CircSATB2＿015 Regulates Human Vascular Smooth Muscle Cell Phenotypic Switch And Vascular Intimal Hyperplasia

Background:Cardiovascular diseases remain the leading cause of death worldwide.Abnormal proliferation and migration of vascular smooth muscle cells(VSMCs)play a vital role in the development of a variety of cardiovascular diseases such as atherosclerosis,venous graft failure,restenosis after angioplasty and pulmonary hypertension(PAH).VSMCs,located in the middle layer of the vessel walls,are highly specialized cells responsible for regulating vascular tone,which in turn regulates blood distribution and pressure.Unlike most terminally differentiated cells,VSMCs can switch between the contractile phenotype and synthetic phenotype,that is,the phenotypic switch of VSMCs,in response to external stimuli,including growth factors,cytokines,mechanical forces.Platelets-derived growth factor(PDGF)-BB,mainly released by vascular endothelial cells and platelets after injury,has been reported to regulate a variety of transcription factors and signaling pathways to promote proliferation and migration of VSMCs.PDGF-BB is recognized as one of the most effective stimuli of phenotypic switch of VSMCs.However,the molecular mechanism under the PDGF-BB induced phenotypic switch remains unclear.Circular RNAs(circ RNAs),a class of regulatory non-coding RNAs,is connected end to end by exons or introns,forming a unique circular structure,which is more stable against the degration by RNase,compared with traditional linear RNA.Emerging evidences indicate that circ RNAs play an important role in the regulation of physiological and pathological processes including development,aging,cancer and etc.Previously reported that circ RNAs can act as molecular sponge of micro RNAs(mi RNAs)to modulate the m RNA or protein level of the downstream target genes.Besides,circ RNAs also play a variety of functions such as protein scaffolding,transcriptional regulation,and peptide translation.Recent years have witnessed the rapid progress of circ RNAs research on cardiovascular diseases.For example,circ MAP3K5 regulates PDGF-BB mediated VSMC differentiation by modulating mi R-22-3p/TET2 pathway.Circ ITCH can sponge mi R-330-5p and upregulate the expressions of SIRT6,survivin and SERCA2 a,alleviating the adriamycin-induced cardiotoxicity.However,to date,studies on circ RNAs regulating VSMC phenotypic transformation are relatively few.Moreover,most VSMCs related circ RNAs are considered as mi RNA sponges in the cytoplasm,and the role of circ RNAs in post-transcriptional regulation is still unclear.Therefore,it is urgent to explore new circ RNAs related to VSMC phenotypic switch and fully reveal its downstream regulatory mechanism,which predicts great diagnostic and therapeutic value for vascular proliferative diseases such as atherosclerosis and PAH.In this study,we use high-throughput RNA sequencing to screen and analyze the differentially expressed circ RNAs in human aortic smooth muscle cells(Ao SMCs)stimulated by PDGF-BB.Bioinformatics analysis and expression verification experiments were used to identify the target circ RNA,and the biological functions such as regulating VSMC proliferation,migration and phenotypic switch and in-depth regulatory mechanisms were further studied.Part I: Screening and identification of circular RNA circSATB2＿015Methods:1)Linear and ribosomal RNA(r RNA)-free high-throughput sequencing was performed to screen the differentially expressed circ RNAs in PDGF-BB induced phenotypic switch model of Ao SMCs,and enrichment analysis was futher conducted;2)QRT-PCR was used to verify the expression of the candidate circ RNAs in PDGFBB or 10% FBS induced proliferation model and SMDM differentiation model;3)The circular structure of the target circ RNA was verified by c DNA/g DNA assay,Sanger sequencing,actinomycin D assay and RNase R digestion assay;4)Genetic information,conservation and translation potential