Published online before print March 22, 2007, doi: 10.1161/01.RES.0000265065.26744.17
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© 2007 American Heart Association, Inc.
Molecular Medicine |
Dicer Dependent MicroRNAs Regulate Gene Expression and Functions in Human Endothelial Cells
From the Department of Pathology (Y.S., J.S.P.), Department of Pharmacology (C.F.-H., W.C.S.), Yale University School of Medicine, New Haven, Conn.
Correspondence to William C. Sessa, Boyer Center for Molecular Medicine, Room 436, Yale University School of Medicine, New Haven, CT 06536. E-mail william.sessa{at}yale.edu
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Abstract |
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Dicer is a key enzyme involved in the maturation of microRNAS (miRNAs). miRNAs have been shown to be regulators of gene expression participating in the control of a wide range of physiological pathways. To assess the role of Dicer and consequently the importance of miRNAs in the biology and functions of human endothelial cells (EC) during angiogenesis, we globally reduced miRNAs in ECs by specific silencing Dicer using siRNA and examined the effects on EC phenotypes in vitro. The knockdown of Dicer in ECs altered the expression (mRNA and/or protein) of several key regulators of endothelial biology and angiogenesis, such as TEK/Tie-2, KDR/VEGFR2, Tie-1, endothelial nitric oxide synthase and IL-8. Although, Dicer knockdown increased activation of the endothelial nitric oxide synthase pathway it reduced proliferation and cord formation of EC in vitro. The miRNA expression profile of EC revealed 25 highly expressed miRNAs in human EC and using miRNA mimicry, miR-222/221 regulates endothelial nitric oxide synthase protein levels after Dicer silencing. Collectively, these results indicate that maintenance and regulation of endogenous miRNA levels via Dicer mediated processing is critical for EC gene expression and functions in vitro.
Key Words: endothelium • Dicer • miRNA • angiogenesis
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Introduction |
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MicroRNAs (miRNAs) are short noncoding RNAs that have been identified in a variety of organisms and have been shown to regulate gene expression.1,2 In mammalian cells, these small RNAs (
22 nt) are transcribed as parts of longer molecules that are processed in the nucleus into hairpins RNAs by the protein Drosha.3,4 These premiRNAs are then transported to the cytoplasm, via an exportin 5-dependent mechanism, where they are digested by a second, doubled-stranded specific ribonuclease called Dicer.5,6 The mature miRNAs are incorporated into a ribonucleoprotein complex7,8 or RISC complex,9 that mediates the downregulation of target gene activity by translational inhibition or target mRNA degradation, resulting in reduced levels of the corresponding protein or transcript, respectively.7,10,11 miRNAs have been implicated in the control of a wide range of physiological pathways12,13 such as development, differentiation, growth and metabolism.14–17 Moreover, tissue-specific patterns of miRNAs are providing insights into their possible functions. Many miRNAs exhibit striking organ specific expression patterns, or even expression restricted to single tissue layer within an organ18 and different miRNAs have been specifically cloned from heart, brain, embryonic stem cells and pancreatic islet cells.19–23
To dissect the significance of miRNAs in mammalian biology, several groups have disrupted the Dicer gene in mice24,25 and the loss of Dicer resulted in embryonic lethality, demonstrating that Dicer is necessary for normal mouse development. Other reports using conditional knockout approaches have demonstrated that Dicer plays essential roles in the maintenance of hair follicles,26 lung epithelium morphogenesis,27 T cell differentiation,28 whereas it is dispensable for some T cell lineage-specific gene expression programs.29 One report suggested the embryonic lethality observed in Dicerex
mice was because of defective blood vessel formation and maintenance. These anatomical defects were associated with altered expression of vascular endothelial growth factor (VEGF), its receptors KDR (VEGFR2) and FLT-1 (VEGFR1) and the angiopoietin receptor, Tie-1. These data suggest that Dicer exerts its function in the processing of miRNAs during embryonic angiogenesis and regulates the expression levels of critical angiogenic regulators.24 More recently, the expression of miRNAs in cultured human endothelial cells (EC) has recently been explored and the miRNA, miR-221/2 is necessary for the expression of c-Kit and Stem Cell Factor induced migration of EC.30
Angiogenesis is the development of new blood vessels from existing vascular structures and is a highly coordinated, multistep process. It is believed that EC migration, proliferation, differentiation and structural rearrangement of cells into patent vessels are crucial events for this process.31–33 However, the role of miRNAs in the biology and responses of EC during angiogenesis are incompletely understood. In the present work, we have globally reduced miRNAs in ECs by specifically reducing Dicer levels using siRNA and have examined several phenotypic responses in vitro. The knockdown of Dicer in ECs alters the expression of several important regulators of endothelial biology and angiogenesis, such as TEK/Tie-2, KDR/VEGFR2, endothelial nitric oxide synthase (eNOS) and IL-8. Moreover, Dicer knockdown increases activation of the eNOS pathway but reduces proliferation and cord formation of EC in vitro. Collectively, these results indicate that the reduction in miRNA levels via Dicer silencing strongly impacts EC functions suggesting a critical role for miRNAs in angiogenesis and EC remodeling.
