| Atherosclerosis is one of the major causes leading to mortality of dysfunctional cardiovascular events all around the world.The atherosclerotic lesions are mostly characterized by the transformation of macrophages to foam cells through uptake of lipoprotein-derived cholesterols,which secrete various inflammatory cytokines in the arterial intima,suggesting a critical role for macrophages in the development of atherosclerosis.Micro RNAs(miRNAs),a group of endogenous non-coding RNAs of ~22nucleotides,have been identified as important negative regulators at the posttranscriptional level.Most recently,several miRNAs have been found to impact the lipid metabolism and inflammatory response by our laboratory and others.It has been proposed that two isoforms of the miR-27 family,miR-27 a and-27 b present in the macrophage cell lines,may participate in the initiation and progression of atherosclerosis as we recently reviewed.MiR-27 was found to inhibit adipocyte differentiation,which is closely associated with the onset of obesity,and also participate in lipid metabolism in the liver.In addition,miR-27 a was detected with twofold lower expression to LPS-induced inflammatory response in the RAW264.7 cells,indicating that the targets of miR-27 a are involved in inflammatory response.These findings suggest that miR-27 may have a close relationship with atherosclerosis.Lipoprotein lipase(LPL),which is expressed and secreted by parenchymal cells in muscle and adipose tissues,could bind to the capillary endothelium and hydrolyze triglyceride(TG)core of circulating TG-rich chylomicrons(CM)and very low-density lipoproteins(VLDL)into free fatty acids(FFA)and glycerol.LPL is considered as a protector against atherosclerosis by reducing atherogenic lipoproteins in a variety of tissues,such as the heart,skeletal muscle,adipose and aorta.However,none of these actions is thought to involve macrophage LPL,which has been suggested to have a proatherogenic role.The expression of macrophage LPL was enhanced markedly in the serum from familial hypercholesterolemia patients.Furthermore,Azumi et al.demonstrated that the expression of LPL in macrophages has been associated with atherosclerotic lesions.This conclusion has been substantiated by the observation that macrophage-specific expression of human LPL promotes the development of atherosclerosis in apo E knockout mice and rabbits.Interestingly,macrophage LPL knockout(MLPLKO)mice using cre-lox P gene targeting did not show any changes in plasma LPL activities or lipoprotein levels but had a decrease in cholesterol ester foam cell formation and diet-induced atherosclerosis.Furthermore,our group and others revealed that the inhibition of macrophages-derived LPL expression has beneficial effects on lipid metabolism and inflammatory response,further supporting a proatherogenic role for macrophage LPL.One possible explanation for those properties of LPL expressed by macrophages is that LPL may contribute to the retention of atherogenic lipoproteins for subsequent cellular uptake and gene expression of inflammatory factors acting as a non-enzymatic molecular bridge between lipoprotein receptors and proteoglycans in subendothelial spaces.Therefore,manipulating macrophage LPL to reduce lipid accumulation and inflammatory response has been an important therapeutic goal of atherosclerosis.KLF14 is a member of zinc finger transpiration factor KLF family,participated in the improvement of atherosclerosis related diseases,including diabetes,dyslipidemia,obesity and coronary heart disease.Recently,several large studies demonstrated that KLF14 has been identified with playing a critical regulator in lipid profile.Genome-wide association studies(GWAS)indicated that KLF14 acts as a master trans-mediator of adipose gene expression,and affects concentration or size of plasma LDL,HDL and VLDL.Working independently in Huang P et al found that the T allele carriers of KLF14 gene had signifcantly lower HDL cholesterol(HDL-C)levels and ratio of Apo AI to Apo B than noncarriers.Those studies indicated that KLF14 may regulate Atherosclerosis related serum lipid profile.The potential mechanism underlying the function of KLF14 as a novel regulator