| Principal function of vascular smooth muscle cells(VSMCs)within adult blood vessels is contraction and regulation of vessel tone and diameter.Contractile VSMCs express differentiation marker genes,such as those encoding smooth muscle ?-actin(?-SMA),SM-myosin heavy chain(MHC),and SM22?.Serum response factor(SRF)reportedly activates multiple smooth muscle marker genes by recruiting several other cofactors,and transforming growth factor-?1(TGF-?1)signaling is involved in the regulation of contractile gene expression,such as ?-SMA.Neuregulin-1(NRG-1)is a member of the epidermal growth factor(EGF)family,and different isoforms can be produced from the NRG-1 gene by alternative splicing.Its transmembrane isoform includes an extracellular domain with an EGF-like sequence and a highly conserved intracellular domain(NRG-1-ICD).After this isoform releases a bioactive fragment containing the EGF-like receptor binding domain by proteolytic cleavage,the NRG-1-ICD translocates into the nucleus to regulate the gene expression probably via physically interacting with some unidentified transcription factors.Moreover,the NRG-1-ICD forms specific complexes with cytoplasmic proteins,including LIM kinase.Several lines of evidence support an important role for the NRG-1 in cardiovascular physiology and disease.First,NRG-1 is expressed in vascular endothelial cells,and its receptors are localized to the underlying smooth muscle cells.Second,treatment of cultured VSMCs with NRG-1 significantly decreases PDGF-stimulated proliferation and migration.Third,NRG-1 regulates myocardial performance and participates in the hemodynamic homeostasis of the cardiovascular system.However,the roles of NRG-1-ICD in the regulation of VSMC function in the context of TGF-?1 signaling remain unclear.Circular RNAs(circ RNAs)represent a novel class of non-coding RNA generated by back splicing.They regulate gene expression in eukaryotes by acting as cytoplasmic micro RNA sponges and RNA-binding protein sequestering agents as well as nuclear transcriptional regulators.In the vascular tissues,circ RNA c ZNF292 is expressed in endothelial cells and controls angiogenic sprouting through altering transcription factor activity.circ ANRIL is formed in VSMCs and macrophages in human atherosclerotic plaques and conferres atheroprotection through regulating ribosome biogenesis.However,little is known about the expressional regulation and the biological function of circ RNAs in the context of VSMC function.In the present study,we determined whether and how NRG-1-ICD and circ ACTA2 regulate VSMC function in the context of TGF-?1 signaling.Part? NRG-1 expression is induced by TGF-?1 in VSMCs,and its intracellular domain participates in cytoskeleton organizationObjective: To demonstrate the expression and roles of NRG-1 induced by TGF-?1 in VSMCs,and the function of NRG-1-ICD in VSMCs.Methods: 1 Immunofluorescence staining,Western blotting,and RT-PCR assays were performed to examine the expression of NRG-1 in VSMCs.2 Phalloidin staining for actin stress fibers was performed in an adenoviral vector encoding GFP-NRG-1,and confocal microscopy was performed with a Confocal Laser Scanning Microscope System(Leica).3 Cell contraction assay detected the NRG-1 influenced Ach-induced contraction of HASMCs.4 Cell immunofluorescence analysis was applied to detect the co-localization of NRG-1-ICD and ?-SMA.5 Co-immunoprecipitation assay was done to examine the interactions between NRG-1 and ?-SMA,NRG-1-ICD and ?-SMA.6 GST pull-down assay was performed to further reveal whether TGF-?1 influenced the relationship between NRG-1/NRG-1-ICD and ?-SMA in vitro.7 An in situ proximity ligation analysis(PLA),a method for visualizing individual protein interactions in cells.To further determine NRG-1 is related to its interaction with ?