1、miR-143 Activation Regulates Smooth Muscle and Endothelial Cell Crosstalk in Pulmonary Arterial HypertensionLin Deng#1,Francisco J.Blanco#1,Hannah Stevens1,Ruifang Lu1,6,Axelle Caudrillier1,Martin McBride1,John D McClure1,Jenny Grant1,Matthew Thomas2,7,Maria Frid3,Kurt Stenmark3,Kevin White1,8,Anita
2、 G.Seto4,Nicholas W.Morrell5,Angela C Bradshaw1,Margaret R.MacLean1,and Andrew H.Baker11Institute of Cardiovascular and Medical Sciences,University of Glasgow,Glasgow,G12 8TA,UK2Novartis Institutes for BioMedical Research,Horsham UK3Division of Critical Care Medicine/Cardiovascular Pulmonary Researc
3、h Laboratories,Department of Pediatrics and Medicine,University of Colorado Denver,Aurora,CO 80045,USA4MiRagen Therapeutics,Inc,Boulder,CO5Division of Respiratory Medicine,Department of Medicine,Addenbrookes Hospital,University of Cambridge School of Clinical Medicine,Cambridge,CB2 0QQ,UK6Kings Brit
4、ish Heart Foundation Centre,Kings College London,125 Coldharbour Lane,London SE59NU,United Kingdom7AstraZeneca R&D Mlndal,R&D|Respiratory,Inflammation and Autoimmunity(RIA)Innovative Medicines,Building AC461,SE-431 83 Mlndal,Sweden8Novartis Institutes for BioMedical Research,Inc.,250 Massachusetts A
5、venue,Cambridge,MA 02139,United States#These authors contributed equally to this work.AbstractRationaleThe pathogenesis of PAH remains unclear.The four microRNAs representing the miR-143 and miR-145 stem loops are genomically clustered.ObjectiveTo elucidate the transcriptional regulation of the miR-
6、143/145 cluster,and the role of miR-143 in PAH.Methods and ResultsWe identified the promoter region that regulates miR-143/145 miRNA expression in pulmonary artery smooth muscle cells(PASMCs).We mapped PAH-related signalling pathways,including estrogens receptor(ER),liver X factor/retinoic X recepto
7、r(LXR/RXR),TGF-(Smads),and hypoxia(HRE)that regulated levels of all pri-miR stem loop transcription and resulting miRNA expression.We observed that miR-143-3p is selectively Address correspondence to:Dr.Andrew H.Baker,Institute of Cardiovascular and Medical Sciences,University of Glasgow,Glasgow,G12
8、 8TA,UK.Tel No:+44 0141 330 1977,Fax No:+44 0141 330 3360,Andrew.H.Bakerglasgow.ac.uk.DISCLOSURES No conflicts of interest,financial or otherwise,are declared by the authors.Europe PMC Funders GroupAuthor ManuscriptCirc Res.Author manuscript;available in PMC 2016 April 23.Published in final edited f
9、orm as:Circ Res.2015 October 23;117(10):870883.doi:10.1161/CIRCRESAHA.115.306806.Europe PMC Funders Author Manuscripts Europe PMC Funders Author Manuscriptsupregulated compared to miR-143-5p during PASMC migration.Modulation of miR-143 in PASMCs significantly altered cell migration and apoptosis.In
10、addition,we found high abundance of miR-143-3p in PASMCs-derived exosomes.Using assays with pulmonary arterial endothelial cells(PAECs)we demonstrated a paracrine pro-migratory and pro-angiogenic effect of miR-143-3p enriched exosomes from PASMC.Quantitative PCR and in situ hybridisation showed elev
11、ated expression of miR-143 in calf models of PAH as well as in samples from PAH patients.Moreover,in contrast to our previous findings that had not supported a therapeutic role in vivo,we now demonstrate a protective role for miR-143 in experimental PH in vivo in miR-143/and antimiR143-3p-treated mi
12、ce exposed to chronic hypoxia in both preventative and reversal settings.ConclusionsmiR-143-3p modulated both cellular and exosome-mediated responses in pulmonary vascular cells,while inhibition of miR-143-3p blocked experimental PH.Taken together these findings confirm an important role for the miR
13、-143/145 cluster in PAH pathobiology.KeywordsmicroRNA;pulmonary hypertension;exosomes;transcriptional regulation;cell migration;smooth muscle cell;endotheliumINTRODUCTIONPulmonary arterial hypertension(PAH)is a rare,severe and progressive disease with an estimated prevalence of 15 cases per million1
14、.The pathogenesis of PAH includes sustained vasoconstriction and abnormal progressive fixed vascular remodelling.This is accompanied by endothelial dysfunction and activation of fibroblasts and smooth muscle cells2.PAH may be initiated by loss of endothelial integrity and dysfunction resulting in ex
15、posure of underlying cells to circulating factors,leading to proliferation and apoptosis resistance in the adventitia,smooth muscle media and the formation of a neointima3.Clinically,PAH is subdivided into several groups,including idiopathic(IPAH),heritable(HPAH),and PAH associated with other diseas
16、es(APAH).Female gender is considered a risk factor per se for all PAH subtypes,since it is more frequent in women than men4.Most cases of HPAH(70%),and some IPAH cases(approximately 20%),are caused by mutations in the bone morphogenetic protein type II receptor gene(BMPR2)that impairs BMP signalling
17、 pathway via Smad1,Smad5 and Smad8,and leads to an increased activity of TGF-pathway via non-canonical and canonical Smad2/3 signalling5.However,since PAH is incompletely penetrant,BMPR2 mutations alone may not be sufficient to cause disease,so that a second hit including other genetic and/or enviro