of the target circ RNA were analyzed by searching databases including circ Base,circ Bank and UCSC,and cell expression specificity of the target circ RNA was determined by q RT-PCR;5)The cytoplasmic distribution of the target circ RNA was determined by RNA fluorescence in situ hybridization(FISH)and nucleocytoplasmic RNA separation of Ao SMCs;6)By HE staining,RNA-FISH and immunofluorescence(IF),the expression profile of the target circ RNA in the vascular tissues of patients with human coronary heart disease(CHD)was determined.Results:1)Through high-throughput sequencing screening,71 circ RNAs with differential expression in Ao SMCs under PDGF-BB stimulation were obtained(P < 0.05;Fold change≥ 1.5;up-regulated,40;down-regulated,31),and 12 candidate circ RNAs were screened by cluster analysis,conservation evaluation,GO enrichment analysis,KEGG and Reactome signal pathway analysis;2)QRT-PCR showed that hsa＿circ＿0003915(hsa＿circ SATB2＿015)was the most obviously down-regulated in PDGF-BB induced VSMC phenotypic switch model.In addition,a time-dependent downregulation of hsa＿circ＿0003915 was induced by both PDGF-BB and 10% FBS,while SMDM triggered time-dependent upregulation of hsa＿circ＿0003915;3)Hsa＿circ＿0003915 was amplified in c DNA by backsplice junction primer,while no amplification product was detected in g DNA.The splice sequence of hsa＿circ＿0003915 was detected by Sanger sequencing.Actinomycin D assay and RNase R enzyme assay showed that hsa＿circ＿0003915 had a longer half-life than linear RNA and could withstand the degradation by exonuclease;4)Hsa＿circ＿0003915 and mmu＿cir＿008822 were highly conserved in sequence(93%),with predicting open reading frame(ORF)and internal ribosomal entry site(IRES).Hsa＿circ＿0003915 was highly expressed in vascular cells including Ao SMCs,HUVECs,PASMCs;5)Nucleocytoplasmic RNA separation experiment showed that hsa＿circ＿0003915 was mainly located in cytoplasm(84.1% in cytoplasm and 15.9% in nucleus),which was further verified by RNA-FISH;6)RNA-FISH results showed that hsa＿circ＿0003915(circ SATB2＿015)was downregulated in coronary tissues of patients with CHD.Conclusion:Through high-throughput circ RNA sequencing,followed by bioinformatics,expression verification,circular formation experiments and molecular characterization analysis,we identified a significantly downregulated circ RNA,named circ SATB2＿015,in PDGF-BB stimulated Ao SMC phenotypic switch models and atherosclerotic vascular tissues.Circ SATB2＿015 is formed by back splicing of the 4-7 exon of SATB2 gene(NM＿001172509-2)on chromosome 2q33.1,with a total length of 531 bp.Circ SATB2＿015is enriched in cytoplasm and the sequence is highly conserved in human,mice and rats.Part II: Circ SATB2＿015 regulates the proliferation,migration and differentiation of Ao SMCs,inhibiting vascular intimal hyperplasiaMethods:1)Si RNA and overexpressing adenovirus of circ SATB2＿015 were designed,synthesized and transfected in Ao SMCs,and the knockdown and overexpression efficiency of circ SATB2＿015 was verified by q RT-PCR;2)Beyo Click TM EDU-594 assay was used to evaluate the effect of circ SATB2＿015knockdown or overexpression on Ao SMC proliferation;3)Scratch healing assay was used to evaluate the effect of circ SATB2＿015 knockdown or overexpression on the horizontal migration of Ao SMCs;4)Transwell migration assay was used to evaluate the effect of circ SATB2＿015knockdown or overexpression on the vertical migration of Ao SMCs;5)QRT-PCR and western blot were conducted to dectect the effect of circ SATB2＿015knockdown or overexpression on the expression levels of differentiation markers in Ao SMCs;6)A mouse carotid artery guidewire injury model was established,circ SATB2＿015overexpression adenovirus was perfused at the injury site,and the effect of circ SATB2＿015on intimal hyperplasia after injury was evaluated by HE staining.Results:1)Expression of circ SATB2＿015 was down-regulated to below 0.1 using circ SATB2＿015-si RNA-1,and