-SMA.Results: 1 TGF-?1 induces NRG-1 expression in HASMC.We first demonstrated that NRG-1 was expressed in arteries of the C57BL/6 mouse and was located in VSMCs,as evidenced by immunofluorescence staining with antibodies against NRG-1 and smooth muscle ?-actin(?-SMA),as well as by co-localization of NRG-1 with ?-SMA,a VSMC marker.Western blot and RT-PCR analyses also revealed that NRG-1 m RNA and protein were abundantly expressed in rat and human VSMCs,as well as in human cardiac microvascular endothelial cells.Because it is known that the expression of VSMC marker genes was regulated by TGF-?1 and PDGF-BB,we sought to determine whether TGF-?1 and PDGF-BB affected NRG-1 expression in VSMCs.q RT-PCR and Western blot analyses showed that TGF-?1 treatment increased,while PDGF-BB treatment decreased the expression of NRG-1 in a time-dependent manner,suggesting that NRG-1 expression facilitates TGF-?1-induced VSMC differentiation.2 NRG-1-ICD participates in cytoskeleton organization.To clarify the roles of NRG-1 in VSMCs,HASMCs were infected with an adenoviral vector encoding GFP or GFP-NRG-1 and stained for the actin cytoskeleton with TRITC-phalloidin.The results showed that the actin filaments were recruited into thick and long actin bundles in the NRG-1-overexpressed HASMCs,and their depolymerization induced by cytochalasin(CB)was markedly decreased,suggesting that NRG-1 participates in F-actin formation and stabilization of actin filaments.Because it is known that NRG-1 contains a highly conserved intracellular domain(ICD)and an extracellular domain with an EGF-like sequence(ECD),which is cleaved to release an N-terminal extracellular fragment,we sought to know which domains mediate the stress fiber formation.Thus,we treated HASMCs with HRG-?1(N-terminal extracellular fragment)and found that HRG-?1 treatment did not significantly affect the actin cytoskeleton.Conversely,NRG-1-ICD overexpression markedly enhanced the stress fiber formation.These results clearly suggest that NRG-1 mediates the cytoskeleton organization through its ICD domain.Moreover,NRG-1 overexpression significantly enhanced Ach-induced contraction of HASMCs 3 TGF-?1 increases the interactions of NRG-1/NRG-1-ICD with ?-SMA.To further determine whether NRG-1-ICD-mediated cytoskeleton organization is related to its interaction with ?-SMA,cell immunofluorescence analysis shows that NRG-1-ICD and ?-SMA markedly increased in HASMC cytoplasm 12 h after TGF-?1 treatment.Co-immunoprecipitation assay and GST pull-down assay showed that TGF-?1 increased the levels of NRG-1 and NRG-1-ICD present in anti-?-SMA immunoprecipitates.We performed an in situ proximity ligation analysis(PLA),a method for visualizing individual protein interactions in cells.As expected,association of endogenous NRG-1 with ?-SMA was observed in cytoplasm upon exposure to TGF-?1.These results indicated that ICD of NRG-1 participates in the cytoskeleton organization via its interaction with ?-SMA.Summary: TGF-?1 treatment increased the expression of NRG-1 in a time-dependent manner.NRG-1 mediates the cytoskeleton organization through its ICD domain.TGF-?1 increases the interaction of NRG-1/NRG-1-ICD with ?-SMA.Part ? circ ACTA2 mediates NRG-1-ICD regulation of ?-SMA expression in VSMCsObjective: To elucidate NRG-1-ICD regulate ?-SMA gene expression and circ ACTA2 can be generated from ?-SMA gene.To clarify whether circ ACTA2 expression is regulated by NRG-1 and circ ACTA2 plays important roles in regulating the TGF-?1/NRG-1-induced ?-SMA expression.Method: 1 Immunofluorescent detection revealed that the location of NRG-1-ICD.2 CHIP analysis was performed to examine the TGF-?1 could enhanced the recruitment of NRG-1-ICD to the first intron of ?-SMA.3 Wild-type and mutant NRG-1-ICD binding site was inserted into p GL3-basic dual luciferase reporter plasmid and transfected into 293 A cells,and dual luciferase reporter assay could measure its activity.4 Western blot revealed that the ?