| BackgroundAtherosclerosis(AS)is the most important pathological change in atherosclerotic vascular disease,characterized by lipid deposition,bleeding and thrombosis,arterial calcification,and arterial wall remodeling.AS is influenced by inflammation,oxidative stress and hemodynamic changes.The mechanism for the occurrence and development of AS was not complete clear.Identification of potential therapeutic targets are important for intervention in plaque formation,stabilization of vulnerable plaques,and reduction of adverse cardiovascular events.Cardiovascular risk factors(hypertension,hyperlipidemia,et.al)damage the vascular endothelium,induce endothelial dysfunction and trigger the formation of atherogenesis.The inflammation and oxidative stress induce thrombus formation,neovascularization and apoptosis of endothelial cells,exacerbate the instability of plaque.Some studies show ER stress(ERS)play roles in the development of atherosclerotic plaques.DKK is a conservative secretory glycoprotein in the evolution.It contains four members(DKK1-4).DKK1 plays its roles via autocrine and paracrine.DKK1 can bind with LRP6,hinder the combination of all Wnts molecules with LRP6,antagonize the Wnt pathway.DKK1 is also combined with cytoskeletal associated protein 4,CKAP4(P63)and activate PI3K/AKT in epithelial cells.Wnt5a can induce ERS,which suggesting that ERS may be regulated by Wnt pathway.DKK1 is noticed in cardiovascular disease.DKK1 increases inflammation in atherosclerotic plaques.Increased serum DKK1 was found in type Ⅱ diabetes mellitus patients,coronary heart disease and acute stroke.The serum DKK1 can reflect the unstable state of atherosclerosis and predict recurrent cardiovascular events independently.DKK1 was upregulated and promoted atherosclerosis by oscillatory shear stress.However,the effect of DKK1 in the occurrence and development of atherosclerosis and endothelial function are poorly understood.Whether DKK1 is secreted by endothelial cells,whether DKK1 promotes plaque apoptosis and ER stress has not been reported.Objectives1.To determine the role of DKK1 in the histology and vulnerability of atheroscleroticplaque in ApoE-/-mice.2.To elucidate the effect of DKK1 on the inflammation,apoptosis and ER stress of atherosclerotic plaque.3.To explore the location of DKK1 in the plaque and the effect of DKK1 on the endothelial dysfunction.Materials and methods1.Lentivirus Construction(Lenti-shDKK1 与 Lenti-DKK1)pGLV3/H1/GFP+Puro(pGLV3)-shDKK1 and LV5-DKK1 were constructed.2.Animal groupApoE-/-mice(n=180,8 weeks old,male,weighing 25-30g)were obtained from Peking University(Beijing,China).Atherosclerotic lesions were induced by being fed with high-fat diet.180 mices were divided randomly into three groups:4 weeks group(n=60),8 weeks group(n=60)and 12 weeks group(n=60).Each group was divided into four subgroups and intravenous injected virus:(1)NS group(n=15):200 μl normal saline;(2)GFP group(n=15):200μl 4×108TU Lenti-GFP;(3)Lenti-shDKK1 group(n=15):200μl 4×108TU Lenti-shDKK1;(4)Lenti-DKK1 group(n=1 5):200μl4×108TU Lenti-DKK1.The transfection efficiency was observed at 2 weeks after transfection.All mice were given euthanasia at 4 weeks after transfection.3.Preparation of blood samples and tissue frozen sectionMice were given euthanasia and the blood was collected and detected:TG,TC,LDL-C,HDL-C and DKK1.The full length of the aorta and heart were conserved in 4%formaldehyde or liquid nitrogen(-80℃).The aortic root was buried with OCT and continuously sectioned(5μm).4.Western BlotThe aortic arch was dissociated by 100μl RIPA per 20mg tissue sample.After centrifugation,the supernatant was taken and boiled.SDS-PAGE electrophoresis,transmembrane and incubation in primary antibodies overnight at 4℃.Bands were incubated