Novel LncRnas Regulating Vascular Smooth Muscle Cell Phenotype Switching And Cardiac Hypertrophy

Background:Hypertension,the elevation of systolic blood pressure,diastolic blood pressure,or both above normal levels（≥130/80 mm Hg）,is common in developed and developing countries.Hypertension is a leading risk factor of stroke,accelerated coronary and systemic atherosclerosis,heart failure,and chronic kidney disease.Vascular remodeling is a major pathological feature under chronic hypertension which is featured by neointima formation,outward hypertrophic,increased media thickness and stiffness.During the development of hypertension-induced vascular remodeling,the phenotype switching of vascular smooth muscle cells（VSMCs）from the differentiated contractile phenotype to the de-differentiated synthetic phenotype takes the central role.But the biological mechanism of VSMCs phenotype switching remains largely unknown.Meanwhile,hypertension is a major risk factor for heart failure.Pressure-overload causes cardiac hypertrophy,which is the core pathological feature of heart failure.So far,the underlying mechanism of cardiac hypertrophy is still limited.Long non-coding RNAs（lnc RNAs）,transcripts longer than 200 nt without protein-coding capacity,are a large subtype of non-coding RNAs with great numbers and essential functions.lnc RNAs exert their functions through the regulation of chromosome territory,transcription,post-transcription,translation,and post-translational modification.lnc RNAs take important roles in both physiological and pathological processes including in cardiovascular system.Though lnc RNAs have been reported to participate in heart development,heart failure,coronary artery disease,and myocardial infarction,the role of lnc RNAs in the hypertension-induced vascular remodeling has not been studied.Also,more lnc RNAs taking part in cardiac hypertrophy remain to be discovered.Therefore,we aim to screen out the functional lnc RNAs during the vascular remodeling as well as pressure overload induced cardiac hypertrophy respectively through high-throughput methods,look into the molecular mechanism and hopefully find out novel therapeutic targets.Part I:The Phenotype Switching Regulator Produces Both A nc RNA and A Short Peptide in Smooth Muscle Cell RegulationMethods:Chapter 1:1.1 To gain better insight to the underlying molecular changes during hypertensioninduced vascular remodeling,we applied transcriptomic analysis using an m RNA-lnc RNA microarray on the thoracic aortae tissue from 24-week-old spontaneously hypertensive rats（SHRs）and the normotensive control Wistar–Kyoto（WKY）rats.A subset of the differentially expressed lnc RNAs was further validated by q RT-PCR.Using RT-PCR,we tested the tissue-specific expression of the lnc RNAs and screen out the lnc RNA Phenotype Switching Regulator（lnc PSR）for further study.RNA FISH was used to test the subcellular localization of lnc PSR in the aorta tissue and VSMCs.1.2 We compared the conservation of lnc PSR between rat,mouse,and human using bioinformatic tools.And test the expression of human lnc PSR in the mesenteric artery specimens from hypertension patients with vascular remodeling.1.3 Using q RT-PCR,we tested the expression level of lnc PSR in the aorta tissue from WKY and SHRs of different ages.Using si RNA,we knocked down lnc PSR in the A10 rat aortic smooth muscle cells to test the change of contractile marker genes（α-SMA,α-MHC,Calponin）expression.Using CCK-8 and Ki-67 staining,we tested the proliferation of A10 cells after lnc PSR knock-down.Using wound-scratching assay and trans-well assay,we tested the migration of A10 cells after lnc PSR knock-down.1.4 We constructed the lnc PSR whole body knock out rat using CRISPR/Cas9 and using carotid artery balloon injury to induce the VSMCs phenotype switching in vivo.H&E staining was used to test the neointima formation.Ki-67 staining was used to test