The Effect And Mechanism Of Circular Rna CircEsyt2 In Atherosclerosis Via Vascular Remodeling

Cardiovascular Disease(CVD)is characterized by the high worldwide morbidity and mortality,which poses great harm to global public health.According to the latest statistic data,at least 330 million Chinese citizens are now under threat of CVD.CVD related deaths make up about 40 percent of overall deaths among citizens,far surpassing those resulted from tumor and other diseases.And atherosclerosis(AS)induced cardiovascular disease(ASCVD)like coronary artery disease,myocardial infarction and stroke,is a leading cause of CVD.AS and its complications have been a major factor endangering our national health,imposing heavy burden on economy and society.AS-led stenosis of blood vessels involves mostly the main and medium sized arteries,resulting in long-term ischemia of associated organs and systemic dysfunction or organic injury.Dyslipidemia is an independent risk factor contributing to AS.Notably,oxidized low-density lipoprotein(ox-LDL)initiates endothelial injury and release of bioactive substances,promoting vascular remodeling via stimulating over proliferation,migration and phenotypic switch of vascular smooth muscle cells(VSMCs)and resulting in formation of atherosclerotic plaques.Currently,lipid-lowering drugs have become fundamental in delaying instead of blocking the progression of disease,suggesting the limited understanding of known mechanisms.The environmental and genetic factors intimately interact in the pathogenesis of AS,and it may add to conventional understanding of AS pathogenic mechanism of studying it from the epigenetic perspective.NoncodingRNA(ncRNA)represents an essential group of epigenetic regulator;microRNA(miRNA)and long noncodingRNA(lncRNA)are the most researched ones with definite association with AS.CircularRNA(circRNA),the rising star in life science,has been highlighted for its unique structure and ubiquitous biofunction,and studies are intensifying of its role in the physiology and pathology of cardiovascular system.Therefore,we are seeking to explore AS-related circRNA and investigate its pathological mechanism,aiming to expanding current knowledge of how circRNA functions as well as providing novel clues to an effective therapy in counteracting AS development.PartⅠ.The effect and mechanism of circularRNA circEsyt2 in atherosclerosis via vascular remodelingMethods:1.Identification of circularRNA circEsyt2 and exploration of its biological effects in atherosclerosis.1.1 The atherosclerosis(AS)animals were cultivated by a high fat diet in the Apo E knockout(Apo E-/-)background mouse strain.High-throughput sequencing and circRNA profiling analysis of aortic plaques indicated 10 significantly up-regulated candidates,whose expressions were confirmed by qRT-PCR.And the top rated circularRNA(circRNA)circEsyt2,which was named after its host gene,was selected for further study.1.2 Characterize circEsyt2.To compare the stability of circular and linear structure,we assessed the expressions of circEsyt2 and linearRNAs(GAPDH,Esyt2)following RNase R and actinomycin D treatment,respectively.Examine the tissue expression as well as the intracellular distribution of circEsyt2 within mouse vascular smooth muscle cell(VSMC).Evaluate the similarity of mouse and human circEsyt2 sequences and analyzed the conservation of circEsyt2 across species.1.3 To characterize circEsyt2 in atherosclerotic vasculature,we performedRNA fluorescence in situ hybridization(FISH)and immunofluorescence(IF)to locate circEsyt2 in arterial wall,and detected its expression in animal(mouse aorta)and clinical specimen(human coronary artery).The expression difference was determined between normal(or mild CAD)samples in comparison with AS(or severe CAD)ones,and the major cell type within vascular wall was clarified with involvement in circEsyt2 mediated potential atheroprone biofunction.1.4 To characterize circEsyt2 in vascular remodeling,we quantified circEsyt2 in the C57BL/6J background mouse strain receiving wire injury in carotid artery by qRT-PCR,which was confirmed by FISH experiment in VSMC-lineage tracing mouse.1.5 Infect C57BL/6J mice with adeno-associated virus(AAV)specifically silencing or overexpressing circEsyt2 into wire-injured carotid arteries.Efficiency and specificity of viral delivery were validated by qRT-PCR and FISH-IF experiment.Inspect the alterations in neointimal formation within the injured carotid artery upon