with second antibodies and recorded by luminescent image analyzer.5.General oil red O stainingThe aorta was cleaned under the general microscope and opened longitudinally.The aorta was stained by Oil red O for 2h in the room temperature,immersed in hot water and pressed to flat.6.Histopathology and ImmunohistochemistryCryosections were stained with HE,oil O,picosirius red,and Masson.Crysections were incubated in different primary antibodies and second antibodies for immunohistochemistry.7.ImmunofluorescenceTo investigate the distribution of DKK1 in endothelial cells(Endomucin)and macrophage(MOMA-2).Cryosections were fixed,permeabilized,blocked,and incubated in two primary antibodies overnight,washed with PBS,and incubated in FITC-and TRITC-conjugated antibodies for 1h.Nuclei were stained by DAPI.Cryosections were photoed under fluorescence microscope.8.Co-cultureThe human umbilical vein endothelial cells(HUVECs)were transfected with Lenti-GFP-DKK1 in the up-chamber,while HUVECs,human aortic smooth muscle cells(HASMCs)and macrophages were cultured in the down-chamber respectively.After co-culture 24h,cells were photoed by the fluorescence microscope.9.Immunofluorescence+TUNEL co-localization stainingCryosections were immunofluorescence stained with endomucin,then TUNEL stained(fixed,TdT enzyme reaction,anti digoxigenin conjugate reaction).Nuclei were stained with DAPI and observed under fluorescence microscope.10.Aorta ring assayThe aorta was taken and placed on a pressure device.After stimulation,10μmol/L NE and 10-3～10-5μmol/LAch stimulated the vessels successively.The tension value was recorded by PowerLab.11.ELISAThe fasting peripheral blood was collected for 5ml.The serum was isolated and detected the concentrations of DKK1,ADM A and ET-1 by ELISA Kit.12.Statistical analysisData are presented as mean±SD and analyzed by SPSS 16.0.Unpaired t test was used for comparison of two samples.ANOVA analysis and Bonferroni post-hoc analysis were used for the comparison among many samples.The correlation analysis used Pearson correlation analysis.P<0.05 means statistical sense.Results1.The general condition of animalsThere were no significant differences in body weight and serum lipids(TG,TC,HDL-C and LDL-C)in four subgroups(Group NS,Group GFP,Group shDKK1 and Group DKK1).2.Detection of lentivirus transfection efficiency and expression efficiency in vivoAfter 2 weeks of lentivirus transfection in 12-week Group,NS group,LV3 group and LV5 group were compared.GFP fluorescence was showed in the aortic root plaques in GFP group,which indicates that the transfection was safe and effective.DKK1 level was detected in the 12-week Group.Compared with NS Group and GFP Group,the protein level of DKK1 was downregulated in shDKK1 Group and increased in DKK1 Group(P<0.05).Compared with NS Group and GFP Group,the serum expression of DKK1 was downregulated in shDKK1 Group and increased in DKK1 Group(P<0.05).3.DKK1 enlarges the size of plaque and increases the vulnerability of plaqueOil red O staining and HE staining showed that the area of plaque significantly decreased in shDKK1 Group(P<0.05),and increased in DKK1 Group(P<0.05)in 4-week Group,8-week Group and 12-week Group.In 4-week Group,8-week Group and 12-week Group,smooth muscle content and fiber cap thickness were higher significantly,macrophages and lipid content were reduced in shDKK1 Group(P<0.05).The vulnerability index was lower significantly in shDKK1 Group(P<0.05).Smooth muscle content and fibrous cap were reduced,macrophage and lipid content were increased in DKK1 Group(P<0.05).The vulnerability index was higher in shDKK1 Group(P<0.05).However,the collagen content among the four groups was similar in 4-week Group and 8-week Group(P>0.05).The collagen content was higher