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Materials and Methods |
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Quantitative real-time PCR, gene expression analysis by PCR array, Quantitative RT-PCR for miRNA, miRNA-array analysis, Western blot analysis, NO release, flow cytometry analyses, cell number assessment, crystal violet staining method, immunofluorescence microscopy, cord formation assay, migration experiments and statistical analysis are described in the online data supplement available at http://circres.ahajournals.org.
Cell Culture
Human umbilical vein endothelial cells (HUVECs) were isolated from discarded umbilical veins by collagenase digestion, under protocols approved by Yale, cultured onto tissue culture dishes with 0.1% gelatin in M199 (Invitrogen) containing 20% FBS (Hyclone), 50 µg/mL endothelial cell growth supplement (ECGS with heparin, BD Biosciences), penicillin-streptomycin; and L-glutamine (Invitrogen). HUVECs were used in passage 1 or 2. EA.hy.926 cells (endothelial cell line) were grown in high glucose Dulbecco‘s modified Eagle‘s medium (DMEM; Invitrogen) supplemented with 10% FBS, penicillin-streptomycin, L-glutamine and HAT (Sigma) at 37°C in a humidified atmosphere of 5% CO2.
RNA Interference
To silence Dicer, we used the published sequences of siRNA34 (siRNA Dicer 2 and 3) plus an additional one whose target sequence was 5'-AAGGGCACCCATCTCTAATTA-3' (siRNA Dicer 1). A scrambled siRNA was used as a negative nonsilencing control (NS) (5'-AATTCTCCGAACGTGTCACGT-3'). All the siRNA sequences were purchased from Qiagen. For cell transfection, EA.hy.926 cells and HUVECs were plated before transfection at a density of 25,000 to 35,000 cells/cm2, respectively. Individual siRNAs for Dicer 25 nM each (75 nM total) or nonsilencing siRNA (75 nM) were mixed with Oligofectamine and Opti-MEM (Invitrogen) and added to the cells. The cells were incubated for 8 hours at 37°C, and then HUVEC or EA.hy.926 growth medium, respectively, was added. In other experiments, cells were cotransfected with miRIDIAN miRNA mimics (Dharmacon). In all these expreriments control samples were treated with and equal concentration of a control mimic negative control.
Northern Blot Analysis for miRNA
Total RNA (10 µg) was resolved in a 15% polyacrylamide gel and blotted to a Nylon membrane Hybond-N+ (Amersham Biosciences). DNA oligonucleotides for hsa-miR-16, hsa-let-7a, hsa-miR-222, hsa-miR-31, hsa-miR-499 and hsa-miR-107 were 5'-C G C C A A T A T T T A C G T G C T G C T A-3', 5'-A A C T A T A C A A C C T A C T A C C T C A 3', 5'-G A G A C C C A G T A G C C A G A T G T A G C T-3', 5'-C A G C T A T G C C A G C A T C T T G C C-3', 5'-T T A A A C A T C A C T G C A A G T C T T A A-3' and 5'-T G A T A G C C C T G T A C A A T G C T G C T-3' respectively, complementary to the mature miRNA were end-labeled with [
-32p] ATP and T4 polynucleotide kinase (New England Biolabs) to generate high-specific activity probes. The oligonucleotide sequence for 5SrRNA was 5'-C A G G C C C G A C C C T G C T T A G C T T C C G A G A T C A G A C G A G A T-3'. Hybridization was carried according Express Hyb (Biorad) protocol. Probes were washed twice for 10 minutes at 25°C in 4 xSSC/0.5% SDS.