18、nmental factors,may be required for the clinical manifestation of PAH6.Triggers for disease may include inflammation,hypoxia and shear stress or vascular injury.MicroRNAs(miRNAs)are small non-coding RNAs that negatively regulate gene expression by recognizing the 3-untranslated regions(3-UTR)of targ
19、et coding mRNAs and abolishing their expression by blocking translation or accelerating their degradation7.Coding sequences of miRNA are distributed across the entire genome and can be classified by their location in either intragenic,typically within an intron sequence(mirtrons),or intergenic regio
20、ns.An Deng et al.Page 2Circ Res.Author manuscript;available in PMC 2016 April 23.Europe PMC Funders Author Manuscripts Europe PMC Funders Author Manuscriptsindependent promoter region drives expression of intergenic miRNAs.Thus,most intergenic miRNAs are transcribed by RNA polymerase II and the basa
21、l transcriptional machinery8.Although little is known about the transcriptional regulation of miRNA expression,this process has been proposed to be orchestrated by the cellular pool of transcription factors that interact with the promoter region,similarly to protein coding genes.Recent studies have
22、developed bioinformatics algorithms to predict the miRNA core promoters,focused on the upstream sequence of the precursor(pri-miRNA)9.In the particular case of intergenic miRNA,the promoter region is predicted to be 20 kb upstream of the pri-miRNA and exhibits similar conservation patterns to the pr
23、omoters of protein coding genes.Nonetheless,only a few papers have reported experimental evidence that validate those regions as core promoters.In blood vessels,one of the most studied miRNA expressed by vascular smooth muscle cells(VSMC)is the miR-143/145 cluster,which has a pivotal role in VSMC di
24、fferentiation and disease10-12.MiR-145 expression has been reported to control VSMC phenotype,restoring the contractile phenotype in atherosclerotic plaques13 and coronary collateral growth in the metabolic syndrome14.Expression of the miR-143/145 cluster is decreased in conditions associated with a
25、cute and chronic vascular stress,such as aortic aneurysms12 and coronary artery disease15.MiR-145-5p is able to control vascular neointimal lesion formation16.Up-regulation of miR-145-5p was observed in pulmonary artery VSMCs and in lung tissue from patients with idiopathic and heritable pulmonary a
26、rterial hypertension(PAH)17.However,the transcriptional regulation of the cluster has not been defined with respect to mediators of PAH nor has the role of miR-143 been addressed in PAH.Interestingly,despite their predominantly intracellular localization,miRNAs have been recently found in extracellu
27、lar compartments,such as exosomes18.These exosomes represent a specific subtype of secreted membrance vesicles of 30-130 nm formed through the fusion of multivesicular endosomes with the plasma membrane19.Exosomes convey a wide array of molecules such as proteins and nucleic acids,including mRNAs an
28、d miRNAs,and have emerged as regulators of cell-cell communication and paracrine signaling mediators during physiological and pathological processes in various diseases20,21.Here,we studied the transcriptional regulation of the miR-143/145 cluster and its contribution to the development of PAH.METHO
29、DSCell culture methods and reagents17,cloning the miR-143/145 proximal promoter and reporter vectors analysis RNA extraction,reverse transcription and TaqMan qPCR Analysis17 and in situ hybridisation,Smad3 decoy assays,isolation of exosomes from cell media,cell migration22 and proliferation23 assays
30、,Western blot and immunohistochemistry analyses24,Animal housing and experimentation including chronic hypoxia exposure and hemodynamic measurements24,and statistical analysis were carried out as previously reported elsewhere and are described in the expanded Methods section in the online data suppl
31、ement,available online at http:/circres.ahajournals.org.Deng et al.Page 3Circ Res.Author manuscript;available in PMC 2016 April 23.Europe PMC Funders Author Manuscripts Europe PMC Funders Author ManuscriptsRESULTSIdentification and cloning of the MIR143HG promoterWe first sought to define the transc
32、riptional regulation of the miR-143/145 cluster.According to miRStart data and using the human genome assembly GRCh38,the transcription start site(TSS,position+1)is likely to be located at position 149406877 in the plus strand of chromosome 5,22,041 bp upstream of the pri-miR143 precursor.Supporting
33、 this TSS,we identified a local TATA-box at position(26)(TATAAG),as well as 2 likely CAAT-boxes(CCAAT)at positions(94)and(54),and an E-box at position(113)(CACGTG)(Figure 1A).We selected a GC-rich(58.5%)region of 1.5 kb spanning from position(1354)to(+202),since the alignment with the homologous reg