in shDKK1 Group and reduced in DKK1 Group in 12-week Group(P<0.05).4.DKK1 increases the inflammatory factors in the plaqueIn 4-week Group,the levels of MMP2 and MMP9 decreased in shDKK1 Group and increased in DKK1 Group(P<0.05).There was no difference in adhesion molecules(ICAM1 and VCAM1)and inflammatory factors(TNF-α,MCP-1,IL-1β,IL-6)between the four groups(P>0.05).In 8-week Group,the expression of MMP2,MMP9,TNF-α,IL-1β and IL-6 decreased in shDKK1 Group and increased in DKK1 Group(P<0.05).There were no differences in the levels of ICAM1,VCAM1 and MCP-1(P>0.05).In 12-week Group,the expression of ICAM1,VCAM1,TNF-α,MCP-1,IL-1β,IL-6,MMP2 and MMP9 decreased in shDKK1 Group and increased in DKK1 Group(P<0.05).5.DKK1 increases the apoptosis and ERS in the plaqueIn 4-week Group,8-week Group and 12-week Group,the expression of Bax,cleaved caspase-3 and caspase-12 decreased in shDKK1 Group and increased in DKK1 Group(P<0.05).Bel-2 increased in shDKK1 Group and reduced in DKK1 Group(P<0.05).6.Location of DKK1 and intercellular interaction in atherosclerotic plaquesThe co-localization staining showed that the expression of DKK1 consistent with the location of endothelial cells in 12-week-NS Group of aortic root,while it is not seen abundant expression in the location of macrophage.After HUVECs were transfected with Lenti-GFP-DKK1,GFP fluorescence appeared in the co-cultured cells(HUVECs,HASMCs and macrophages).DKK1 was involved in ox-LDL induced inflammatory in macrophage.Exogenous DKK1 could inhibit HASMCs’proliferation.7.DKK1 affects the vasodilatation of vascular endotheliumThe vasodilatation assay was carried out in 4-week Group.Compared with GFP Group,the maximum diastolic rate was increased in shDKK1 Group and descreased in DKK1 Group(P<0.05).8.DKK1 affects apoptosis in atherosclerotic plaquesTUNEL and colocalizaton staining was carried out in 12-week Group.Compared with NS and GFP Group,the apoptosis decreased in shDKK1 Group and increased in DKK1 Group(P<0.05).Apoptosis mainly occurred in endothelial cells.9.Positive correlation between DKK1 and endothelial cell dysfunction related molecules ADMA and ET-1 in the human serumThe serum levels of DKK1,ADMA and ET-1 in patients with acute myocardial infarction(AMI)were significantly increased,compared with normal persons and unstable angina(P<0.05).The level of DKK1 was positively correlated with ADMA and ET-1(P<0.05).Conclusions1.DKK1 increased the vulnerability and promoted the occurrence and development of atherosclerotic plaque.2.DKK1 increased the inflammation,ER stress and apoptosis in the atherosclerotic plaque.3.DKK1 promotes endothelial dysfunction and intercellular interaction in vivo.BackgroundEndothelial cell injury is one of the atherosclerogenesis mechanism.Previous study found that ox-LDL is uptaked by LOX-1,activated several signal pathways(ER stress,Wnt/β-catenin pathway),induced endothelial cell dysfunction and promoted AS development.Therefore,ox-LDL is the initiating step of AS.The mechanism of ox-LDL in endothelial cells is important for searching potential target in clinical intervention of AS.In previous,we found that(1)the silence of DKK1 delay the occurrence and development of AS,overexpression of DKK1 might accelerate the occurrence and development of plaque;(2)endothelial cells were the main expression location of DKK1 in vivo;(3)DKK1 regulates endothelial diastolic function in the early plaques,and affects the ER stress and apoptosis in the advanced plaques.Therefore,we focus on endothelial cells and study the mechanism of DKK1 in endothelial cell.DKK1 is a secretory glycoprotein of the DKK family.Various studies of tumor found that DKK1 might inhibit the classical Wnt/β-catenin pathway,upregulate PCP/JNK non-classical Wnt pathway and