the proliferation of VSMCs in vivo.Chapter 2:2.1 We analyzed the potential ORFs in lnc PSR using bioinformatic tools,and compare the conservation among rat,mouse,and human.We also predicted the secondary structure of the ORF using bioinformatic tool.2.2 We made the specific antibody against this ORF（Arteridin）.Using WB,we tested the endogenous of Arteridin among different tissues of the rat.We constructed the overexpression vectors with tags to test the expression of Arteridin in HEK293 cells.Using immunostaining we tested the subcellular expression of endogenous and overexpressed Arteridin in rat A7r5 vascular smooth muscle cells.2.3 We constructed Arteridin KO rat using CRISPR/Cas9 without disturbing the expression of lnc PSR transcription.Carotid artery balloon injury was used to induce the VSMCs phenotype switching in vivo.H&E staining was used to test the neointima formation.Human Arteridin was overexpressed in rat A7r5 cells to test the functional conservation of Arteridin.And immunohistobiochemisty was used to test the expression of Arteridin in the mesenteric artery specimens from hypertension patients with vascular remodeling.2.4 We overexpressed lnc PSR full-length in A10 cells and tested the expression of contractile marker genes.Using different vectors,Arteridin-FLAG,Arteridin ATG deletion,lnc PSR full-length,lnc PSR full-length ATT mutant,and Arterdin Remix-FLAG to tell whether the phenotype switching function of lnc PSR comes from the transcript or Arterdin protein.Finally,we used RNA-seq to test the transcriptome of A10 cell with lnc PSR knock-down,Arteridin-FLAG overexpression,and lnc PSR full-length ATT mutant.Chapter 3:3.1 Using Arteridin co-IP followed by LC-MS,we find out the interacting protein of Arteridin In A10 cells.Using co-IP to validate the interaction between Arteridin and YBX1.Constructing YBX1 domain deletion vectors and using co-IP to find out which domain of YBX1 is responsible for the interaction with Arterdin.3.2 Using immunostaining to test the colocalization of Arteridin and YBX1 in A7r5 cells with Arteridin-FLAG and YBX1-HA overexpression.And using WB to test the nuclear translocation of YBX1 after Arteridin overexpression.3.3 To test the functional relationship between Arteridin and YBX1,we first used si RNA to knock-down YBX1 and used adenovirus to overexpress YBX1 in A7r5 cells,and tested the contractile genes expression.We then used TGF-β to treat A7r5 cells and detected the expression of lnc PSR and YBX1.Finally,we overexpressed Arteridin-FLAG with or without knock-down of YBX1 in the A7r5 cells and tested the contractile genes expression.Chapter 4:4.1 Using Arteridin Ch IP-seq to find out the DNAs bound by Arteridin in A10 cells with or without Arteridin overexpression and in rat aorta tissue.4.2 Using dual-luciferase reporter assay to test the transcriptional auto-regulation of Arteridin to lnc PSR.4.3 Using RIP,we tested the interaction between lnc PSR transcription with Arteridin and YBX1.Results:Chapter 1:Vascular smooth muscle cell dominant lnc RNA PSR is necessary for the VSMCs phenotype switching.1.1 Using lnc RNA-m RNA micro-array,we screened out 39 differentially expressed lnc RNAs in the aorta tissue of 24 weeks-old WKY and SHRs.And using q RT-PCR,we validated 6 lnc RNAs.Using RT-PCR,we tested the tissue specific expression of lnc RNAs and found rat lnc RNA NR₀27983（LOC680254 gene）was dominantly expressed in aorta tissue.We named this lnc RNA PSR.Using lnc PSR specific probe we tested the cell type expression in the aorta tissue with FISH and found lnc PSR was mainly expressed in the vascular smooth muscle cells of the middle layer of the vascular wall.In the VSMC,lnc PSR expresses both in the cytoplasm and nucleus.1.2 Using the UCSC and ENSEMBL database,we found lnc PSR is conserved among rat,mouse,and human.Using q RT-PCR,we found lnc PSR was also upregulated in the mesenteric artery tissue of