-SMA expression after overexpression or knockdown NRG-1-ICD.5 ?-SMA gene expression was detected by q RT-PCR in NRG-1-ICD overexpression.6 RT-PCR with Sanger sequencing and q RT-PCR with RNase R treatment to amplify total RNAs and the circ RNA of ?-SMA.7 q RT-PCR detects circ ACTA2 and ?-SMA m RNA expression after CRISPRi-mediated genomic blockage of NRG-1-ICD binding site in ?-SMA introns.8 circ ACTA2 expression was detected by q RT-PCR in both TGF-?1 treatment and NRG-1-ICD overexpression,singly or in combination.9 RNA interference was used to knock down the expression of both circ ACTA2 and ?-SMA m RNA,or only circ ACTA2.10 Western blot analyses revealed that the overexpression or knockdown of the circ ACTA2.Results: 1 NRG-1-ICD regulates the ?-SMA expression in HASMC.Multiple lines of evidence suggest that NRG-1-ICD may translocate into nucleus to regulate the expression of certain genes.We found that in basal condition without TGF-?1 stimulation,NRG-1-ICD was predominantly localized in the cytoplasm in HASMCs.After TGF-?1 stimulation for 12 h,NRG-1-ICD was markedly translocated into nuclei from cytoplasm.To identify NRG-1-ICD target genes in HASMCs,we performed a chromatin immunoprecipitation-sequencing(CHIP-seq).Analysis of CHIP-seq data showed the binding of NRG-1-ICD to sequences in the first intron of ?-SMA gene.Consistent with this,CHIP-q PCR further confirmed that there is NRG-1-ICD-binding site in the first intron of ?-SMA gene,and it is located approximately 5642 bp/-5787 bp downstream of the transcription start site,and TGF-?1 treatment significantly increased the binding of NRG-1-ICD to this site.Next,luciferase reporter vectors containing NRG-1-ICD-binding sequence of ?-SMA gene or its mutant were constructed and luciferase assay was performed.In these experiments,co-transfection of NRG-1-ICD expression vector with the luciferase reporter significantly increased luciferase activities,while mutation of NRG-1-ICD-binding site reduced luciferase activities..To further validate the role of NRG-1-ICD in TGF-?1-induced ?-SMA expression,Gain-and loss-of-function experiments for NRG-1-ICD were performed.In these experiments,NRG-1-ICD overexpression increased,while knockdown of NRG-1-ICD by si RNA decreased TGF-?1-induced ?-SMA expression.Unexpectedly,NRG-1-ICD overexpression did not significantly affect ?-SMA m RNA levels 2 NRG-1-ICD induces the generation of circ ACTA2.The results that NRG-1-ICD upregulated ?-SMA protein expression but did not affect m RNA level prompted us to consider the possibility that NRG-1-ICD might regulate ?-SMA expression at post-transcriptional level through micro RNAs or circular RNAs(circ RNAs).We hypothesized that NRG-1-ICD upregulated ?-SMA expression probably through circular RNA.To test this,we used convergent and divergent primers,respectively,to amplify total RNAs and the circ RNA of ?-SMA(circ ACTA2)by RT-PCR with Sanger sequencing and q RT-PCR with RNase R treatment.Fortunately,we found a novel 730 bp circ RNA,termed circ ACTA2,whose sequences are complementary to ?-SMA m RNA.In further experiments,we designed another divergent primers,in which nucleotide bases of 5'end were partially overlapped,to identify full-length circ ACTA2.Reverse transcription followed by RT-PCR and sequencing verified the presence of circ ACTA2.To clarify whether NRG-1-ICD regulated circ ACTA2 expression,we designed a single-chain guide RNA(sg RNA)complementary to the NRG-1-ICD binding site in the first ?-SMA intron to conduct CRISPR interference(CRISPRi),sterically repressed transcription by blocking transcriptional initiation or elongation with a catalytically dead Cas9.q RT-PCR showed that CRISPRi at the NRG-1-ICD binding site significantly reduced circ ACTA2,but not ?