increase ROS,p53,p21,NF-KB and other molecules,which participate in cell proliferation,migration,apoptosis and inflammatory.On the other hand,DKK1 can be combined with CKAP4(P63),activate PI3K/AKT and promote proliferation in epithelial cells.However,it is not clear whether DKK1 of endothelial cells is regulated by ox-LDL,whether DKK1 participates in the regulation of ER stress induced by ox-LDL and whether DKK1 affects endothelial dysfunction.Objectives1.To clarify the expression of DKK 1 in ox-LDL induced HUVECs.2.To explore the role of DKK 1 in the HUVECs’ apoptosis,proliferation,migration and tube formation induced by ox-LDL.3.To investigate the role of ER stress and Wnt pathway in DKK1’s regulation of HUVECs apoptosis.Methods1.In vitro ox-LDL interventionThe cells were exposed to 0,0.5,1,3,6,12 hours ox-LDL(150μg/ml)and 0,25,50,100,150,200μg/ml ox-LDL(6h).Cells and supernatant were collected and detected the expression of DKK 1 by using qPCR,Western Blot and ELISA.2.ELISAThe cells were stimulated by ox-LDL and replaced with serum-free medium.The supernatant was collected and centrifuged in 1000 4℃ for 20 minutes.DKK1 concentration in the cell supernatants was detected by ELISA Kit(R&D Systems).3.Transient TransfectionDKK1 siRNA,CHOP siRNA,IRE1α siRNA,ATF6 siRNA and NC siRNA were synthesized by Shanghai GenePharma Biotechnology Company.Cells were transfected with lipo3000.Different stimulations were given.Collections and detections were taken 24h after transfection.4.Quantitative real-time PCRTotal RNA was extracted by use of Trizol(Invitrogen),transcribed into cDNA by a cDNA synthesis kit(TAKARA),and performed qPCR by TAKARA SYBR Green Mix kit.The primer sequences were synthesized by Shanghai GenePharma Biotechnology Company.5.Western Blot(WB)HUVECs were harvested and lysed using RIPA.Protein was separated on SDS-PAGE,transferred into PVDF membrane,blocked in 5%BSA,incubated with primary and second antibodies.The bands were photoed by luminescent image analyzer.6.Cell seed on slidePreparation of collagen:mix double distilled water(80ml)and acetic acid(92.2μl),adding 1ml rat tail collagen.1ml collagen were dripped on the slide for 1 hours,then collected the collagen and dried the slide.HUVECs were seeded on these glass slides.7.TUNELThe detailed protocol was according with the manufactures’ instructions(S7101,Millipore).Cells apoptosis was detected by the fluorescence microscope.Quantification involved calculating positive cell rate.8.PE/7-AAD flow cytometryThe detailed protocol was according with the manufactures’ instructions(BD PE Annexin V apoptosis detection kit).Cells are washed,trypsin digested and collected.Each tube adds 100μl substrate,5μl PE and 5μl 7-AAD.The mixture soft vortex for 15min and detect by flow cytometry.Analysis and statistics by FLOW JO software.9.Detection of roles of DKK1 in regulating endothelial cells functionHUVECs were transfected DKK1 siRNA and NC siRNA and stimulated ox-LDL for 6 h.(1)To detect the proliferation,the EdU assay was used.(2)To detect migration,the cell wound scratch assay and Transwell assay were used.(3)To detect ROS production,ROS assay was used.(4)To detect the tubule formation,tube formation assay was used.10.Statistical analysisData are presented as mean±SD and analysised by SPSS 16.0.Unpaired t test was used for comparison of two samples.AN OVA analysis was used for the comparison among many samples.P<0.05 means statistical sense.Results1.ox-LDL increased the expression of DKK1 in HUVECsHUVECs were stimulated by ox-LDL(150μg/ml)at different time.Compared with 0 hours,DKK1 mRNA,protein and secretion levels were increased(P<0.05).Compared with 0μg/ml,DKK1 mRNA,protein and secretion levels were increased(P<0.05).2.DKK1 participated in the apoptosis of HUVECs