocol of Hwang et al,13 followed by RNA isolation using miRNeasy Kits (Qiagen). For measuring mRNAs or lncRNAs, 100- to 1000-ng total RNA was reverse transcribed using MulV reverse transcriptase (Life Technologies) and random hexamer primers (Thermo Scientific, Waltham, MA) in a 20-μL reaction. cDNA was used as template for quantitative real-time reverse transcriptase-polymerase chain reaction (PCR) using Fast SYBR Green (Applied Biosystems, Forster City, CA) and an Applied Biosystems StepOnePlus machine. Human ribosomal P0 (RPLP0) mRNA was used for normalization. Primer sequences are listed in the Methods in the Online Data Supplement. Analysis of relative gene expression levels was performed using the formula 2–ΔCT with ΔCT=CT(target gene)–CT(control).
Mouse Retinal Angiogenesis Model
All animal experiments were conducted according to the principles of laboratory animal care and according to the German national laws. The studies have been approved by the local ethical committee (Regierungspräsidium Darmstadt, Hessen). MALAT1–/– mice were described previously.14
MALAT1+/+ or MALAT1–/– pups were euthanized at postnatal day 5. The efficient depletion of MALAT1 in MALAT1–/– mice was validated by quantitative reverse transcriptase-PCR analysis from lung RNA. Retinas were dissected and stained with biotin-labeled isolectin-B4 (1:200; Vector Laboratories Inc, Burlingame, CA) or rabbit antiphosphohistone H3 antibody (1:100; Millipore), followed by streptavidin secondary antibody or Alexa Fluor–labeled antirabbit secondary antibodies (1:400; Invitrogen), respectively, as described previously.15 Whole-mount retinas were visualized by confocal microscopy on the laser scanning microscopes LSM780 (Zeiss) and SP2-FCS (Leica) using 10×, 20×, and 25×/0.8 (Imm) Plan-Neofluar objectives. Four individual retinal flaps per mouse retina were assessed.
Hindlimb Ischemia Mouse Model
C57BL/6 mice were purchased from Charles River (Sulzfeld, Germany). LNA GapmeR Ctrl (20 mg/kg; Exiqon) or LNA GapmeR MALAT1 (20 mg/kg; Exiqon) were injected intraperitoneally before, after surgery, and after 14 days. Hindlimb ischemia and subsequent laser-Doppler perfusion measurement and assessment of capillary density were performed as described.16
Microarray Experiments and Analysis
Human GeneChip Exon 1.0 ST arrays (exon arrays; Affymetrix) were used to access the molecular signatures on the loss of MALAT1. The microarray experiment was performed following the manufacturer’s protocol. CEL files were uploaded to the noncoder Web interface17 and analyzed at the levels of genes and exons. To derive differentially expressed exons, a 2-fold threshold (either inclusion or exclusion of an exon) and P<0.05 were applied to siMALAT1 knockdown when compared with the control. The Database for Annotation, Visualization, and Integrated Discovery bioinformatics resources18 were used for the analysis of Gene Ontology terms.
Data were analyzed with GraphPad Prism 5 using unpaired Student t tests when comparing 2 conditions or 1-way ANOVA with Bonferroni correction for multiple comparisons. A significance level of P<0.05 was considered significant. Data are presented as mean with error bars depicting the SEM.
Characterization of LncRNA Expression in Endothelial Cells
To determine the expression patterns of noncoding RNAs in endothelial cells, we performed deep sequencing of poly A-selected RNA (Online Table I). This experiment showed that in HUVECs, ≈56% of the total RNA comprises noncoding RNA (Figure 1A). Of the noncoding RNAs, 7% are annotated as NAT, 7% as long intergenic noncoding RNAs, and 42% as other noncoding RNA. Analysis of expression levels identified various lncRNAs that showed expression levels comparable with endothelial coding genes, such as endothelial nitric oxide synthase or vascular endothelial growth factor (VEGF) receptor 2 (Figure 1B). The sequences of many lncRNAs are not well conserved between species, but 5 of the highest expressed lncRNAs, namely linc00493, MALAT1, maternally expressed 3 (MEG3), taurine upregulated gene 1 (TUG1), and linc00657, showed considerable sequence conservation between mouse and human (Online Figure I/data not shown).