circEsyt2 manipulation.Check the difference in cell proliferation by Ki-67 immunofluorescent staining and expression of phenotypic switch markers by Western blot and IF experiment as well.1.6 To clarify the regulatory role of circEsyt2 in VSMC,we achieved in vitro silencing of circEsyt2 by transfecting siRNA into HASMC and mouse VSMC.Cell proliferation was evaluated by CCK-8 and Ed U incorporation assay.Migratory capacity was detected by wound healing and Transwell assay.Apoptosis was examined by flowcytometry.And phenotypic switch of VSMC was assessed by Western blot analysis of related markers(α-SMA,Calponin and Myh11).2.CircEsyt2 directly binds to PCBP1 and regulates its intracellular distribution.2.1 Via sequence-based prediction of circEsyt2-binding proteins using bioinformatics cat RAPID,the top-ranked protein poly(r C)binding protein(PCBP1)was distinguished.2.2 To testify the direct binding of circEsyt2 and PCBP1,we performedRNA pulldown experiment to capture the real poly(r C)binding proteins using the biotin-labeled circEsyt2 probe,followed by Western blot and mass spectrum analysis of PCBP1 enrichment.Reciprocally,RIP experiment was conducted to harvest PCBP1-bindingRNAs using the specific PCBP1 antibody.And circEsyt2 abundance was confirmed by qRT-PCR.Binding specificity was analyzed by RIP experiment of PCBP1-binding circEsyt2 before and after circEsyt2 silencing.Co-localization of circEsyt2 and PCBP1 were confirmed by FISH-IF experiment.2.3 Intracellular distribution of PCBP1 was determined by IF staining of circEsyt2-silenced both VSMC and wire-injured carotid artery.Check the protein level of PCBP1 in cytoplasmic and nuclear fractions by Western blot analysis as well.3.CircEsyt2 regulates VSMC proliferation through modulating p53β expression.3.1 Transcriptome high-throughput sequencing and gene profiling analysis were performed in circEsyt2-silenced VSMCs and aimed to identify differentially expressed genes concerning cell proliferation.PUMA and NOXA were confirmed in vitro,and were enriched in p53 pathway,indicating a regulatory role of circEsyt2 in p53 pathway.3.2 We analyzed the total protein,acetylation and alternative splicing level of p53 post to circEsyt2 depletion by qRT-PCR and Western blot analysis,and found circEsyt2 affects the biogenesis of a p53 splicing variant p53β independent of transcriptional,translational and acetylated regulation.3.3 Ascertain the pivotal role of p53β by assessing the effects of down-regulating circEsyt2 in VSMC proliferation upon p53β manipulation.4.CircEsyt2-PCBP1 interaction in the generation of p53β variant.4.1 Calculate the binding possibility of PCBP1 and pre-mRNA p53 using the bioinformatic tool and confirm this prediction by RIP and FISH-IF experiment in vitro.4.2 Based on the notion that PCBP1 facilitates gene splicing by recruiting spliceosome auxiliary factor U2AF65 to the pre-mRNA,we quantified the U2AF65-binding pre-mRNA p53 upon intervention of circEsyt2 and PCBP1 respectively or collaboratively,aiming to demonstrate that circEsyt2 regulates PCBP1-dependent p53 gene splicing.4.3 We further validated the regulation of circEsyt2-PCBP1 interaction in p53 splicing by detecting the amount of PCBP1-binding pre-mRNA p53 following circEsyt2 silencing by RIP experiment.4.4 Inspect the regulatory role of circEsyt2 in p53β generation upon PCBP1 manipulation for the purpose of illustrating the essential role of PCBP1 in the circEsyt2-modulated gene splicing.Results:1.According to the circRNA profiling analysis and confirmative experiment in tissue,we discovered and identified a significant up-regulated circRNA within mouse aortic plaques circEsyt2,which was named after its host gene.2.Compared to linear transcripts(GAPDH and Esyt2),circEsyt2 exhibits highly structural stability as evidenced by its undisturbed expression following the exonuclease RNase R-mediated digestion and actinomycin D-induced blockade of gene transcription.CircEsyt2 was pervasively found in main types of mouse organs with relatively high expression in aorta within cardiovascular system.In the subcellular level,circEsyt2 predominates in the cytoplasm of VSMC.A highly similarity was found of mouse and human circEsyt2 sequences following alignment.3.CircEsyt2 expresses ubiquitously in several major cell types consisting of arterial wall(smooth muscle cell,endothelia,adventitious fibroblast and macrophage),and highly up-regulates in mouse aortic plaques and coronary arteries of severe CAD