by ox-LDLsiRNA knockdown of DKK1 attenuated the apoptosis by ox-LDL(P<0.05),reduced the expression of Bax and cleaved caspase-3 by ox-LDL,and increased the expression of Bcl-2 by ox-LDL(P<0.05).3.DKK1 participates in ER stress of HUVECs by ox-LDLCompared with control group,both ox-LDL and rDKK1 could upregulate ER stress proteins,such as p-eif2,CHOP,IRE1,XBP1s,GRP78 and ATF6 levels(P<0.05).SiRNA knockdown of DKK1 significantly suppressed these protein levels(P<0.05).4.DKK1 promotes the apoptosis of HUVECs via ER stressHUVECs are pretreated with 4-PBA or salubrinal for 1 hours,or transfected with CHOP siRNA,IRE1α siRNA or ATF6 siRNA to inhibit the corresponding molecules.Compared with rDKK1 group,interference groups(4-PBA,salubrinal,CHOP siRNA and IRE1α siRNA)alleviated the Bax and cleaved caspase-3,increased the expression of Bcl-2,attenuated the apoptosis rate(TUNEL and PE/7-AAD flow cytometry assay)(P<0.05).There was no significantly difference in the expression of Bax,cleaved caspase-3 and Bcl-2 in the ATF6 siRNA group(P>0.05).Meanwhile,the results in the Lenti-DKK1 group were consistent with rDKK1 group(P<0.05).5.DKK1 promotes apoptosis and ER stress in HUVECs via activating JNK pathwayDKK1 siRNA downregulated p-JNK,p-ERK and p-P38,while Lenti-DKK1 upregulated p-JNK,p-ERK and p-P38(P<0.05).Pretreatment with JNK inhibitor SP600125,the levels of Bax,cleaved caspase-3 and ER stress(CHOP and GRP78)were attenuated,the expression of Bcl-2 was increasing(P<0.05).However,the expression of Bax,Bcl-2 and ER stress(CHOP and GRP78)have no differences in pretreatment with SB203580 and U0126(P>0.05).6.DKK1 induced HUVECs apoptosis through upregulation of IRE1α via antagonizing Wnt/β-cateninCompared with DKK1 group,the expression of GRP78,IRE1α,Bax and cleaved caspase-3 was inhibited(P<0.05)and the expression of Bcl-2 was increased in pretreatment of IM-12(Wnt pathway agonist)(P<0.05).However,the expression of CHOP and JNK have no differences(P>0.05).Compared with DKK1 siRNA group,GRP78,IRE1α,Bax and cleaved caspase-3 was increased(P<0.05),and the expression of Bcl-2 was reduced in pretreatment of FH535(Wnt pathway inhibitor)(P<0.05).The expression of CHOP and JNK have no differences(P>0.05).7.DKK1 also participates in ox-LDL mediated endothelial cell migration,tubule formation and ROS production,but has no obvious influence on the proliferation of endothelial cells(1)Compared with control group,the proliferation of endothelial cells was decreased in ox-LDL group.However,DKK1 siRNA has no obvious influence on the proliferation of HUVECs(P>0.05).(2)Compared with control group,the migration,ROS production and tubule formation of HUVECs was increased in ox-LDL group.DKK1 siRNA attenuated these functions of HUVECs(P<0.05).Conclusions1.DKK1 was upregulated by the stimulation of ox-LDL.2.DKK1 participates in ox-LDL induced endothelial cell apoptosis,migration,tubule formation and ROS production.3.DKK1 induces apoptosis of endothelial cells through activation of ER stress via JNK and Wnt/β-catenin.BackgroundThe ox-LDL is recognized by LOX-1 and participates in inflammatory reaction,oxidative stress,apoptosis and proliferation,regulates specific genes at transcriptional,post-transcriptional and post-translational levels,ultimately induces endothelial cell dysfunction and atherosclerosis(AS).DKK1 is a secretory glycoprotein of the DKK family.There are several upstream mechanism of DKK1,including histone modification,transcriptional changes,posttranscriptional changes and posttranslational modifications(phosphorylation,glycosylation).However,the upstream mechanism of the expression of DKK1 in ox-LDL induced endothelial cells is unclear.Ets-1 is a member of the Ets family(E26 transformation-specific),possesses a conserved domain(EBS)to