-SMA m RNA,levels relative to the control.Both TGF-?1 treatment and NRG-1-ICD overexpression,singly or in combination,significantly increased the expression of circ ACTA2 in cultured VSMCs.These results prompted us to investigate the function of circ ACTA2 in VSMCs.3 circ ACTA2 regulates the ?-SMA expression induced by TGF-?1.Initially,RNA interference was used to knock down the expression of both circ ACTA2 and ?-SMA m RNA.We designed two si RNAs: one si RNA targeting the backsplice sequence for circ ACTA2,another targeting sequence in a circularized exon shared by both linear and circular species.As expected,si RNA(si-circ ACTA2)targeting the backsplice sequence knocked down only circ ACTA2 but did not affect the expression of ?-SMA m RNA.si RNA(si-both)targeted to exon sequences shared by both the linear and circular species effectively knocked down circ ACTA2 and ?-SMA m RNA expression.Western blot analyses revealed that the overexpression or knockdown of the circ ACTA2 markedly increased or decreased ?-SMA protein level,respectively.Likewise,Knockdown using si-both RNA also dramatically decreased ?-SMA expression.In the further studies,the overexpression of circ ACTA2 further increased ?-SMA protein level induced by TGF-?1,while knockdown of circ ACTA2 partly abrogated TGF-?1-induced ?-SMA expression.Summary: Collectively,these data clearly suggest that circ ACTA2 plays important roles in regulating the TGF-?1/NRG-1-ICD-induced ?-SMA expression.Part? circ ACTA2 acts as a mi R-548f-5p sponge to regulate ?-SMA expression and regulates VSMC functionObjective: To explore the function of circACTA2 regulate of ?-SMA,and the role of it in HASMC.1 Wild-type and mutant circ ACTA2 was inserted into pmir-GLO dual luciferase reporter plasmid and transfected into 293 A cells,and dual luciferase reporter assay could measure its activity.2 We used biotin-labeled circ ACTA2 to pull down micro RNAs and biotin-labeled mi R-548f-5p to pull down circ RNAs.3 In situ hybridization was performed to co-locate the expression of circ ACTA2 and mi R-548f-5p.4 Wild-type and mutant 3' UTR of ?-SMA was inserted into pmir-GLO plasmid,transfected into 293 A cells and measured its activity by dual luciferase reporter assay.5 We transfected HASMCs with mi R-548f-5p mimic and anti-mi R-548f-5p to detect the ?-SMA protein level.6 Western blot detected the ?-SMA protein expression after NRG-1-ICD was overexpressed,circ ACTA2 was knocked down and silenced the mi R-548f-5p.7 Cell contraction assay detected the NRG-1-ICD? circ ACTA2 and mi R-548f-5p influenced Ach-induced contraction of HASMCs.Method:Results: 1 circ ACTA2 sponges with mi R-548f-5p and inhibits its activity.Because circ RNAs function as mi RNA sponges,and in the present study we verified the expression of circ ACTA2 in HASMCs,we next predicted mi RNA targets of circ ACTA2 and studied the circ ACTA2/mi RNA interaction.The heteroduplex analysis based on RNAhybrid and mi Randa showed that circ ACTA2 contains the sequences complementary to mi R-548f-5p seed sequence.We hypothesized that circ ACTA2-associated mi RNA may potentially inhibit the luciferase activity,presumably owing to the mi RNA-mediated activation of deadenylation and subsequent exonucleolytic degradation.Supporting this hypothesis,insertion of the circ ACTA2 sequence immediately downstream of the luciferase reporter gene caused downregulation of luciferase activity by mi R-548f-5p,while mutation of mi R-548f-5p target site in the circ ACTA2 sequence abrogated the inhibitory effect of mi R-548f-5p on reporter gene expression.In further study,we used biotin-labeled circ ACTA2 to pull down micro RNAs that is complementary to circ ACTA2 sequences from VSMC lysates.q RT-PCR results showed a more than 20-fold enrichment of mi R-548f-5p in the circ ACTA2-pulled down fraction compared with the negative control.Consistently,when biotin-labeled mi R-548f-5p was used to pull down circ ACTA2,mi R-548f-5p also increased dramatically the enrichment of circ ACTA2 in the mi R-548f-5p-pulled down fraction compared with the negative control.The results of co-localization experiments were consistent with circ ACTA2 interacting with mi R-548f-5p.In addition,TGF-?1 treatment and NRG-1-ICD overexpression,singly or in combination,significantly reduced mi R-548f-5p level.2 mi R-548f-5p inhibits ?