The Mechanism Of Circ-Sirt1 Inhibiting Nuclear Translocation Of NF-?B And Its Significance In Vascular Inflammation

Objective: Atherosclerosis and vascular degenerative disease are the main causes resulting in cardiovascular disease,and the inflammatory response is considered as a major factor in atherosclerosis and vascular degenerative disease.In this study,we used circular non-coding RNA circ-Sirt1 deriving from the exon of SIRT1 gene as the research object.The regulatory effect of circ-Sirt1 in the proliferative process of inflammation induced VSMCs was confirmed in vitro and in vivo through establishment of carotid artery stenosis model in rat.Method: The wild-type SD rat was used to establish carotid artery stenosis model.The relationship between circ-Sirt1 and vascular inflammatory injury was detected by real-time fluorescence quantitative PCR and Western blot assays.The proteins interacted with circ-Sirt1 were identified by Rip and RNA pull-down assays combined with Western blot.The effects of circ-Sirt1 on transcriptional regulator of NF-?B p65 activation and nuclear translocation were investigated via Western blot and fluorescence in situ hybridization.Results:1 circ-Sirt1 is involed in the phenotypic switching of VSMCs1.1 circ RNAs deriving from SIRT1 gene were selected by RT-PCRcircBase retrieval revealed that SIRT1 host gene could produce 11 circ RNAs in human genome.We detected the expression of 11 circ RNAs in human VSMCs by RT-PCR,and found that hsa_circ₀093887 was highly expressed.Circular SIRT1 isoform?hsa_circ₀093887?located at chr10: 69647174-69669199 in human genome was derived from circularization of the exon-2 to exon-7 of the SIRT1 gene.Through designing additional divergent primers,circ-Sirt1 presence was verified by RT-PCR in rat VSMCs,and its length was 927 nt.The sequencing result was completely in accordance with circ-Sirt1 sequence as shown in circ Base.Multiple sequence alignment indicated that the sequence of human circ-Sirt1 was 93% homologous with that of rat.1.2 circ-Sirt1 is widely distributed in tissues and cellsThe distribution of circ Sirt1 was detected in diverse tissues and cells of rat and human using q RT-PCR,and showed that circ-Sirt1 was expressed in several rat tissues,including heart,liver,spleen,brain,artery,and vein,as well as in human umbilical vein endothelial cells?HUVECs?and vascular smooth muscle cells?HASMCs?,indicating that the circ-Sirt1 was widely distributed and might be necessary for the homeostasis of VSMCs.We next investigated the relationship between the expression of circ-Sirt1 and the phenotypic switching of VSMCs.We showed that circ-Sirt1 was significantly decreased in VSMCs treated with PDGF-BB or TNF-?,and increased in ATRA-induced VSMCs.Furthermore,we also found that circ-Sirt1 was down-regulated in exosomes secreted from dedifferentiated VSMCs,suggesting that circ-Sirt1 may be involved in phenotypic switching of VSMCs.2 circ-Sirt1 is involved in the regulation of inflammatory response in vivo2.1 The expression of circ-Sirt1 is decreased during neointimal formationTo investigate the expression of circ-Sirt1 in process of inflammatory response in vivo,rat carotid artery stenosis model was established.We found that the level of circ-Sirt1 was gradually decreased in both the plasma and the carotid arteries at 7,14 and 28 days in a rat balloon injury model compared with the sham.We next collected renal arterial specimens from patients with hypertension or not who underwent nephrectomy.The hypertensive renal arterial wall displayed a neointimal hyperplasia.Using in situ hybridization,we showed that the level of circ-Sirt1 expression in human renal arterial neointima was lower than that in the normal controls,accompanied with increased expression of pro-inflammatory cytokines VCAM-1,ICAM-1 and MCP-1.This finding was also validated by q RT-PCR analysis.To determine whether the expression of circulating circ-Sirt1 was associated with atherosclerosis progression in human,we examined the plasma circ-Sirt1 level by q RT-PCR assay,and showed that circ-Sirt1 was significantly decreased in the plasma fraction of peripheral blood in patients with coronary artery disease.Collectively,these results suggest that circ-Sirt1 is potentially involved in the pathogenesis of vascular diseases,and may act as a novel potential biomarker for detecting patients with atherosclerosis.2.2 circ-Sirt1 is involved in the regulation of inflammatory response of VSMCsTo further confirm the mediation effect of circ-Sirt1 on the inflammatory response in VSMCs,we knocked down and overexpressed circ-Sirt1,and found that circ-Sirt1 decreased TNF-?