recognize(GGAA/T).The activity of Ets-1 is affected in transcription,posttranscription and posttranslation levels,which are different in different cells.Ets-1 could promote angiogenesis,migration,inflammatory and apoptosis in HUVECs.Ets-1 may be a transcription factor of DKK 1,by the famous softwears(JASPAR and ALGGEN).As predicted,there are multiple possible sites to bind with Ets-1.It is unclear that whether Ets-1 regulates DKK1 and affects the function of endothelial cells.MiRNA is a class of noncoding single-stranded small RNA molecules that can specifically pair with the 3’ untranslated region(3’-UTR)of target gene mRNA to inhibit the expression of target genes through translational repression or mRNA degradation.One miRNA can target one or more genes,and the regulatory mechanism of miRNA may be different in different cells.Studies shows that miRNA plays an important role in AS and endothelial cell function.Both DKK1 and Ets-1 are miR33a-5p’s target genes by the famous softwears,such as:MicroRNA.org.、Starbase、Targetscan.At present,miR33a-5p may be related to macrophage lipid metabolism and tumor cell’s proliferation.Previous study found that DKK1 was also regulated by miR217,miR33a,miR33b,miR103a,miR93,miR106a in the diabetic cardiomyopathy.However,the mechanisms of miR33a-5p in endothelial cells and the mechanism of miR33a-5p to regulate Ets-1 and DKK1 are unclear.Objectives1.To investigate the role of Ets-1 in the ox-LDL induced DKK1 expression in HUVECs;2.To investigate the role of miR33a-5p in the upregulation of DKK1 by ox-LDL in HUVECs;3.To explore the role of CBP/P300 and c-jun in the upregulation of DKK1 by ox-LDL in HUVECs and its relationship with Ets-1.Methods1.Cell cultureHuman umbilical vein endothelial cells(HUVECs)(passages 4 to 7)and 293T cells were resuscitated and cultured at 5%CO2 at 37℃.HUVECs are cultured in ECM,293T cells are cultured in DMEM medium with high glucose containing 10%fetal bovine serum(FBS)and 1%penicillin/streptomycin.2.Construction of Plasmid The wild and mutated 3’-UTR of the DKK1 gene(pGL3-DKK1-3’-UTR-WT&pGL3-DKK1-3’-UTR-MUT),the wild and mutated 3’-UTR of the Ets-1 gene(pGL3-Ets-1-3’-UTR-WT&pGL3-Ets-1-3’-UTR-MUT),the full-length of DKK1 promotor(PO),serially truncated DKK1 promotors(P1-P9),deleted fragment(ATGGAAT)of DKK1 promotor(PO-del),PCDNA3.1-Ets-1 plasmid and PCDNA3.1-c-jun plasmid were constructed.3.TransfectionEts-1 siRNA,c-fos siRNA,c-jun siRNA,miR33a-5p mimic,miR33a-5p inhibitor and plasmids were transfected with lipo3000.Different stimulations were given.Cells were collected and detected in 24-48h after transfection.4.Quantitative real-time PCRTotal RNA was collected by Trizol(Invitrogen).Reverse transcribed into cDNA by a Prime Script RT reagent kit(TaKaRa Bio,Japan).Real time PCR was performed by use of TAKARA SYBR Green Mix kit.Primers were synthesized by Shanghai GenePharm Company.5.Western Blot(WB)HUVECs were lysed using RIPA.Protein was separated on SDS-PAGE,transferred into PVDF membrane,blocked in 5%BSA,incubated with primary and second antibodies.The bands were photoed by luminescent image analyzer.6.Cell immunofluorescence stainingAfter fixed with 4%paraformaldehyde and penetrated with 0.5%Triton X-100 PBS,cells were incubated with first and second antibody.Stain nucleus and seal slide for fluorescent detection by fluorescence microscope.7.Luciferase reporter assaysAfter plasmid transfection of HUVECs and 293T cells 48h,cells were digested by 1×1ysis buffer,collected supernatant and detected by microplate reader with Luc Buffer Ⅰ and Luc Buffer Ⅱ.8.In vitro EMSAThe nucleoprotein was extracted by the nuclear protein extraction kit and quantified by the BCA kit.The cold wild or mutant primers were mixed with the reagent in EMSA kit,which