Therefore, we next focused on these 5 lncRNAs and confirmed their expression in endothelial cells derived from various human vascular beds by quantitative reverse transcriptase-PCR (Figure 1C). With the exception of MEG3, which was significantly lower in arterial when compared with venous or microvascular endothelial cells, all other lncRNAs were expressed at similar levels in the endothelial cells tested (Figure 1C). Because lncRNAs may exhibit different functions depending on their subcellular localization, we additionally determined the levels in nuclear versus cytoplasmic extracts. The known lncRNAs TUG1, MEG3, and MALAT1 were highly enriched in the nuclear fraction, whereas levels of the to date unexplored linc00657 and linc00493 are higher in the cytoplasm (Figure 1D).
To assess whether the expression of the selected lncRNAs is regulated by physiological stimuli, we exposed HUVECs to hypoxia (0.2% O2). These experiments revealed that of the 5 conserved highly expressed lncRNAs, MEG3 and MALAT1 were profoundly upregulated by hypoxia with a significant induction at 24 hours (Figure 1E). Linc00657 and TUG1 were also significantly increased to ≈1.5-fold, whereas linc00493 expression was not affected (Figure 1E).
MALAT1 Knockdown Modulates Endothelial Cell Function In Vitro
Because MALAT1 was highly expressed and most profoundly increased by hypoxia, we further explored the function of MALAT1 in endothelial cells by silencing MALAT1 expression with siRNAs. siRNA treatment resulted in a reduction of total MALAT1 levels when compared with scramble controls RNAs (Figure 2A). Importantly, nuclear localized MALAT1 was also significantly downregulated by siRNA treatment (Figure 2A), despite the observation in a recent report that siRNAs may not target nuclear RNAs.19 Silencing of MALAT1 increased basal endothelial cell migration and sprouting as assessed by an in vitro spheroid angiogenesis assay (Figure 2B and 2C) and scratched wound assay (Figure 2D and 2E). However, when sprouting was stimulated with VEGF, MALAT1 siRNA treatment did not further increase the outgrowth of spheroids (Figure 2F). Yet, the MALAT1 siRNA-treated spheroids showed an interesting phenotype (Figure 2G). Although endothelial cells migrated a similar distance in both groups, the siRNA MALAT1-treated spheroids showed a significantly higher number of discontinued sprouts (Figure 2G and 2H; Online Figure II), indicating that the extension of the sprouts, which is mediated by proliferation of stalk cells, is disturbed. Indeed, subsequent analysis of endothelial cell proliferation revealed a significant reduction in cell number in MALAT1 silenced endothelial cells (90±4% compared with control siRNAs after 48 hours; P<0.05). The reduced cell number was associated with a significant inhibition of cell cycle progression, and MALAT1 silencing reduced the number of cells in S-phase under basal and hypoxic conditions, and after VEGF-stimulation (Figure 2I and 2J; Online Figure III). Cell death as measured by the number of propidium iodide–positive cells and caspase activity, which is indicative of apoptotic cell death, was slightly, but not significantly, upregulated in MALAT1 silenced cells (PI+ cells: 125±13%; caspase activity: 151±26% compared with control siRNAs).
