Mechanisms Of Circ-Sirt1 Upregulated The Expression Of SIRT1 In Vascular Smooth Muscle Cells

Objective:As a defense response of vascular system to damage factors,the inflammation and proliferation of vascular smooth muscle cells(VSMCs)have been considered as important etiological factors during the development of cardiovascular diseases.circRNAs are a special class of endogenous non-coding RNAs,which have covalent closed loop structure.With extensive roles,circRNAs can act as miRNA sponge to regulate the stability of mRNA in post-transcriptional level.In addition,it can form protein-RNA complex to regulate downstream signaling pathways and to promote the transcription of linear parent genes.Recent studies have shown that circRNAs are involved in regulation of the cellular proliferation,apoptosis,aging and differentiation.In this study,we investigated the mechanisms of circ-Sirt1 upregulated the expression of SIRT1 in VSMCs.Methods:The overexpression or the knockdown of circ-Sirt1 were performed in cultured VSMCs.The assays of RIP,RNA pull-down,FISH and luciferase reporter gene were used to determine the involvement of circ-Sirt1 in the post-transcriptional modification of SIRT1.The inflammation model of VSMCs was established by TNF-α stimulation.The relationship between circ-Sirt1 and the activity of NF-κB p65 was detected using ChIP,oligonucleotide pull-down assays and luciferase reporter gene assay,respectively.Western blot and coimmunoprecipitation(Co-IP)assay were used to identify the effect of circ-Sirt1 mediated SIRT1 expression on the nuclear translocation and deacetylation level of NF-κB p65.The carotid balloon injury model was used to investigate the relationship between the circ-Sirt1 overexpression and the VSMC proliferation and neointimal formation in vivo.Results: 1 Molecular mechanism of the upregulation of SIRT1 by circ-Sirt11.1 circ-Sirt1 upregulated the expresssion of SIRT1According to the previous studies,circ-Sirt1 was mainly distributed in the cytoplasm with high expression specifically.To evaluate the regulatory function of circ-Sirt1 to SIRT1 expression,the overexpression of circ-Sirt1 with adenovirus vector or the knockdown of circ-Sirt1 with siRNA were performed in cultured VSMCs followed by the assays of qRT-PCR and Western blot.Our data showed that circ-Sirt1 positively regulate the expression of SIRT1 at post-transcriptional level.1.2 The interaction between circ-Sirt1 and miR-132/212-3PThe bioinformatics predict showed that circ-Sirt1 contained 3 potential sites interacted with miR-132/212-3p.The results of immunoprecipitation(RIP)assay showed that circ-Sirt1 was specifically enriched by Ago2 antibody but not the control Ig G.To test the miRNA sponge action of circ-Sirt1,biotin-labeled or FAM-labled miR-132/212-3p probe was used to pull down circRNAs,and revealed more circ-Sirt1 in the pulled down sediments.Similarly,miR-132/212-3p were also enriched in circ-Sirt1-pulled down sediments.Furthermore,we also observed the same results using RNA in situ hybridization.It revealed the interactions between circ-Sirt1 and miR-132/212-3p.These data indicate that circ-Sirt1 directly binds to miR132/212-3p.1.3 circ-Sirt1 relieves the inhibitory effect of mi R132/212-3p on SIRT1 mRNAAccording to the bioinformatics prediction that circ-Sirt1 contained 3 potential sites interacted with mi R-132/212-3p,we speculated the SIRT1 expression can be regulated through the competitive binding of circ-Sirt1 and miR-132/212-3P.To confirm our hypothesis,full length of circ-Sirt1 was cloned into pGL3 luciferase reporter vector and the recombined vector(LUC-circ-Sirt1)was co-transfected miR-132/212-3P mimics into 293 A cells.Compared with the control miRNA mimics,miR-132/212-3p mimics significantly reduced the activity of LUC-circ-Sirt1 in HEK293 A.When the 3’UTR of SIRT1 containing binding sites of miR-132/212-3P and its mutants were cloned into the luciferase vector(LUC-SIRT1 3’UTR)and co-transfected with miR-132/212-3p mimic into HEK293 A cells,a significant reduction in luciferase activity was observed in presence of miR-132/212-3p mimics,whereas the mutants in binding sites targeting the seed sequences of miR-132/212-3p abolished this repression.These data indicate that circ-Sirt1 can regulate the expression of SIRT1 though the binding to miR132/212-3p.In addition,the suppressive effect of miR-132/212-3P on SIRT1 3’UTR was abolished by the overexpression of circ-Sirt1.These data indicate that circ-Sirt1 directly binds to miR132/212-3p,and relieves the inhibitory effect of miR132/212-3p on SIRT1 mRNA.2 circ-Sirt1 mediated SIRT1 expression