incubated 30min at room temperature.The mixture was separated on nondenatured PAGE-gel,transferred into NC membrane.DNA was crosslinked.NC membrane was blocked and photoed.9.In vivo chromatin immunoprecipitationChIP assay was performed by using a ChIP kit,according manufacturer’s protocols.Protein was cross-linked to DNA and digested by enzyme to shear DNA to lengths 200bps.Shear chromatin were incubated with different antibodies and incubated with magnetic beads at 4℃ overnight for 2h.Puried immunoprecipitated chromatin fragments from IP samples were tested by qPCR and agarose gel electrophoresis.The qPCR was used for quantitative analysis of ChIP enrichment efficiency and expression analysis.10.lmmunoprecipitation1μl RIPA lysised cells for 30min and centrifugated 12000g 4℃ for 30min.The supernatant incubated with 1μg antibody 4℃ overnight.The pretreatment of Protein A agarose beads added into the supernatant,and incubated 4℃ for 4h.The mixture were centrifugated in 3000rpm 4℃ for 3min.The deposit was added with 1×SDS loading buffer,degenerated in 99℃ for 10min and detected by WB.11.Detection of miR33a-5p/Ets-1 in the role of DKK1 in regulation of the endothelial cells’ function(1)To detect roles of miR33a-5pregulates endothelial cells’ biological function in ox-LDL,cells were divided into the following groups:①NC group,②NC+ox-LDL group,③miR33a-5p mimics group,④miR33a-5p mimics+ox-LDL group,⑤NCI group,⑥miR33a-5p inhibitor group.(2)To detect roles of miR33a-5p/Ets-1 in endothelial cells’ function in ox-LDL,cells were divided into the following groups:①NC group,②miR33a-5p mimics group,③NC+Ets-1 group,④miR33a-5p mimics+Ets-1 group,⑤NCI group,⑥miR33a-5p inhibitor group,⑦Ets-1 siRNA group,⑧Ets-1 siRNA+miR33a,5p inhibitor.(3)To detect roles of Ets-1/DKK1 in endothelial cells’ function,cells were divided into the following groups:①NC group,②NC+Ets-1 group,③DKK1 siRNA group,④DKK1 siRNA+Ets-1 group.(4)To detect roles of CBP/P300 in endothelial cells’ function in Ets-1,cells were divided into the following groups:①DMSO group,②CBP/P300 inhibitor group,③DMSO+Ets-1 group,④CBP/P300 inhibitor+Ets-1 group.HUVECs were conducted the following experiments:(1)TUNEL assay was used to detect the apoptosis rate;(2)Cell scratch test and transwell were used to detect migration of HUVECs;(3)Tubule formation was used to detect angiogenesis.12.Statistical AnalysisAll data were shown as mean±SD and analyzed by SPSS 16.0..Statistical analysis method involved independent Student’t test for two groups and one-way ANOVA for multiplecomparisons.P<0.05 was considered statistically significant.Results1.Ets-1 participates in the ox-LDL-induced upregulation of DKK1 in HUVECsox-LDL upregulated Ets-1 expression in HUVECs,compared with Oh group(P<0.05).The nuclear translocation of Ets-1 increased after ox-LDL stimulation.Ets-1 siRNA downregulated the expression of DKK1 induced by ox-LDL(P<0.05).2.Ets-1 increased the transcriptional activity of the DKK1 promoterHUVECs were transiently cotransfected with P0 and pRL-TK vector.DLR assay showed that ox-LDL significantly increased DKK1 promoter activity.293T cells were co-transfected with P0,pRL-TK and PCDNA3.1-Ets-1 or Ets-1 siRNA respectively.DLR assay showed Ets-1 significantly enhanced DKK1 promoter activity(P<0.05)and Ets-1 siRNA decreased DKK1 promoter activity(P<0.05).293T cells were co-transfected with P0,pRL-TK and PCDNA3.1(control group),or co-transfected with P0-P9,pRL-TK vector and PCDNA3.1-Ets-1(P0 group-P9 group).DLR assay showed DKK1 promoter activity was increased in P0 group(P<0.05),and reduced from PI group to P9 group(P<0.05).EMSA assay revealed that ox-LDL significantly increased the DNA binding activity of Ets-1.Ets-1 might bind with the-2006bp--2000bp of the DKK1 promoter.ChIP revealed