infection with overexpression adenovirus(100 MOI)could increased the expression level of circ SATB2＿015 to 90 times.Both si RNA and overexpressed adenovirus did not affect the expression level of host gene SATB2;2)Beyo Click TM EDU-594 assays showed that knockdown or overexpression of circ SATB2＿015 could promote or inhibit the proliferation ability of Ao SMCs,respectively;3)Scratch healing assays showed that knockdown or overexpression of circ SATB2＿015could promote or inhibit the horizontal migration of Ao SMCs,respectively;4)Transwell migration assays showed that circ SATB2＿015 knockdown or overexpression could promote or inhibit the vertical migration of Ao SMCs,respectively;5)QRT-PCR and western blot demonstrated that knockdown or overexpression of circ SATB2＿015 can inhibit or promote the expression of α-smooth muscle actin(α-SMA,ACTA2),calponin 1(CNN1)and smooth muscle protein 22-α(SM22-α,TAGLN)in Ao SMCs;6)Perfusion of adenovirus overexpressing circ SATB2＿015 could significantly inhibit the phenotypic switch of VSMCs and alleviate intimal hyperplasia after carotid artery guidewire injury in mice.Conclusion:By transfecting si RNA or overexpressing adenovirus,circ SATB2＿015 was knocked down or overexpressed in Ao SMCs.The results showed that circ SATB2＿015 can inhibit the proliferation and migration of Ao SMCs,promote the switch of Ao SMCs to differentiated phenotypes.Overexpression of circ SATB2＿015 can significantly inhibit the intimal hyperplasia in mice treated with carotid artery guidewire injury in vivo.Part III: Circ SATB2＿015 promotes Ao SMC differentiation by regulating mi R-9-5p/myocardin pathwayMethods:1)Mi Randa software and mi RBase database were used to prediect the potential mi RNAs binding to circ SATB2＿015,and primers of candidate mi RNAs were designed using tail addition or stem loop method;2)QRT-PCR was used to verify the expression of target mi RNA in Ao SMC proliferation and differentiation models;3)QRT-PCR was used to evaluate the effect of circ SATB2＿015 knockdown or overexpression on the expression of target mi RNA;4)Circ RNA-mi RNA FISH assay was conducted to verify the co-location of circ SATB2＿015 and the target mi RNA in Ao SMCs;5)RNA binding protein immunoprecipitation assay(RIP)was performed to determine whether Ago2 protein could bind circ SATB2＿015 or the target mi RNA;6)RNA-RNA pull-down experiment,using the biotin-labeled RNA probe of circ SATB2＿015,was performed to verify the direct binding of circ SATB2＿015 to the target mi RNA;7)Mimic and inhibitor of the target mi RNA were designed,synthesized,and transefected in Ao SMCs,and the efficiency of overexpression or knockdown was determined by q RT-PCR;8)Double luciferase reporter gene experiment,using the synthesized circ SATB2＿015wild-type or mutant double luciferase reporter plasmid,was perfomed to verify the binding site of circ SATB2＿015 to the target mi RNA;9)Beyo Click TM EDU-594 assay was used to evaluate the effects of target mi RNA mimic and inhibitor on the proliferation of Ao SMCs;10)Scratch healing assay was used to evaluate the effects of target mi RNA mimic and inhibitor on Ao SMC migration;11)QRT-PCR and western blot were conducted to evaluate the effects of target mi RNA mimic and inhibitor on the expression levels of Ao SMCs-related differentiation markers;12)Mi RPath B,Targetscan and mi Rwallk website were used to predict the downstream genes of target mi RNA;13)QRT-PCR and western blot were conducted to evaluate the expression regulation of mi RNA on downstream target genes;14)Dual luciferase reporter gene experiment,using the synthesized wild-type and mutant dual luciferase reporter plasmids containing the 3’UTR binding sites of the target gene,was perfomed to verify the binding relationship between mi RNA and downstream target gene;15)QRT-PCR and western blot were conducted to evaluate the regulation of circ SATB2＿015 on the downstream target gene.Results:1)Circ SATB2＿015 was