-induced expression of VCAM-1,ICAM-1 and MCP-1 at the m RNA and protein levels in VSMCs,whereas knockdown of endogenous circ-Sirt1 increased the expression of these pro-inflammatory cytokines.These results suggest that circ-Sirt1 is a negative regulatory factor of vascular inflammatory response.2.3 circ-Sirt1 inhibits NF-?B p65 nuclear translocation in VSMCsNF-?B is an important transcriptional regulator in inflammatory response in vivo,and plays a central role in the process of inflammation activation.In vitro studies have shown that overexpression of circ-Sirt1 inhibits the inflammatory response in VSMCs.To investigate the regulatory mechanism of circ-Sirt1 on the inflammatory response,we used Western blot to analyze the cytoplasmic and nuclear protein from VSMCs.The results showed that nuclear translocation of NF-?B p65 induced by TNF-? was significantly inhibited in VSMCs with overexpression of circ-Sirt1.Conversely,knockdown of endogenous circ-sirt1 using specific si-circ-Sirt1 enhanced it.These results suggest that circ-Sirt1 inhibits inflammatory response of VSMCs through inhibition of NF-?B activation.2.4 circ-Sirt1 inhibits vascular inflammatory response in humanTo further confirm the inhibitory effect of circ-sirt1 on vascular inflammation,human renal artery tissues were cultured in vitro,and infected with Ad-circ-Sirt1.We showed that exogenous circ-Sirt1 significantly inhibited TNF-?-induced expression of VCAM-1,ICAM-1 and MCP-1 at m RNA level in human renal artery tissue infected with Ad-circ-Sirt1,compared with the Ad-Vector group.3 The molecular mechanism of circ-Sirt1 inhibiting the activation of NF-?B3.1 The subcellular localization of circ-Sirt1 is in the cytoplasmBy the cytoplasmic and nuclear fractions of q RT-PCR and fluorescence in situ hybridization,we found that circ-Sirt1 mainly distributed in the cytoplasm.We also validated the effects of circ-Sirt1 on nuclear translocation of NF-?B p65 by in situ hybridization and protein immunofluorescent staining.In the Ad-Vector infected VSMCs,NF-?B p65 was located in the nucleus after treatment with TNF-?.However,overexpression of circ-Sirt1 resulted in a cytoplasmic sequestration of NF-?B p65 under the same conditions.These results suggest the possible interactions between circ-Sirt1 and NF-?B p65.3.2 circ-Sirt1 interacts with NF-?B p65To prove the interaction between circ-Sirt1 and NF-?B p65,we used Rip assay to determine whether circ-Sirt1 could bind to NF-?B p65 in VSMCs.We showed that circ-Sirt1 physically interacted with NF-?B p65 in VSMCs.To further verify this finding,lysates from Ad-Vector and Ad-circ-Sirt1 infected VSMCs were incubated with a biotinylated DNA probe designed to specifically detect circ-Sirt1.We then tested the interactions of circ-Sirt1 with NF-?B p65 in VSMCs using the probe to pull down NF-?B p65,and Western blot validated the interactions of circ-Sirt1 with NF-?B p65.Moreover,this interaction was enhanced in the cells infected with Ad-circ-Sirt1,and decreased in VSMCs following knockdown of circ-sirt1.In addition,we also examined the interactions between circ-Sirt1 and NF-?B p65 in human VSMCs under the same conditions.We found that overexpression of circ-Sirt1 significantly enhanced the interactions of circ-Sirt1 with NF-?B p65 in human VSMCs by Rip and RNA pull-down assay.These data suggest that circ-Sirt1 directly interacts with NF-?B p65,and at least partly sequestrates it from the cytoplasm into the nucleus upon TNF-? stimulation.3.3 circ-Sirt1 does not affect the phosphorylation and degradation of I?B?It has been known that I?B? interacts with NF-?B in resting state, making it inactive in the cytoplasm.In view of the inhibitory effects of endogenous I?B? on NF-?B p65,we tested the potential interactions between circ-Sirt1 and I?B?.However,the probe of circ-sirt1 could not precipitate I?B? protein by RNA pull-down assay.Moreover,infection with Ad-circ-Sirt1 did not affect the phosphorylation and degradation of I?B? upon TNF-? stimulation compared with Ad-Vector infected control group.The results indicate that circ-Sirt1 is a new RNA inhibitor for NF-?B signal pathway.3.4 circ-Sirt1 exists two NF-?B binding consensus sequencesTo identify the possible sites of circ-Sirt1 binding to NF-?B p65,we used computational approaches to assess the likelihood of protein-RNA interaction.cat RAPID,a predictor of protein-RNA binding,predicted 327-378 nt and 726-777 nt of circ-Sirt1 binding to NF-?B p65.To verify the prediction,the blocking oligos that were complimentary to the circ-Sirt1 sequence were transfected into VSMCs.We showed that the blocking oligos did not affect the level of circ-Sirt1,and resulted in a decreased interaction of NF-?B p65 with circ-Sirt1 in the pull-down assay.Similarly,the blocking oligos abolished the circ-sirt1 probe pulling down NF-?B p65.Conclusions:1.The expression of circ-Sirt1 is associated with vascular inflammatory response and phenotypic switching of VSMCs,and may act as a novel biomarker for detecting patients with atherosclerosis.2.circ-sirt1 mainly expresses in the cytoplasm,and inhibits inflammatory response in VSMCs via directly interacting with and sequestrating NF-?B p65 in the cytoplasm.3.circ-Sirt1 is a new endogenous circRNAs inhibitor for NF-?B signaling,and has a synergistic effect with I?B? in VSMCs.