that ox-LDL significantly increased the binding activity between Ets-1 and promotor of DKK1.293T cells were co-transfected with P0(or PO-del),pRL-TK and PCDNA3.1-Ets-1.DLR assay showed DKK1 promoter activity of PO-del group was decreased(P<0.05).3,c-jun participates in the ox-LDL-induced upregulation of DKK1 in HUVECsox-LDL upregulated c-jun and c-fos expression in HUVECs(P<0.05).siRNA transfection downregulated the expression of c-jun or c-fos,and inhibited the upregulation of DKK1 by ox-LDL(P<0.05).The nuclear translocation of c-jun increased after ox-LDL stimulation.293T cells were co-transfected with P0,pRL-TK and c-jun siRNA.DLR assay showed c-jun siRNA decreased DKK1 promoter activity(P<0.05).293T cells were co-transfected with P0,pRL-TK and PCDNA3.1(control group),or co-transfected with P0-P9,pRL-TK and PCDNA3.1-c-jun(P0 group-P9 group).DLR assay showed DKK1 promoter activity of P0 group was increased(P<0.05),while the activity of DKK1 promoter was significantly reduced from P1 group to P9 group(P<0.05).4.CBP/P300 participates in the ox-LDL-induced upregulation of DKK1 in HUVECsCBP/P300 inhibitor descreased the upregulation of DKK1 by ox-LDL(P<0.05).The co-IP assays were performed after stimulation of ox-LDL.The results showed that Ets-1,CBP and c-jun interacted with each others.293T cells were co-transfected with P0 and pRL-TK.DLR assay showed CBP/P300 inhibitor significantly decreased DKK1 promoter activity(P<0.05).After co-transfected with PCDNA3.1-c-jun and PCDNA3.1-Ets-1,DKK1 promoter activity was increased compared with co-transfection with PCDNA3.1-Ets-1 alone.After co-transfection with PCDNA3.1-c-jun and P0-del,DKK1 promoter activity was decreased compared with P0+PCDNA3.1-c-jun group.5.miR33a-5p directly inhibited the expression of Ets-1 in HUVECs by ox-LDLox-LDL downregulated miR33a-5p expression in HUVECs(P<0.05).Dicer siRNA transfection upregulated the expression of DKK1 and Ets-1.Mimics of miR33a-5p transfection downregulated the expression of DKK1 and Ets-1,while miR33a-5p inhibitor transfection upregulated the expression of DKK1 and Ets-1(P<0.05).Co-transfection of miR33a-5p mimics with Ets-1 3’UTR luciferase reporter plasmids resulted in a significant repression of luciferase reporter gene expression in 293T cells.Transient co-transfection of miR33a-5p inhibitor with Ets-1 3’UTR luciferase reporter plasmids resulted in an opposite result.However,DLR showed that miR33a-5p did not directly binded to 3’UTR of DKX1.6.MiR33a-5p/Ets-1/DKK1 participates in the function of HUVECs by ox-LDLCompared with NC+ox-LDL group,endothelial apoptosis,migration and angiogenesis in miR33a-5p mimics+ox-LDL group was alleviated(P<0.05).Compared with NCI group,miR33a-5p inhibitor group induced endothelial cell apoptosis,migration and angiogenesis.Compared with miR33a-5p mimics group,endothelial cell apoptosis,migration and angiogenesis in miR33a-5p mimics+Ets-1 group was elevated(P<0.05).Compared with miR33a-5p inhibitor group,miR33a-5p inhibitor+Ets-1 siRNA group alleviated endothelial cell apoptosis,migration and angiogenesis.Compared with NC group,endothelial apoptosis,migration and angiogenesis in NC+Ets-1 group was augmented(P<0.05).Compared with NC+Ets-1 group,DKK1 siRNA+Ets-1 group alleviated endothelial cell apoptosis,migration and angiogenesis.Compared with Ets-1 group,CBP/P300 inhibitor+Ets-1 group alleviated endothelial cell apoptosis,migration and angiogenesis(P<0.05).Conclusions1.Ets-1 participates in the upregulation of DKK1 in HUVECs by ox-LDL;2.MiR33a-5p/Ets-1,c-jun and CBP mediated the ox-LDL induced DKK1 upregulation in HUVECs;3.MiR33a-5p/Ets-1-CBP/DKK1 pathway participates in the apoptosis,migration and angiogenesis of HUVECs by ox-LDL. |
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