Because silencing of MALAT1 by siRNA induced an interesting switch from a proliferative to a promigratory state of the endothelial cells resulting in an aberrant vessel sprouting under proangiogenic conditions, we next aimed to confirm the above observations using a different approach to silence MALAT1. Therefore, we used LNA GapmeRs, which are single-stranded oligonucleotides that consist of a DNA stretch flanked by LNA nucleotides. Basepairing with the targeted lncRNA in the nucleus induces degradation by an RNAse H-dependent mechanism (Figure 3A). Transfection with GapmeRs directed against MALAT1 at concentrations ranging from 1 to 50 nmol/L silenced human MALAT1 in HUVECs in a dose-dependent manner (Figure 3B). Inhibition of MALAT1 expression by GapmeRs significantly induced angiogenic sprouting of HUVECs under basal conditions but did not further increase VEGF-stimulated angiogenic sprouting (Figure 3C and 3D). Moreover, silencing of MALAT1 by GapmeRs for 48 hours significantly reduced the number of cells (84±4%; P<0.05) and reduced progression through the cell cycle (Figure 3E), thus confirming the results achieved by siRNA-induced silencing of MALAT1 expression.
MALAT1 Regulates Angiogenesis In Vivo
Because MALAT1 silencing induces a complex switch in endothelial cell function, namely increased migratory and basal sprouting capacity but inhibition of cell cycle progression, we next aimed to address the consequences of these effects for vessel formation in vivo. To this end, we determined vascularization of the neonatal retina at postnatal day 5 in mice lacking MALAT1 expression (MALAT–/–; Figure 4A). MALAT1–/– mice showed a delayed vessel extension in the retina when compared with wild-type littermates (Figure 4B and 4C). Moreover, a reduction of the vessel density, particularly in the front of the vasculature, was observed in MALAT1–/– mice when compared with wild-type littermates (Figure 4B and 4D). Interestingly, the number of proliferating endothelial cells, as identified by phosphohistone H3 staining, was significantly reduced (Figure 4E and 4F). However, the number of filopodia was not different in MALAT1–/– when compared with wild-type mice (103±5%; P=0.95).
To determine whether MALAT1 is required for postnatal neovascularization, we inhibited MALAT1 by GapmeRs in vivo. Intraperitoneal injection of GapmeRs directed against MALAT1 significantly and efficiently suppressed MALAT1 in control and ischemic muscle tissue at day 21 after induction of hindlimb ischemia (Figure 4G). Inhibition of MALAT1 significantly inhibited blood flow recovery as determined by laser-Doppler imaging and capillary density analysis (Figure 4H and 4I).
These data confirm the results of the above in vitro studies that silencing of MALAT1 profoundly impairs endothelial cell proliferation, which leads to a block in vessel outgrowth in vitro and in vivo.
MALAT1 Modulates the Expression of Cell Cycle Regulators
To gain insights into the mechanism by which MALAT1 regulates endothelial cell function, we performed microarray experiments using RNA of MALAT1 siRNA-treated HUVEC when compared with control siRNA-treated HUVEC. Bioinformatics pathway analysis revealed that genes involved in cell cycle (P=0.0003) and DNA replication (P=0.021) were most significantly regulated (Online Figure IV). As illustrated in Figure 5A, various critical cell cycle regulatory genes, such as cyclins (Cyc or CCN) A2, B1, and B2 and cyclin-dependent kinase 1 were downregulated by >2-fold (Online Table II), whereas the cell cycle inhibitor p21 (CDKN1A) was induced. The control of cell cycle regulatory genes by MALAT1 was further confirmed by quantitative reverse transcriptase-PCR, demonstrating that particularly S-phase cyclins CCNA2, CCNB1, and CCNB2 were significantly downregulated (Figure 5B). Moreover, the cell cycle inhibitory genes p21 and p27Kip1 were significantly increased on silencing of MALAT1 (Figure 5B).
As MALAT1 was previously shown to regulate splicing, we next assessed the expression of splicing-related genes, which seemed not to be regulated after MALAT1 silencing (Online Figure V). Furthermore, bioinformatics analysis showed that only few genes in which splicing was significantly regulated and conventional PCR analysis of selected potentially alternatively spliced genes also showed no evidence of alternative splicing (Online Figure VIA and VIB).