of lipid metabolism is related to transcriptional activation of hepatic SK1 expression with the consequent up-regulation in S1 P lipid signaling.Interesting,Genetic variants newly identified in the KLF14 were implicated not only in lipid metabolism regulated genes expression but also in the etiology of atherosclerotic cardiovascular disease(ASCVD).Furthermore,hepatic-specific KLF14 gene deletion markedly decreased plasma HDL-C levels and cholesterol efflux capacity,and subsequent accelerated atherosclerotic lesion development as result of elimination effect of KLF14-mediated apo A-I promoter activation in mice.However,those previous studies focused on KLF14 of effects on hepatic lipometabolism,whether its effects and mechanism on inflammation-mediated atherosclerosis is not yet clear.Therefore,to further explore the role of extrahepatic KLF14 in inflammatory response and lipid metabolism in macrophages may shed new light on the diagnosis and intervention treatment strategy of atherosclerosis.To date,however,it is still not clear whether KLF14-mediated upregulation of miR-27 which targets macrophage LPL induced lipid accumulation and inflammatory response plays a role in the development of atherosclerosis through in vitro and vivo.In the present study,first,we predict that miR-27a/b could coincidently target LPL m RNA by bioinformatics analysis and demonstrate that miR-27a/b repressed the expression of endogenous LPL and KLF14-mediated promoter activation upregulated miR-27 a in preliminary experiments,so we put forward a working hypothesis that KLF14-mediated upregulation of miR-27 may down-regulate the expression of endogenous LPL through binding directly to the LPL m RNA 3’UTR,and then inhibit inflammatory cytokine reaction and lipid accumulation in macrophages,playing a protector against atherosclerosis.Then,in this study with gain-and loss-of-function experiments,we investigated the roles of KLF14/miR-27 in lipid metabolism and inflammatory response and atherosclerotic lesion formation in vitro or in vivo.KLF14-mediated Enhancing miR-27a/b function protected apo E knockout(KO)mice from atherosclerosis as a result of obvious reduction of LPL expression,lipid uptake,and pro-inflammatory cytokine secretion.Part I: MiR-27 Inhibits Inflammatory Response and Lipid Accumulation in MacrophagesObjective: To explore the potential roles of miR-27 in inflammatory cytokines secretion and lipid accumulation in ox-LDL-treated macrophages transfected with miR-27 mimics or inhibitors.Methods: 1.Human THP-1 and mouse RAW264.7 cells were induced to differentiate into macrophages.2.The expression levels of endogenous miR-27a/b were examined in both macrophage cells incubated with ox-LDL for 48 h using real-time quantitative PCR.3.Both cells were transfected with miR-27a/b mimic or inhibitor,chemically synthesized so as to effectively mimic and inhibit the expression of mature miR-27a/b,respectively.4.Then the quantitation of secreted pro-inflammatory cytokines,including interleukin-1β(IL-1β),interleukin-6(IL-6),monocyte chemoattractant protein1(MCP-1)and tumor necrosis factor α(TNF-α)in the media supernatants,was performed by enzyme-linked immunosorbent assay(ELISA).5.Dil-ox LDL binding assay was applied to examine the effect of miR-27a/b on lipid uptake in both ox-LDL-stimulated macrophages.6.High-performance liquid chromatography assays(HPLC)assay was conducted for the detection of free cholesterol(FC),cholesterol ester(CE)and total cholesterol(TC)in both macrophages.Results: 1.Results of incubation with ox-LDL showed a significant lower expression of miR-27a/b than that without ox-LDL in THP-1 and RAW264.7 macrophages.2.The Dil-ox LDL binding activity was significantly diminished in both macrophages treated with miR-27a/b mimic,when compared with control cells.Conversely,miR-27a/b inhibitor enhanced its binding activity.3.Transfection of both macrophages with miR-27a/b mimic was shown a significant decrease in the levels of IL-1β,IL-6,MCP-1 and TNF-α.Conversely,transfection with miR-27a/b inhibitor,those cytokines were markedly increased.4.As shown in the data of HPLC,those cells transfected with miR-27a/b mimic showed a significant reduction in the decreased