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Results |
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We initially assessed the expression of Dicer protein in EC via Western blotting. Dicer levels were readily detectable in HUVECs and were not changed after treatment (for up to 8 hours) with vascular endothelial growth factor (VEGF) a well known regulator of angiogenesis (Figure 1A). VEGF induced the phosphorylation of eNOS on serine 1179 as a positive control for VEGF action. To investigate the role of endogenous Dicer on EC functions, we developed a siRNA approach to reduce levels of Dicer in EC. As shown in Figure 1B, transfection of ECs (EA.hy.926 cells and HUVECs) with Dicer siRNA duplex (at 75 nM) reduced the expression of Dicer (via qRT-PCR) in both cell types in a time-dependent manner, with a maximal reduction at 48 hour (Figure 1B and 1C). The expression of Drosha, a ribonuclease implicated in the biogenesis of miRNAs was not affected (data not shown). Western blotting of cell extracts confirmed the reduction of Dicer expression was still evident at the protein level after 72 hour in both EC cultures (Figure 1D and 1E). Given that Dicer is the main regulator of miRNA biogenesis, we analyzed the expression of two miRNAs expressed in the majority of the cells including HUVECs (30 namely, hsa-miR-16 and hsa-let-7) before and after 60 hour of knockdown of Dicer. Analysis of these endogenous miRNA levels after Dicer silencing resulted in a substantial decrease in the levels of both miRNAs when analyzed by Northern blotting and qRT-PCR (Figure 1F, left panel for Northern and right panel in Figure 1G for qRT-PCR). The knockdown of Dicer in HUVECs did not affect cell morphology (by crystal violet staining), vWF localization in discrete cytoplasmic granules or VE-cadherin localization at the cell junctions, indicating no overt signs of endothelial cell injury or damage (Figure 1H).
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To examine potential functions attributed to miRNAs in ECs, we analyzed the effects of Dicer knockdown on the expression profile of several genes by PCR Arrays. EA.hy.926 cells were transfected with Dicer-specific siRNA or the nonsilencing siRNA for 60 hour and the levels of several angiogenesis and vascular remodeling genes were assessed. As seen in Figure 2A, the expression levels of several growth factors receptors were upregulated such as TEK (Tie-2), KDR (VEGFR2), adhesion molecules and proteins implicated in matrix remodeling including COL18A1 (Collagen, typeXVIII, 1), EDG1 (the endothelial differentiation sphingolipid G protein-coupled receptor 1) for the bioactive lipid, sphingosine 1- phosphate (S1P), ANPEP (Aminopeptidase N), ENG (Endoglin), and SERPINF1 (-2 antiplasmin) were upregulated, whereas PLAU (Urokinase Plasminogen activator) was downregulated. The expression of distinct chemokines and cytokines, IL-8, IL1ß, CXCL1, and CXCL3, implicated in inflammation and angiogenesis were also downregulated. Likewise several other angiogenesis related genes such as the protein kinase AKT1, the transcription factor Id3 (inhibitor of DNA binding 3) and the growth factors, ANGPT2 (Angiopoietin 2) and ANGPTL4 (Angiopoietin-like 4) were also regulated after the silencing of Dicer. The upregulation of gene expression in response to Dicer silencing were additionally confirmed by semi-quantitative Western blotting. As seen in Figure 2B in both EA.hy926 cells (left) and HUVECs (right), the loss of Dicer increased the protein levels of Tie-2, eNOS and AKT proteins, but did not change the levels of ERK or Hsp-90 (supplemental Figure IA). The increased protein levels of Tie-2, eNOS and AKT were also observed at different time points after Dicer silencing (Figure 2C). The expression of other proteins was assessed by flow cytometry. As seen in Figure 2D, the expression of KDR/VEGFR2 (first panel) was increased, whereas as the levels of VEGF (second panel) and PECAM-1 (third panel) were unchanged consistent with the mRNA data. Next, we examined the levels of the angiopoietin receptor, Tie-1, altered in Dicer1/2 embryos.24 The protein levels were increased without a significant increase in mRNA levels (supplemental Figure IA and IB available at http://circres.ahajournals.org). Collectively, our data indicate that the reduced expression of miRNAs by means of the knockdown of Dicer altered the expression of several critical regulators of EC function.