Here, we demonstrate that many lncRNAs are highly expressed in endothelial cells, including the well-conserved lncRNAs TUG1, MEG3, and MALAT1. We further report a novel, to date unknown function of MALAT1 in endothelial cells and showed that inhibition of MALAT1 induces a switch of the endothelial cell phenotype to a promigratory but antiproliferative state that resulted in impaired endothelial cell proliferation in vitro and in vivo and reduced retinal vessel growth.
The highly expressed lncRNAs MALAT1, TUG1, MEG3, and linc00657 were all further augmented by the exposure of endothelial cells to hypoxia, whereas linc00493 was not. TUG1 regulates retina differentiation20 and controls the proliferation of tumor cells,11 but its function in the vasculature is unknown. MEG3 exhibits a tumor suppressive function, and MEG3–/– mice show an increased expression of VEGFA in the brain.21,22 Whether the deletion of MEG3 leads to an endothelial cell intrinsic angiogenesis defect is unclear. The function of linc00657 and linc00493 is currently unknown. Although both transcripts are annotated as lncRNAs, they are localized in the cytoplasm, implicating that further studies are mandatory to exclude their function as protein-coding RNAs.
MALAT1 was initially discovered as a tumor-associated lncRNA and was reported to regulate splicing and epigenetic control of gene expression.11,12 The function of MALAT1 in the vasculature has not been studied, but the high expression of MALAT1 observed in cultured endothelial cells is consistent with previous results of whole-mount in situ hybridization in zebrafish, which showed a strong staining in the vasculature.23 The data of the present study additionally demonstrate that inhibition of MALAT1 in cultured endothelial cells enhanced sprouting but blocked cell cycle progression in vitro. Although the cell cycle inhibitory activity that we observed after silencing of MALAT1 expression in vitro and in vivo is consistent with previous findings in tumor cells and fibroblasts,11,24 the increased sprouting activity was unexpected because previous studies showed that inhibition of MALAT1 in tumor cells severely compromised the motility and migration of tumor cells.25,26 In contrast, in the present study, a promigratory activity was observed in 2 different in vitro assays (namely spheroid outgrowth assay and the scratched wound assay) after depleting MALAT1 expression by either siRNAs or GapmeRs. Interestingly, an increase in sprouting after silencing of MALAT1 was only observed under basal conditions, whereas no additional increase in sprouting was detectable after VEGF stimulation. This phenotype is likely because of an inhibition of endothelial cell proliferation that prevents the expansion of stalk cells and results in the discontinuation of sprouts. Overall, our data suggest that MALAT1 controls the phenotypic switch from migration to proliferation in endothelial cells, a typical aspect of endothelial cell biology.27
The antiproliferative effect observed after inhibition of MALAT1 is consistent with a profound dysregulation of cell cycle genes observed by gene expression profiling. Previous studies showed that MALAT1 is required for the recruitment of coactivators by polycomb 2 to the promoters of cell cycle control genes in HeLa cells,11 where it serves as a scaffold and physically recruits cell cycle genes into nuclear subdomains. This is associated with an increase in transcriptionally active histone marks and a decrease in repressive marks. In tumor cells, particularly the expression of CCNE1 and retinoblastoma protein proteins was downregulated on MALAT1 depletion. However, in our study, a different set of cell cycle regulators, namely members of the S-phase cyclins A and B, was modulated in endothelial cells. Interestingly, the change in expression of cell cycle regulatory genes in endothelial cells after MALAT1 depletion closely resembled a recently reported gene expression profile of human diploid fibroblasts, which also showed a profound reduction of CCNA2 and cyclin-dependent kinase 1 expression after silencing of MALAT1.24 These data indicate that the mechanisms by which MALAT1 controls cell cycle progression may be different in tumor cells when compared with somatic cells.