hypertension patients with vascular remodeling.1.3 lnc PSR is upregulated only in the aorta tissue of 24-week-old SHRs but not in the 4 or 14 weeks old SHRs,indicating the function during vascular remodeling.Using si RNA we knocked down lnc PSR in the A10 VSMCs,resulting in the upregulation of contractile genes at both m RNA and protein level.Using CCK8 and Ki-67 staining,we found the proliferation of A10 cells was reduced significantly at both baseline and PDGF-BB stimulation.Using wound-scratching and trans-well assay,we found the PDGF-BB induced migration of A10 cells was reduced.1.4 Using CRISPR/Cas9,we constructed the lnc PSR whole-body KO rat and used balloon injury to induce the phenotype switching in vivo,found lnc PSR KO reduced the neointima formation.And Ki-67 staining showed VSMCs proliferation was attenuated in vivo.Chapter 2:lnc PSR encodes a protein Arteridin.Both the lnc PSR transcript and Arteridin Protein induce VSMCs phenotype switching.2.1 Using ORF finder and ATGpr online tools,we identified a 117 aa potential ORF in lnc PSR,which is conserved in mouse,while a 106 aa ORF exists in human lnc PSR.2.2 We made the specific antibody targeting the rat 117 aa ORF,and detected the endogenous expression in rat multiple tissues,finding a band of 12.9k Da only expressed in aorta tissue.We named this novel protein Arteridin.We then constructed the overexpression vectors of Arteridin with FLAG or e GFP tag at the 3?.Using WB,we detected the expression of Arterdin-FLAG or e GFP transfected in HEK293 cells,while no expression was detected with the ATG deletion mutants.In A7r5 cells,we found Arteridin mainly locate in the nucleus both at the endogenous level or after overexpression.2.3 To determine the function of Arteridin protein in the vasculature,we generated an Ateridin ablation rat using CRISPR/Cas9 mediated genome editing to introduce a single T insertion at the 39 th nucleotide downstream of the ATG start codon.This insertion created a premature stop codon TAG for the Arteridin ORF and resulted in a total loss of Ateridin peptide,while the lnc PSR transcript was only modestly affected.Attenuated neointima formation was also observed in the Arteridin-KO rats following carotid artery balloon injury.We also overexpressed human Arteridin in rat A7r5,which attenuated the expression of contractile genes,indicating functional conservation of Arteridin.Consistent with the results from rat,immunoblot using an anti-human Arteridin antibody also detected the human Arteridin peptide in tunica media from mesenteric artery tissue,and the signal was significantly elevated in the hypertensive patients correlated with the status of vascular remodeling.2.4 Overexpression of the PSR full-length c DNA transcript in A10 cells significantly downregulated the contractile phenotype genes.To determine the specific contribution of coded peptide vs.lnc RNA from PSR gene,we expressed Arteridin ORF with deletion of the start codon,ATG to ATT mutation,a synthetic c DNA construct which encodes the same rat Arteridin but with more than 30% alterations in the nucleotide sequences.Compared to the empty vector controls,the expression of Arterdin-FLAG-del ATG,lnc PSR-ATT,and Arteridin-Remix-FLAG all significantly reduced the expression of the contractile genes,α-SMA and calponin.2.5 We performed global transcriptome analysis in A10 cells after PSR knock-down,Arteridin-FLAG overexpression or lnc PSR-ATT mutant overexpression.By comparing with the untreated controls,a significant overlap in differentially expressed genes was identified between lnc PSR-ATT mutant and Arteridin-FLAG expressing cells.Furthermore,these differentially regulated genes were also significantly altered in the lnc PSR knock-down cells but in the opposite direction.Therefore,both lnc PSR non-coding transcript（nc RNA）and Arteridin protein can regulate a common set of genes in VSMCs.By GO analysis for all the