predicted with two binding sites to hsa-mi R-9-5p using mi Randa software,and the two binding sites were conserved in human and mice;2)PDGF-BB could induce time-dependent upregulation of mi R-9-5p,while SMDM induced time-dependent downregulation of mi R-9-5p;3)Knockdown or overexpression of circ SATB2＿015 could promote or inhibit the expression of mi R-9-5p in Ao SMCs,respectively;4)Circ RNA-mi RNA FISH co-localization assay showed that circ SATB2＿015 was colocated with mi R-9-5p in the cytoplasm of Ao SMCs;5)RIP results showed that both circ SATB2＿015 and mi R-9-5p were significantly enriched in the Ago2-RIP group compared with Ig G group;6)RNA-RNA pull-down experiment showed that compared with the negative probe group,circ SATB2＿015 and mi R-9-5p were significantly enriched in the circ SATB2＿015biotin probe group;7)Transfection of mi R-9-5p mimic or inhibitor could significantly increase or decrease the expression level of mi R-9-5p in Ao SMCs;8)Dual luciferase reporter gene assay showed that mi R-9-5p could bind to the predicting two binding sites on circ SATB2＿015;9)Beyo Click TM EDU-594 assays showed that mi R-9-5p mimic or inhibitor could promote or inhibit the proliferation of Ao SMCs,respectively;10)Scratch healing assays showed that mi R-9-5p mimic or inhibitor could promote or inhibit the horizontal migration of Ao SMCs,respectively;11)QRT-PCR and western blot showed that mi R-9-5p could inhibit the expression of differentiation markers,including ACTA2,CNN1,and TAGLN in Ao SMCs;12)Mi RPath B,Targetscan and mi Rwallk websites showed that myocardin(MYOCD)was the potential target of mi R-9-5p with two binding sites,and mi R-9-5p could negatively regulate the expression of MYOCD in Ao SMCs;13)Dual luciferase reporter assay showed that mi R-9-5p could directly bind to the3’UTR of MYOCD(site 1,1756-1763;site 2,5225-5231);14)QRT-PCR and western blot verified that knockdown or overexpression of circ SATB2＿015 could inhibit or promote the expression of MYOCD in Ao SMCs.Conclusion:1)RIP,RNA pull-down and dual luciferase reporter assay reveals that circ SATB2＿015can sponge mi R-9-5p and negatively regulate its expression;2)Mi R-9-5p can promote the proliferation and migration of Ao SMCs and inhibit the switch of Ao SMCs to differentiated phenotypes;3)Mi R-9-5p can sponge the downstream MYOCD by targeting its 3’UTR and block its m RNA and protein expression.Taken together,circ SATB2＿015 negatively regulates the expression of mi R-9-5p through the molecular sponge mechanism,thereby releasing the inhibitory effect of mi R-9-5p on downstream MYOCD,and promoting the switch of Ao SMCs into differentiated phenotypes.Part IV: Circ SATB2＿015 suppresses Ao SMC proliferation by inhibiting the SUMOylation of YBX1 and blocking the transcriptional activation of CDK1Methods:1)RNA-protein pull down assay,using biotin-labeled RNA probe of circ SATB2＿015,was conducted to obtain the proteins directly bound to circ SATB2＿015,followed by silver staining,protein mass spectrometry and enrichment analysis;2)Cat RAPID and RBPmap websites were used to predict the binding site between circ SATB2＿015 and YBX1;3)QRT-PCR and western blot were conducted to evaluate the regulatory role of PDGFBB and circ SATB2＿015 on RNA and protein levels of YBX1 in Ao SMCs;4)Cycloheximide(CHX)and MG132 treatments were performed to evaluate the effect of circ SATB2＿015 on YBX1 stability and ubiquitination degradation;5)SUMO SP 2.0.and Uniprot website was used to predict the SUMOylation sites of YBX1,and Cat RAPID website was used to predict the binding site of circ SATB2＿015 and SUMO2;6)Western blot was used to detect the effect of circ SATB2＿015 on SUMO2 protein level in Ao SMCs;7)Western blot was used to detect whether YBX1 or SUMO2 protein was enriched in the pull-down product of circ SATB2＿015 probe;8)RIP was conducted to determine whether circ SATB2＿015 was enriched in the RNA product pulled by the YBX1 or SUMO2 antibody;9)RNA-FISH and