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Although the mRNA levels for Tie-1 and eNOS did not significantly increase after Dicer knockdown (supplemental Figure IA and Figure IIA available in the online data supplement), the levels of both proteins were consistently elevated (see Figure 3A, using different amounts of protein loaded with densitometric evaluation below the blots). To examine whether the increase in eNOS protein influenced NO production, the levels of NO (quantified by the stable breakdown product nitrite NO2–) were measured. As seen in Figure 3B (top panels), basal (10 hour accumulation) and stimulated (ATP; 10 µmol/L or S1P; 400 nM treatment for 30 minutes) NO release was increased following the knockdown of Dicer. Thus, the loss of Dicer dependent miRNAs does not significantly change eNOS mRNA levels but increases the levels of eNOS protein and NO production suggesting miRNA regulation of eNOS translational efficiency.
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To characterize the importance of miRNAs for additional EC functions in vitro, we examined the effects of Dicer silencing on three angiogenic phenotypes of EC, namely proliferation, migration and cord formation on reduced growth factor containing Matrigel. In both EC types, silencing of Dicer had a negative effect on cell proliferation. As seen in Figure 4A, knockdown of Dicer inhibited cell proliferation after 48 to 72 hour treatment with Dicer siRNA and similar results were obtained using the crystal violet staining for quantification (not shown). The effects of Dicer knockdown on cell proliferation was not associated with diminished cell viability, as indicated by absence of the subG1 population, or because of cell cycle arrest (Figure 4C). Instead, it was a result of a cell cycle delay from G1 to S phase indicated by a diminished incorporation of BrdUrd35 (Figure 4B inserts). In addition, the decrease in cell proliferation was not associated with activation of a dsRNA-interferon response (data not shown). Next, we examined the effects of Dicer silencing on EC migration in both EC types. Knockdown of Dicer reduced basal migration in EA.hy.926 cells, but did not significantly reduce VEGF or S1P induced migration in either EC type (see supplemental Figure II). However, knockdown of Dicer resulted in significant impairment of the cord formation under basal conditions and after stimulation with serum or VEGF (Figure 5A and quantified in graphs) in both EC types.
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Next, we determined the miRNA expression profile in both EC types. (Table; see also supplemental Table I in the online data supplement). In brief, miRNAs were isolated from exponentially growing HUVECs (3 cords pulled together for one sample repeated with an additional 3 cords) and EA.hy.926 cells (experimental duplicates collected at different time points) and duplicates of each sample run on miRNA microarrays (see Material and Methods). Background was subtracted from all samples and data normalized to the level of let-7A (which was 1 to 1.5-fold above background). The Table shows the 25 miRNAs giving the strongest hybridization signals in HUVECs compared with the same miRNAs in EA.hy.926 cells. The levels of certain miRNA identified in the arrays, were validated by Northern blotting using 5SrRNA as a loading control. Specifically, the expression of hsa-miR-222, hsa-miR-31, hsa-miR-107 and "pred00211" (predicted miRNA) recently described as hsa-miR-499,36 was confirmed by Northern blotting and compared with the expression of these miRNAs in other human cells (Figure 6). These results indicate that hsa-miR-222 and hsa-miR-499 are highly expressed in EC and VSMC in comparison to hsa-miR-31 (preferentially expressed in VSMC) and hsa-miR-107 (highly expressed in monocytes) suggesting cell specific expression patterns of miRNAs in vascular cells.