promotes the NF-κB p65 inactivation through deacetylation 2.1 circ-Sirt1 inhibits the activation of NF-κB p65We found that overexpression of circ-sirt1 did not completely block TNF-α-induced nuclear translocation of NF-κB p65.Moreover,the decreasing of nuclear p65 level was less than that of pro-inflammatory factors,implying that the activity of nuclear NF-κB p65 may be reduced.To verify this,DNA binding and transcriptional activity of NF-κB were analyzed in VSMCs stimulated with TNF-α using Ch IP and oligonucleotide pull-down assays,respectively.We showed that overexpression of circ-Sirt1 completely abolished TNF-α-induced binding of NF-κB to the DNA elements.To confirm the decreasing in transactivation potential of NF-κB,we constructed circ-sirt1 expression vector pcDNA-circ-Sirt1.The inhibitory effect of circ-Sirt1 on the nuclear NF-κB activity was further supported by the observation obtained in reporter gene assay via transiently co-expressing pcDNA-circ-Sirt1 with a luciferase reporter driven by a 6 tandem-repeat NF-κB element in HEK293 A,suggesting that circ-Sirt1 resulted in a decreased NF-κB activity..2.2 circ-Sirt1-mediated SIRT1 expression promotes the deacetylation of p65To elucidate the mechanism underlying circ-Sirt1 decreasing nuclear NF-κB activity,the acetylation of NF-κB p65(Ac-p65)was tested in VSMCs with and without overexpression of circ-sirt1 upon TNF-α treatment.We showed that TNF-α-induced Ac-p65 was mainly expressed in nucleus.Overexpression of circ-sirt1 markedly suppressed nuclear Ac-p65 expression.Using coimmunoprecipitation(Co-IP)assay,we showed that SIRT1 interacted with NF-κB p65 in nucleus of VSMCs.Compared with Ad-Vector infected control,overexpression of circ-Sirt1 enhanced the interactions between SIRT1 and NF-κB p65 upon TNF-α stimulation,accordance with up-regulated SIRT1 expression and decreased NF-κB p65 acetylation.Collectively,these results indicate that circ-Sirt1 enhances SIRT1 expression,contributing to deacetylation and decreased transcriptional activity of NF-κB p65.2.3 circ-Sirt1 inhibits vascular inflammation in vivoTo verify whether circ-Sirt1 inhibits the neointimal hyperplasia in vivo,Ad-Vector or Ad-circ-Sirt1 was infected into the rat carotid arteries after balloon injury.We showed that circ-Sirt1 expression significantly increased in the carotid arteries infected with Ad-circ-Sirt1,accompanied by markedly decreased expression of VCAM-1,ICAM-1 and MCP-1.The neointimal hyperplasia was examined by the intima to media ratio,and alleviated in Ad-circ-Sirt1 infected arteries compared with the controls on days 14 after balloon injury.These findings suggest that circ-Sirt1 may be a potential target for the treatment of atherosclerosis and vascular diseases.3 circ-Sirt1 inhibits the proliferation of VSMCs though the suppression of c-Myc nucleus translocation 3.1 The inhibitory effect of circ-Sirt1 on VSMC proliferationTo determine the effect of circ-Sirt1 on VSMC proliferation,the proliferative phenotype of VSMCs was induced by the treatment of PDGF-BB(20 ng/mL).We found that the level of circ-Sirt1 as well as SIRT1 mRNA was gradually decreased in VSMCs after the treatment of PDGF-BB,compared with the control.Cell counts and MTT assay showed a decreased cell numbers following the overexpression of circ-Sirt1.The expression of PCNA,a marker of proliferation,was inhibited under the same conditions.These data suggest that circ-Sirt1 inhibits the proliferation of VSMCs.3.2 The interaction between circ-Sirt1 and c-MycRPISeq software was first used to analyze some proteins associated with proliferation that may interact with circ-Sirt1.The interaction between circ-Sirt1 and c-Myc was further verified using RIP and RNA pull-down.3.3 circ-Sirt1 inhibits c-Myc nuclear translocationWestern blots showed that the nucleus translocation of c-Myc induced by PDGF-BB was significantly inhibited in VSMCs with overexpression of circ-Sirt1.The DNA binding activity of c-Myc was also significantly inhibited in VSMCs under the same conditions.The data indicated that circ-sirt1 suppresses the proliferation of VSMCs induced by PDGF-BB via inhibiting nuclear translocation of c-Myc.Conclusion:1 circ-Sirt1 acts as a miRNA sponge to relieve the inhibitory effect of miR132/212-3p on SIRT1 mRNA,and increases the expression of SIRT1 protein.2 circ-Sirt1-mediated SIRT1 expression promotes the deacetylation of p65.3 circ-sirt1 inhibits the proliferation of VSMCs induced by PDGF-BB though directly interaction with c-Myc and sequestration it in cytoplasm.4 circ-Sirt1 is a new endogenous circRNA inhibitor for NF-κB signaling in VSMCs.