patients.Likewise,circEsyt2 expression is significantly promoted in the vascular remodeling of wire-injured mouse carotid arteries.Above all,altered circEsyt2 expression is confirmed within VSMCs.4.Arterial silencing of circEsyt2 suppressed neointimal formation of injured carotid arteries,reduced Ki-67(cell proliferation indicator)positive cells and enhanced vascular phenotypic switch markers(α-SMA and Myh11).Consistently,overexpression of circEsyt2 stimulated neointimal formation,increased Ki-67 positive cells and down-regulated related markers.Silencing circEsyt2 in vitro decreased cell proliferation and migration but enhanced apoptosis and marker expressions.5.Via bioinformatic prediction and confirmative experiments(RNA pulldown,mass spectrum and RIP),we validated of the direct binding of circEsyt2 and PCBP1.6.PCBP1 was found to decrease in cytoplasm but increase in nucleus by circEsyt2 depletion both in vitro and in vivo,revealing that circEsyt2 inhibits nuclear trafficking of PCBP1 by binding PCBP1 in the cytoplasm and therefore modulates intracellular distribution of PCBP1 by serving as “molecular switch”.7.Silencing circEsyt2 promotes alternative splicing of p53 gene into p53β,suppressing VSMC proliferation through modulating cell proliferation related genes within p53 pathway.8.PCBP1 directly binds to pre-mRNA p53,and positively regulates p53 gene splicing into p53β via facilitating the binding of U2AF65 and pre-mRNA p53 and improving splicing activity.9.Intervention of PCBP1 impedes the regulation of circEsyt2 on p53β expression.And circEsyt2-PCBP1 interaction modulates p53β biogenesis by altering nuclear trafficking of PCBP1.Conclusion:1.CircEsyt2 is an important atherogenic factor.2.CircEsyt2 modulates vascular remodeling through regulating cell proliferation,migration and phenotypic switch of VSMC.3.CircEsyt2 regulates p53β-dependent VSMC proliferation.4.PCBP1 directly binds to pre-mRNA p53,facilitating alternative splicing of p53 gene into p53β.5.CircEsyt2-PCBP1 interaction within cytoplasm modulates nuclear trafficking of PCBP1,therefore affecting the regulation of PCBP1 on splicing.Myocardial infarction(MI)is a serious complication mostly caused by atherosclerosis with high hospital and outside hospital mortality rate.The progressive loss of cardiomyocyte underlies the underlying pathological manifestation of MI-induced deteriorated cardiac function and end-stage heart failure.Therefore,how to alleviate MI-induced myocardium injury poses a key factor in preventing and treating the incoming heart failure.Multiple G-protein-coupled receptors(GPCRs)have been identified within cardiomyocyte such as β-adrenergic receptor and adiponectin receptor.These receptors facilitate preserving and regulating normal cardiac function in the physiological condition,but pose a pro-apoptotic effect in cardiomyocyte when in the hyperphosphorylated and desensitized state post to MI injury.Phosphorylation of GPCR is generally regulated by G-protein-coupled receptor kinase(GRK)and most often hyperphosphorylation is induced by MI.Among the 7 GRK subtypes,GRK4 highly correlates with pathogenesis of hypertension,whose up-regulated expression or activity impairs the normal natriuresis function of kidney via GPCRs.Moreover,according to a population based study,individuals harboring mutant variants with enhanced GRK4 activity(A142V,A486 V and R65L)predispose to higher three-year all-cause mortality,MI and stroke rate compared to the wild type genotype.Consequently,we decide to make further investigation into the role of GRK4 in MI-induced myocardial injury as well as a primary exploration of the association between GRK4 and circadian rhythm of blood pressure,as hypertension represents a critical factor in myocardial infarction.PartⅡ.The effect and association of G-protein-coupled receptor kinase 4 in/with myocardial infarctionMethods:1.Perform MI surgery in the C57BL/6J background mouse strain.Quantify GRK4 expression in myocardium by qRT-PCR and Western blot analysis.2.Detect the intracellular distribution of up-regulated GRK4 in cardiomyocyte post to MI surgery by immunofluorescent staining.3.Construct cardiac specific GRK4 knockout mice and perform MI surgery.Examine heart infarct size 4 weeks later by Masson staining in order to ascertain the biological effect of GRK4 in MI.4.Echocardiographic assessment of heart function by evaluating LVEF value of cardiac specific GRK4 knockout mice post to MI was performed to testify