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Because hsa-miR-222 and hsa-miR-221 were highly expressed miRNAs in EC, we examined whether specific mimics for these miRNAs could rescue/restore some of the effects produced by Dicer silencing (Figure 7). As seen in Figure 7A, Dicer silencing increased the protein levels of Tie-2 and eNOS as shown above (see Figure 2 and 3
). Transfection with hsa-miR-222 or hsa-miR-221 mimics slightly reduced the increased eNOS protein levels without affecting the levels of Tie-2. However, these mimics could not rescue the defect in EC proliferation because of Dicer silencing (Figure 7C).
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Discussion |
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In this study, the levels of Dicer were reduced using siRNA to ascertain the global role of miRNA function in cultured human ECs. The RNAi-mediated knockdown of Dicer in ECs, resulted in a significant reduction, but not complete loss of mature miRNAs. The incomplete loss of miRNAs likely reflects the long half-life of endogenous miRNAs37 over the relatively short time period of these experiments.34 Under these circumstances, the reduction in Dicer levels changed the pattern of gene expression in ECs presumably via miRNA modulation of steady-state mRNA levels and/or mRNA translation of many gene products.11,38 After silencing Dicer in ECs, we found that different pathways implicated in regulating EC functions were either increased or decreased. The net phenotype of Dicer knockdown in ECs was an increase in NO release, and decrease in both EC growth and cord formation in Matrigel, but a negligible effect on EC migration. The decrease in growth and morphogenesis in ECs with reduced levels of Dicer, is consistent with a recent report showing that Dicer is required for embryonic and yolk sac angiogenesis in mice,24 suggesting that some of the molecular signals and mechanisms that govern the activity of miRNAs in embryonic vascular development are also functional in cultured human EC.
Work in hypomorphic Dicerex1/2 mice has shown that the defects in embryonic angiogenesis were associated with an upregulation of FLK/VEGFR2, FLT/VEGFR1 and downregulation of Tie-1 suggesting that the VEGF signaling pathway was upregulated to compensate for failed angiogenesis and remodeling. In cultured ECs, we also detected a marked upregulation of KDR/VEGFR-2, FLT/VEGFR1 but little changes in VEGF-A or VEGF-C suggesting that upregulation of VEGF receptors is likely a direct effect of the loss of Dicer via miRNA regulation of mRNA stability or translational interference. Accordingly, Tie-2 and Tie-1, were also upregulated, suggesting that the VEGF and angiopoietin pathways are targets for miRNA regulation.39,40 Paradoxically, in cultured ECs the protein levels of Tie-1 were increased whereas in the hypomorphic Dicerex1/2 embryos were diminished.24 Several angiogenesis- and vascular remodeling- related genes such as ANGPT2, ANPEP, SERPINF1, COL18A1, IL-8 and ANGPTL4 were also regulated. However, no consistent pattern of pro- versus antiangiogenic profiles, of the observed genes, was apparent at the time points examined after Dicer knockdown in cultured ECs.
Although both Tie-1 and eNOS mRNA levels were not significantly elevated after Dicer siRNA treatment, the levels of Tie-1 and to a greater extent eNOS protein were consistently higher suggesting miRNA control of protein translational efficiency. eNOS was initially defined as a constitutively expressed enzyme, however recent studies highlight the contribution of both transcription and mRNA stabilization to overall expression levels of eNOS mRNA and protein41,42 and our data supports a translational or posttranslational mechanism of eNOS and Tie-1 regulation by miRNAs. Functionally, the increased levels of eNOS correlated with an increased NO release. NO synthesized by eNOS is necessary for endothelial cell survival, migration and angiogenesis43 in post-natal mice,44 whereas it is dispensable for vascular development.44 Paradoxically, although eNOS and other pro-angiogenic genes were upregulated by reducing the expression of miRNAs after Dicer silencing, EC proliferation and cord forming activity were reduced. This is consistent with an impaired vascular development in Dicerex1/2 mice24 despite compensatory upregulation of FLK/VEGFR2 and FLT/VEGFR1.