differentially expressed genes affected by lnc PSR/Arteridin,the top three enriched biological processes were vasculature development,extracellular matrix organization,and collagen biogenesis,consistent with phenotypic switching regulation.Chapter 3:Arteridin interacts with YBX1 to induce VSMCs phenotype switching.3.1 We analyzed the Arteridin interacting molecules by performing immunoprecipitation of Arteridin from A10 cells with or without Arteridin overexpression followed by mass spectrometry.The top one molecule identified in the Arteridin complex was Nuclease-sensitive Element-Binding Protein 1（YBX1）.The interaction between rat Arteridin and YBX1 was confirmed by co-IP in HEK293 cells.Human Arteridin also binds human YBX1,showing the conservation of this interaction.Using various deletion mutants of HA-tagged YBX1 in co-immunoprecipitation assays,the C terminal domain of YBX1 was determined to be responsible for Arteridin binding.3.2 Arteridin and YBX1 were colocalized in rat A7r5 VSMCs as demonstrated by immunofluorescence confocal imaging and cell fractionation.YBX1-HA was mainly detected in the cytoplasm.However,co-expression of Arteridin induced marked translocation of YBX1 from the cytosol to nucleus,where Arteridin and YBX1 were colocalized in discrete granules.WB was also performed to test the nuclear translocation of YBX1 after Arteridin overexpression.3.3 TGF-β treatment in A7r5 VSMCs down-regulated both lnc PSR and YBX1 expression.Knockdown of YBX1 in A7r5 VSMCs significantly upregulated the contractile genes α-SMA and calponin while overexpression of YBX1 repressed them.These effects phenocopied the effect of Arteridin manipulation in VSMCs.Inactivation of YBX1 abolished the VSMC phenotype switching induced by Arteridin overexpression.Chapter 4:Arteridin auto-regulates transcription of lnc PSR forming a positive feedback loop.4.1 Chromatin immunoprecipitation from rat aortic A10 cells using anti-Arteridin antibody followed by DNA sequencing（Ch IP-seq）identified multiple Arteridin binding peaks in the rat genome.Particularly,two significant peaks were located at the transcriptional start site（TSS）and the second exon of its own gene.4.2 Arteridin expression significantly augmented the transcription of its own as demonstrated by a PSR-promoter-luciferase reporter in HEK293 cells.Using a series of DNA fragments for the reporter assay,we further narrowed the Arteridin dependent transcriptional activity within the PSR gene promoter to a fragment between 223 and 666 bp from the TSS.Importantly,the induction of PSR gene expression could be achieved by either Arteridin or Arteridin-Remix mutant in A10 cells,implying a potential positive feed-back regulation of Arteridin at a transcriptional level independent of its lnc PSR function.4.3 In A10 cells expressing Arteridin-FLAG,YBX1-HA,and domain deletion YBX1-HA,we detected a significant binding of the lnc PSR transcript to Arterdin and YBX1.The Cold Shock Domain（CSD）and the C-terminal domain of YBX1 were required for its interaction with lnc PSR.Conclusion:Our results uncovered a new genetic player（PSR）in vascular remodeling via targeted modulation of vascular smooth muscle cells phenotype switching.More importantly,we uncovered two products from the same PSR gene,an encoded peptide,and a non-coding RNA,exert the same biological effect in vascular smooth muscle cells.The autoregulatory effect of the protein product Arteridin on its own lnc RNA expression implies an intriguing positive-feedback mechanism to enforce target gene regulation during vascular remodeling.This new mode of gene regulation may have general implications in the precision and robustness of gene regulation.Part II:The Long Non-coding RNA Ahit Protects Against Cardiac Hypertrophy through SUZ12-Mediated MEF2 A InactivationMethods:Chapter1:1.1 Using mouse lnc RNA microarray,we compared the differentially expressed genes in mouse