There are conflicting reports about the involvement of MALAT1 in splicing events.12,24,25 In particular, splicing of the oncogenic transcription factor B-myb was proposed to mediate the cell cycle regulatory activity of MALAT1 in fibroblasts.24 B-myb expression was also reduced in endothelial cells after silencing MALAT1, but we did not observe a significant regulation of splicing of B-myb (Online Figure VIA). Moreover, we did not observe a transcriptional regulation of splicing factors as shown by others in fibroblasts24 (Online Figure V) nor did we observe alternative splicing events in potentially alternatively spliced genes identified in our gene expression profiling (Online Figure VIB). Although we cannot fully exclude that deregulated splicing contributes to the observed effects of MALAT1 in endothelial cells, it seems rather unlikely. These data suggest a cell type or context-dependent difference in MALAT1 activity. Additional studies are necessary to define the precise molecular function of MALAT1, which may include >1 specific mode of action.
The findings of the present study may have therapeutic implications. MALAT1 is highly expressed in tumor cells,10 and its inhibition was recently proposed as a novel strategy to block metastasis and tumor growth.25 The present study now demonstrates that MALAT1 expression is augmented by hypoxia in endothelial cells and contributes to the proliferative response of endothelial cells. Therefore, one may speculate that the inhibition of MALAT1 may elicit an antiangiogenic effect in the hypoxic tumor environment that may contribute to a potential therapeutic benefit.
We thank Natalja Lerch, Marion Muhly-Reinholz, and Ariane Fischer for expert technical assistance and Dr Döring for help with exon array experiments.
Sources of Funding
The study was supported by the German Center for Cardiovascular Research (DZHK) supported by the German Ministery of Education and Research to S. Dimmeler and W. Chen, the LOEWE Center for Cell and Gene Therapy (State of Hessen) to S. Dimmeler and M. Zörnig, and the Deutsche Forschungsgemeinschaft (SFB834 to R.A. Boon and S. Uchida). The Georg-Speyer-Haus is funded jointly by the German Federal Ministry of Health and the Ministry of Higher Education, Research and the Arts of the state of Hessen.
In February 2014, the average time from submission to first decision for all original research papers submitted to Circulation Research was 13.8 days.
This article was sent to Ali J. Marian, Consulting Editor, for review by expert referees, editorial decision, and final disposition.
The online-only Data Supplement is available with this article at http://circres.ahajournals.org/lookup/suppl/doi:10.1161/CIRCRESAHA.114.303265/-/DC1.
- Nonstandard Abbreviations and Acronyms
- Cyc or CCN
- human umbilical vein endothelial cells
- long noncoding RNA
- locked nucleic acid
- metastasis-associated lung adenocarcinoma transcript 1
- maternally expressed 3
- natural antisense transcripts
- polymerase chain reaction
- small interfering RNA
- taurine upregulated gene 1
- vascular endothelial growth factor
- Received December 18, 2013.
- Revision received March 5, 2014.
- Accepted March 6, 2014.
- © 2014 American Heart Association, Inc.
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Novelty and Significance
What Is Known?
Long noncoding RNAs (lncRNAs) regulate several cell processes.
The lncRNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is highly conserved throughout evolution and expressed in tumor cells. It regulates tumor cell proliferation, apoptosis, and migration.
What New Information Does This Article Contribute?
A comprehensive analysis of lncRNAs present in endothelial cells.
MALAT1 is highly expressed in endothelial cells and regulates angiogenic sprouting in vitro.
Genetic deletion or inhibition of MALAT1 impairs vascularization and endothelial proliferation in vivo.
Therapeutic angiogenesis is a promising strategy to augment regeneration of ischemic tissue. LncRNAs have been described to play key roles in cellular processes, but a role for lncRNAs in angiogenesis has not been studied. The lncRNA MALAT1 is highly expressed in endothelial cells. It is induced by hypoxia and is required for proliferation of endothelial cells. Silencing with GapmeRs or genetic deletion of MALAT1 reduces neovascularization in mice. Therefore, augmenting MALAT1 could be a potential strategy to promote therapeutic neovascularization.