34、ion from other mammalian species was strong(match 60%).A proximal region of 0.5 kb was also selected from(304)to(+202)for mapping transcriptional activity.Despite some GC-rich regions in the promoter,no putative CpG islands were predicted and experimentally the DNA demethylating reagent 5-Aza-2-deox
35、ycytidine did not affect the pri-miR-143/145 expression either in PASMC or PAEC/HUVEC(Online Figure I).Analysis of TF binding sitesThe in silico analysis of the 1.5kb sequence of miR-143/145 promoter revealed a number of putative binding sites for transcriptional factors(TFs).We treated PASMC with t
36、heir respective ligands,and pri-miR precursors and mature forms were detected by qPCR and luciferase reporter assays.Based on 3 predicted estrogen receptor binding sites at position(1129),(801)and(14),we treated PASMC with estradiol(E2).E2 induced the expression of both pri-miR-143 and pri-miR-145 a
37、fter 24 h(Figure 1B).The respective mature lead strand miRNAs were upregulated correspondingly(Figure 1C).Reporter vectors confirmed the increased transcriptional activation of the miR-143/145 promoter in response to E2(Figure 1D).Using site-directed mutagenesis,we studied which of the 3 ER sites ar
38、e necessary for ER-dependent induction of miR-143.We identified that the sites at positions(801)and(14)are required since the activity of the promoter is not enhanced when these sites are mutated Figure 1E).We also identified several putative binding sites for the retinoic acid receptor,which is wel
39、l known to act in combination with the liver X receptor(LXR/RXR)25.PASMC treated with 9-cis-retinoic acid(9cRA)and/or 22R-hydroxycholesterol(22R)upregulated both precursor and mature forms of the cluster(Figure 1F and 1G).The transcriptional activity of the reporter vectors showed a small but signif
40、icant induction for the minimal promoter(p0.5-luc)but this was more marked for the full-length sequence(p1.5-luc)(Figure 1H).Activity was dependent on the binding sites located at(673),(4)and(+98)(Figure 1I).We also identified 2 putative hypoxia response elements(HRE)and,upon stimulation,we observed
41、 a 2-fold induction in pri-miR-143 expression and moderate induction for pri-miR-145 in PASMC cultured in hypoxic conditions(Figure 1J).Expression of mature miR-143-5p and-3p were upregulated in the same pattern but only miR-145-5p and not miR-145-3p was induced(Figure 1K).Moreover,both minimal and
42、full-length reporters responded to hypoxia(Figure 1L).Mutation analysis confirmed that the HRE site in(113)that overlaps the basal transcription element E-box was critical since the minimal Deng et al.Page 4Circ Res.Author manuscript;available in PMC 2016 April 23.Europe PMC Funders Author Manuscrip
43、ts Europe PMC Funders Author Manuscriptspromoter p0.5-luc maintains the response to hypoxia(Figure 1M).We also searched for STAT sites in the MIR143HG promoter.It was predicted in silico that several consensus sites for STAT5A,STAT5B,STAT1 and STAT3 were in the region between(1354)and(304).None were
44、 located in the minimal 0.5kb region.The miR-143/145 promoter responds to TGF-Several putative binding sites for Smad proteins for both TGF-and BMPs signalling pathways were localised(Figure 1A).TGF-1 increased the expression of precursor and the 4 mature forms of the miR-143/145 cluster(Figures 2A
45、and 2B)in PASMC.Moreover,treatment of cells with SB525334,a specific inhibitor of the TGF-receptor ALK5,completely abolished this induction(Online Figure II).In contrast,BMP4 treatment did not affect the basal levels of the precursor forms or the mature miRNAs(Figure 2A and 2B).We therefore focused
46、on 2 putative Smad3 binding sites.Because TGF-1 induced the transcriptional activity of the full length reporter but not the minimal p0.5-luc construction(Figure 2C),we assayed the Smad3 element at position(592).Site-directed mutagenesis of this Smad3 binding site abolished the activity of the repor
47、ter in response to TGF-1(Figure 2D).Next,using release 138 of the 1000 Genomes Project26 on Ensembl we found 3 single nucleotide polymorphisms(SNPs)affecting the Smad3 binding site at position(592).One of these was outside the consensus motif(rs145177914,CT).However,the remaining two disrupted the S
48、mad3 binding site(rs12517403,TC;and rs116423755,GA).To test the functionality of this,a Smad3 decoy assay was designed using FAM-labelled PTO-ODN bearing the SNPs.The wild type and a scramble sequence were used as controls.A dose-response assay co-transfecting WT and mock probes in PASMC,with or wit
49、hout TGF-1 treatment,demonstrated that increasing WT probe reduces the expression of pri-miR-143 and pri-miR-145,as well as Col1A1 as a TGF-1 positive control,validating this sequence as a Smad3 binding site(Online Figure III).Furthermore,PTO-ODN probes bearing Smad3 SNPs were unable to block the TG
50、F-1 stimulus,confirming the functionality of that Smad3 site(Figure 2E and 2F).Manipulation of miR-143/145 cluster affects PASMCs migrationWe sought to assess the impact of promoter activation on PASMC migration,proliferation and apoptosis.We first sought to assess the effect of wound assay on induc