the effect of GRK4 in cardiac function.5.Distribution of GRK4 variants(A142V,A486 V and R65L)in dipper and non-dipper hypertensive patients using PCR and exon sequencing.Results:1.GRK4 up-regulates in mouse myocardium post to MI surgery.2.Increased GRK4 within cardiomyocyte post to MI surgery was accumulated in nucleus.3.Cardiac specific knockout of GRK4 results in reduced cardiac infarct size post to MI surgery.4.Cardiac specific knockout of GRK4 ameliorates cardiac function impaired by MI surgery.5.GRK4 variants account for up to 84% non-dipper hypertensive patients and positively associate with non-dipper hypertension.Conclusions:1.GRK4 contributes to MI-induced cardiac injury,whose suppression could alleviate myocardium damage caused by MI.2.GRK4 may convey pathological effect through regulating gene transcription within nucleus.3.GRK4 might promote pathogenesis of non-dipper hypertension.A bulk of animal experiments and epidemiologic researches showed that in addition to genetic and environmental factors,exposure to adverse intrauterine conditions during a specific time window during fetal development would incite negative effects to the offspring in a long run,including the improved risk of developing hypertension in the adulthood.According to present studies,exposure in gestational diabetes causes altered programming of the fetus,resulting in vascular dysfunction and pathogenesis of hypertension in the offspring.Thus,it is of critical importance to focus on the lifetime influence and harm brought by the early exposure to a hyperglycemic intrauterine environment.However,it still remains unclarified of the pathogenic mechanism of maternal diabetes/hyperglycemia programmed hypertension in the offspring.Multiple prior results have demonstrated that endoplasmic reticulum stress underlies one of the essential pathological mechanisms of hypertension.Endoplasmic reticulum plays a fundamental and critical role in protein synthesis,folding and transport and is recognized as a primary sensor for detecting cell stress.Impaired endoplasmic reticulum homeostasis and function stimulate endoplasmic reticulum stress,activating so called unfolded protein response in order to restore the homeostasis within endoplasmic reticulum.However,long-lasting activation of this signaling would impair the normal cellular function and result in the onset of chronic diseases such as hypertension.Moreover,endoplasmic reticulum stress in the context of hypertension also engages in the dysfunction of vascular endothelial cells whereby inhibition of endoplasmic reticulum stress could improve the bioavailability of nitric oxygen as well as endothelial cell dependent vascular dilation.Therefore,our work is to further study and illustrate the effect of endoplasmic reticulum stress induced vascular endothelial dysfunction in the maternal diabetes caused hypertension in the offspring.Methods:1.Perform intraperitoneal injection of streptozotocin(STZ)to establish the rat model of gestational diabetes.Detect the plasma glucose concentration,insulin concentration,mean arterial pressure,weight gain and litter size in comparison with control group.2.Examine the blood pressure,heart rate and fasting glucose of the adult offspring from diabetic and control maternal rats.3.Monitor the blood pressure of the adult offspring post to intraperitoneal injection of endoplasmic reticulum stress inhibitors 4-sodium phenylbutyrate(4-PBA)or tauroursodeoxycholic acid(Tudca)from diabetic and control maternal rats.Results:1.Plasma glucose concentration was significantly elevated and insulin concentration was reduced in the gestational rats following STZ treatment compared to the control.No difference was detected in gestational period,mean arterial pressure,weight gain and litter size between the two groups.2.A higher weight and systolic pressure were confirmed in the adult offspring of gestational diabetic rats compared to the control whereas no difference was detected in the heart rate and fasting glucose between the groups.3.Treatment of endoplasmic reticulum stress inhibitors could prevent the signiciant rise of blood pressure in the adult offspring of gestational diabetic rats with none effect in those of control.Conclusions:1.The adult offspring of gestational diabetic rats is more predisposed to hypertension compared to that of control.2.Inhibiting endoplasmic reticulum stress ameliorates the blood pressure of the adult offspring of gestational diabetic rats.