IF experiments were used to evaluate the intracellular localization of circ SATB2＿015 and YBX1;10)Pairwise Structure Alignment software was used to predict the spatial conformation of binding relationship between YBX1 and SUMO2,and co-immunoprecipitation(CO-IP)was conducted to verify the direct binding of YBX1 and SUMO2 protein;11)MG132 treatment was conducted to evaluate the effect of SUMO2 on the stability and ubiquitin degradation of YBX1;12)Nucleoplasmic isolation assay was used to determine the effect of circ SATB2＿015on the nuclear-cytoplasmic transport of YBX1 and SUMO2;13)Jaspar website was used to predict the binding site of YBX1 to CDK1 promoter region;14)YBX1 si RNA and overexpressing plasmid were designed and synthesized.QRTPCR and western blot were conducted to evaluate the the effect of YBX1 knockdown and overexpression on RNA and protein level of CDK1;15)QPCR and western blot were conducted to evaluate the regulation of circ SATB2＿015 on the RNA and protein level of CDK1.Results:1)RNA-protein pull down showed that specific enrichment of proteins was observed in circ SATB2＿015 biotin probe group,compared with the negative probe group,and enrichment analysis showed that the pull-down product is enriched in RNA binding,protein binding and other molecular functions;2)Cat RAPID and RBPmap websites showed that circ SATB2＿015 had the strongest binding ability with YBX1 in the position of 100-400nt;3)The protein level of YBX1 increased in a time-dependent manner under PDGF-BB stimulation,and knockdown or overexpression of circ SATB2＿015 could increase or decrease the YBX1 protein level,while the PDGF-BB and circ SATB2＿015 did not affect the m RNA level of YBX1;4)CHX treatment results showed that circ SATB2＿015 knockdown could prolong the half-life of YBX1 protein in Ao SMCs,and the downregulation of YBX1 protein induced by circ SATB2＿015 overexpression could be blocked by MG132 treatment;5)SUMO sp 2.0.and Uniprot websites showed that YBX1 had SUMOylation sites(SUMO2),and Cat RAPID websites demonstrated that circ SATB2＿015 could bind to SUMO2 protein,with the strongest binding ability between 100 and 300nt;6)Overexpression or knockdown of circ SATB2＿015 did not affect the protein level of SUMO2;7)Western blot showed that YBX1 and SUMO2 were significantly enriched in the pulldown product of circ SATB2＿015 probe;8)RIP results showed that circ SATB2＿015 was significantly enriched in the RNA product pulled by YBX1 or SUMO2 antibody;9)RNA-FISH and IF showed that circ SATB2＿015 was co-located with YBX1 protein in the cytoplasm of Ao SMCs;10)Co-IP results showed that SUMO2 protein were enriched in the YBX1-IP group,indicating the direct binding of YBX1 to SUMO2;11)Western blot confirmed that the protein level of YBX1 was significantly downregulated after SUMO2 knockdown,which could be reversed by MG132 treatment;12)Nucleoplasmic isolation assay showed that when circ SATB2＿015 was knocked down,the levels of YBX1 protein in both cytoplasm and nucleus increased,while the distribution of SUMO2 protein increased in cytoplasm and decreased in nucleus;13)Jaspar website predicted that YBX1 could bind to the promoter region of CDK1;14)QRT-PCR and western blot demonstrated that transfection with YBX1 si RNA or overexpression plasmid could effectively downregulate or upregulate the m RNA and protein level of CDK1;15)QRT-PCR and western blot showed that knockdown or overexpression of circ SATB2＿015 could promote or inhibit the expression of CDK1 in Ao SMCs.Conclusion:1)Circ SATB2＿015 can bind with both YBX1 and SUMO2 protein,inhibiting the stability of YBX1 and promoting its ubiquitination degradation;2)YBX1 can bind to the promoter region of CDK1 in the nucleus to activate its transcriptional activity;3)Circ SATB2＿015 acts as protein scaffold and inhibits the SUMOylation of YBX1,and blocks the transcriptional activation of downstream CDK1,which plays a critical role in regulating Ao SMC proliferation.