heart 2 weeks subjected to pressure-overload by transverse aortic constriction（TAC）.And using q RT-PCR to validate the differentially expressed genes.Screen out a candidate lnc RNA Ahit based on the tissue-specific expression in multiple tissues.Then using bioinformatic tools to analyze the conservation of lnc RNA Ahit among mouse,rat,and human as well as the coding potential of lnc RNA Ahit.1.2 In NRCMs,we knocked down Ahit using si RNA and overexpress Ahit using plasmid,then detect influence on the PE induced cardiomyocyte hypertrophy,including cell surface and hypertrophic genes expression.1.3 In vivo,we knocked down Ahit in mouse heart using AAV9-sh RNA driven by c Tn T promoter,and detect the influence on TAC induced cardiac hypertrophy through echo cardiac function test,WGA cell staining,H&E staining,and Masson trichrome staining of fibrosis.Chapter 2:2.1 In NRCMs knockdown Ahit and use q RT-PCR and WB detect the expression of the neighboring gene MEF2 A.In NRCMS knockdown Ahit together with knockdown MEF2 A to detect whether MEF2 A is necessary for the anti-hypertrophic function of Ahit.2.2 Using RNA FISH and subcellular fractionation to detect the subcellar localization of Ahit in NRCMs.Using cat RAPID to predict the interaction between Ahit and SUZ12.Using RIP and RNA pull-down to validate this interaction.2.3 In NRCMs,using Ch IP-PCR detect the interaction of SUZ12 and H3K27me3 binding to the promoter of the MEF2 A gene with or without Ahit knockdown.Results:Chapter 1:Identification of lnc RNA Ahit and its anti-hypertrophic function.1.1 Using lnc RNA microarray,we identified differentially expressed genes in mouse hearts 2 weeks subjected to TAC compared with sham.After q RT-PCR validation,we found lnc RNA 4833412C05 Rik to be upregulated in the TAC hearts,and expressed more abundantly in heart than other tissue.We named this lnc RNA Ahit.Ahit is conserved among mouse,rat,and human.The homologous lnc RNA in human is LUNAR1.LUNAR1 is upregulated in the serum of patients diagnosed with hypertensive heart disease.1.2 In NRCMs,knockdown of Ahit increased the cell surface enlargement and the expression of hypertrophic marker genes（ANP,BNP,β-MHC）induced by PE.While overexpression of Ahit attenuated the PE-induced hypertrophy.1.3 Using AAV9-sh RNA,we knocked down Ahit in the mouse heart and found an increase of cardiac hypertrophy induced by TAC,evidenced by elevated heart weight,decreased cardiac function,increased cell surface,and increased fibrosis.Chapter 2:lnc RNA Ahit interacts with SUZ12 and inactivates the transcription of MEF2 A.2.1 In NRCMs and mouse heart,knockdown of Ahit increased the expression of MEF2 A.In NRCMs,knockdown of Ahit together with MEF2 A knockdown,rescued the PE induced hypertrophy,indicating a MEF2A-dependent anti-hypertrophic function of Ahit.2.2 RNA FISH and subcellular fractionation showed Ahit to nuclear dominant lnc RNA.Using cat RAPID,we found Ahit has the potential to interact with SUZ12,a core compartment of PRC2.Using RIP and RNA pulldown,we validated this interaction.2.3 Using Ch IP-PCR,we found knockdown of Ahit leads to a decrease of SUZ12 binding to the promoter of the MEF2 A gene.And the H3K21me3 of MEF2 A gene promoter decreased accordingly.Conclusion:Our present work reveals a previously uncharacterized lnc RNA,Ahit that acts as an anti-hypertrophic regulator.Ahit protects against cardiac hypertrophy by modulating chromatin remodeling.Ahit binds with SUZ12,and recruits PRC2 to promote trimethylation of H3K27 on the MEF2 A promoter region.Finally,up-regulated Ahit,induced by stress,inhibits transcription factor MEF2 A expression to prevent the activation of cardiac hypertrophy-related genes,blocking cardiac remodeling.These discoveries provide new insight into the mechanism of cardiac hypertrophy and could have important implications for the treatment of pathological cardiac hypertrophy by targeting lnc RNA.