Based on miRNA microarrays, we detected 40 miRNAs commonly expressed in HUVECs and EA.hy.926 cells (supplemental Table I) and the most abundant 25 shown in Table. Twelve of the 25 highly expressed miRNAs were consistent with a recent study,30 however, our analysis detected an additional 13 miRNAs namely, CAND853_ZF, hsa-miR-107, -181a, -181b, -213, -22, -27, -320, -321, -91, -92 to 1,2, -499 (aka pred 00211) and pred 00209 (see Table and supplemental Table I). The expression of 6 different miRNAs were confirmed by Northern blotting with miR-222 and -499 being highly expressed in EC to a greater extent than in VSMC, whereas, miR-31 was more robustly expressed in VSMC and miR-107 in monocytes. Because miR-221 and 222 were among the highest expressed in ECs, we examined two potential targets regulated by Dicer silencing, namely eNOS and Tie-2, in EC by transfecting with miRNA mimics. In theory, the miRNA mimic should recapitulate the effects of the endogenous miRNA on a given gene product. Interestingly, transfection of miR-221and 222 mimics partially restored the elevated eNOS protein levels, but not Tie-2 levels, after Dicer silencing. However, using different predictive algorithms, miR-221/222 were not found to target the 3' UTR of eNOS and Tie-2; similar results for Tie-2 were found by Poliseno et al.30 This suggests that the partial rescue with the miR-221 and -222 mimics could be indirect via gene expression, translational efficiency or post-translational mechanisms. Indeed, overexpression and knockdown of individual miRNAs should be performed to gain further insight into miRNA regulation of targets reported here. The only example of a cell specific miRNA regulating a specific target in EC has been recently shown. Felli and Poliseno45,30 found high expression of miR-222 and miR-221 in human cord blood-derived CD34+ hematopoietic progenitor cells (HPCs) and HUVECs, respectively. Bioinformatic analysis suggested c-kit as a possible target whose levels correlate inversely with those of hsa-miR-222 and hsa-miR-221 in cultured cells. Forced expression of these miRNAs mimics slowed cell growth concomitant with reduced c-kit protein levels in HPCs,45 or diminished stem cell factor mediated cell migration.30 Although target disruption of individual miRNAs and targeted mutation of putative miRNA-binding sites of candidate genes are necessary to prove specific targets of miRNAs, the redundancy among miRNA regulators may limit the power of such experiments and a thorough understanding of miRNA functions will only emerge from integrated consideration of complementary results.46
The possibility that a single miRNA may target multiple transcripts and that individual transcripts may be subject to regulation by multiple miRNAs amplifies the scope of putative miRNA regulation of mRNA and protein levels in cells. Additionally, it is likely that not all miRNAs function similarly: some may indeed regulate a single gene tightly, whereas others may have a wider influence over gene expression programs associated with selected developmental or physiological states.46 In line with this concept, the antiproliferative effect of Dicer silencing was not rescued by transfection of the miR-222 and 221 mimics suggesting that other individual or multiple miRNAs clusters may regulate specifically EC growth, similar to the lack of the effects of these mimics on growth in HUVECs,30 but not in HPCs, TF-1 cells (Erythroleukemic cell line).45 Because the role of miRNA regulation and function in vascular cells is a new emerging area, additional investigation is needed to study the contribution of individual miRNAs or miRNA clusters in controlling gene expression that governs the angiogenic program in EC.
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Acknowledgments |
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Sources of Funding
This work was supported by grants from the National Institute of Health (R01 HL64793, RO1 HL 61371, R01 HL 57665, PO1 HL 70295, Contract No. N01-HV-28186 (NHLBI-Yale Proteomics Contract) to W.C.S and Programme 3+3 Fellowship from the Centro Nacional de Investigaciones Cardiovasculares (CNIC), Spain to Y. Suárez. C. Fernández-Hernando is supported by a fellowship from the Ministerio de Educación y Ciencia (Spain).
Disclosures
None.
miRNA arrays were performed by Penn Microarray Facility, University of Pennsylvania. The authors thank to Dr Don Baldwin and Robert Benjamin Isset (UPENN) and Keck facility (Yale University) for miRNA array analysis. We are grateful to Jan Pawlicki for helpful comments for miRNA Northern blots.
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Footnotes |
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Original received December 8, 2006; resubmission received February 13, 2007; revised resubmission received March 9, 2007; accepted March 14, 2007.
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