-SMA expression by targeting its 3'-UTR in HASMCs.Interestingly,we found that the 3'-UTR of ?-SMA gene also contains mi R-548f-5p-binding site.To evaluate the effect of mi R-548f-5p on ?-SMA expression,we constructed the wild-type pmir GLO-?-SMA-3'UTR and its mutant pmir GLO-?-SMA-3'UTR-mut and co-transfected 293 A cells with them and mi R-548f-5p mimic.Luciferase assay showed that mi R-548f-5p mimic,but not the control oligonucleotide,could decrease luciferase activity by 60%,whereas the mutation of the mi R-548f-5p-binding site in the ?-SMA 3'UTR completely restored luciferase activity in the presence of the mi R-548f-5p mimic.In further experiments,we transfected HASMCs with mi R-548f-5p mimic and anti-mi R-548f-5p and confirmed that mi R-548f-5p mimic and its antagomir markedly reduced or increased the ?-SMA protein level,respectively,compared with their corresponding control.3 NRG-1/circ ACTA2/mi R-548f-5p axis regulates VSMC contractile function.To investigate the possible relationship between ?-SMA expression and circ ACTA2 or NRG-1-ICD,we overexpressed NRG-1-ICD with adenoviruses encoding NRG-1-ICD and simultaneously knocked down circ ACTA2 in HASMCs with si RNA specific for circ ACTA2.As expected,knockdown of circ ACTA2 markedly decreased ?-SMA expression induced by NRG-1-ICD overexpression compared with con-si RNA-transfected cells.Conversely,circ ACTA2 overexpression further increased ?-SMA protein level induced by NRG-1-ICD overexpression.The results that ?-SMA expression was upregulated by NRG-1-ICD or circ ACTA2 and downregulated by si-circ ACTA2 prompted us to hypothesize that circ ACTA2 is a crucial mediator of NRG-1-ICD-regulated ?-SMA expression.Consistent with this,co-expression of circ ACTA2 and NRG-1-ICD in HASMCs significantly increased the cell contraction induced by Ach compared with cells overexpressing circ ACTA2 or NRG-1-ICD alone.To further clarify the role of mi R-548f-5p in circ ACTA2-regulated ?-SMAexpression,we transfected HASMCs with anti-mi R-548f-5p and si-circ ACTA2 alone or in combination,and demonstrated that silencing of mi R-548f-5p by the corresponding antagomir markedly increased ?-SMA expression,whereas knockdown of circ ACTA2 markedly decreased ?-SMA expression compared with con-si RNA transfected cells.Conversely,circ ACTA2 overexpression significantly enhanced ?-SMA expression,and circ ACTA2 overexpression and silencing of mi R-548f-5p exerted a co-operative effect on ?-SMA expression.These findings suggest that circ ACTA2 can function as a sponge to bind mi R-548f-5p,resulting in increased ?-SMA expression.Similarly,mi R-548f-5p silencing and circ ACTA2 overexpression alone or in combination significantly stimulated VSMC contraction induced by Ach,and a combination of both exerted a co-operative effect,again suggesting that ?-SMA expression is regulated by mi R-548f-5p and circ ACTA2.Summary: Taken together,these data support an important role of NRG-1/circ ACTA2/mi R-548f-5p axis in the regulation of VSMC contractile function.Conclusion:1 NRG-1 expression is induced by TGF-?1 in VSMCs,and its intracellular domain participates in cytoskeleton organization.2 circ ACTA2 mediates NRG-1-ICD regulation of ?-SMA expression in VSMCs.3 circ ACTA2 acts as a mi R-548f-5p sponge to regulate ?-SMA expression.4 NRG-1/circ ACTA2/mi R-548f-5p axis regulates VSMC contractile function. |
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