levels of total cholesterol(TC)and esterified cholesterol(CE),compared with the negative control group.However,miR-27a/b inhibitor significantly enhanced TC and CE.Conclusion: 1.miR-27a/b could negatively affect the inflammatory cytokines secretion of ox-LDL-stimulated macrophages.2.miR-27a/b negative regulates not only cellular lipid uptake,but also lipid composition and content to affect the lipid accumulation in ox-LDL-stimulated macrophages.Part II: MiR-27 Inhibits Aortic Atherosclerotic in Apo E-/-MiceObjective: To further investigate the effects of miR-27a/b on the development of atherosclerosis in apo E-/-mice.Methods: 1.Eight-week-old male apo E-/-mice,fed high-fat diet and randomly allocated to 6 groups(n=10 mice per group),were given a tail vein injection with 80 mg/kg miR-27a/b agomir(AGa and AGb),scrambled agomir negative control(AG-NC),miR-27a/b antagomir(ANa and ANb)or its scrambled antagomir negative control(AN-NC)every4 weeks,respectively.2.After 8 weeks,the mice were anesthetized with 20% urethane,blood was obtained,and tissues were collected for further analysis.3.For en face analysis,the whole aorta was excised from the aortic arch to the common iliac artery and observed with stereoscopic microscope.4.To analyze the atherosclerotic lesions in the aortic sinus,frozen sections of the aortic root were prepared and stained with hematoxylin-eosin(HE)and oil red O.5.Collagen contents were evaluated by Masson’s staining.6.ELISA was applied to detect the level of plasma inflammatory cytokines,including IL-1β,IL-6,MCP-1 and TNF-α.7.Triglyceride(TG),total cholesterol(TC),HDL-C and LDL-C were detected with the automatic biochemistry analyzer.Results: 1.Stereoscopic microscope observation results showed that miR-27a/b agomir(AGa and AGb groups)reduced the quantity and size of plaques in the aortic arch and thoracic aorta region,but miR-27a/b antagomir(ANa and ANb groups)had the opposite effects.2.The atherosclerotic lesions of the whole aorta in en face were reduced in miR-27a/b agomir-treated group,but increased in miR-27a/b antagomir-treated group,when compared with their respective control mice by Oil red O staining.3.The assessment of plaque in the hematoxylin-and eosin-stained cross-sections of the aortic root revealed that the plaque area in miR-27a/b agomir-treated mice was less thanthat those in mice treated with miR-27a/b scrambled agomir.Conversely,the aortic root of miR-27a/b antagomir-treated mice contained more severe lesion than miR-27a/b scrambled antagomir-treated mice.4.Furthermore,severity of lipid deposition in the Oil red O stained cross-sections of the aortic root was alleviated in mice treated with miR-27a/b agomir,but enhanced in mice treated with miR-27a/b antagomir,compared with control groups.5.Masson trichromatic staining results showed that fiber content in the aortic sinus collagen was decreased in AGa and AGb groups,compared with AG-Con group.Compared with the AN-Con control group,the collagen fiber content was increased significantly in ANa and ANb group.6.Treatment of apo E KO mice with miR-27a/b agomir significantly decreased the levels of IL-1β,IL-6,MCP-1 and TNF-α compared with those in control mice,consistent with the regulatory role of miR-27a/b in affecting inflammatory response in vitro.7.MiR-27a/b agomir-treated group significantly increased plasma TG levels,but decreased plasma TC and plasma LDL-C levels compared with mice treated with miR-27a/b agomir negative control.On the other hand,miR-27a/b antagomir decreased plasma TG levels,but increased plasma TC and plasma LDL-C levels in comparison with miR-27a/b antagomir negative control.Conclusion: 1.miR-27a/b inhibits the development of atherosclerotic lesions in apo E-/-mice.2.miR-27 may alleviate plasma levels of inflammatory cytokines and cholesterol.Part III: MiR-27 Inhibits targetly Lipoprotein Lipase Gene to Reduce Inflammatory Response and Lipid Metabolism in MacrophagesObjective: 1.To investigate the potential regulatory effects of miR-27 on LPL 2.To observe the relevant mechanisms underlying miR-27 effects on inflammatory response and lipid accumulation in macrophages after overexpression or silence of LPL.Methods: 1.Online tools and open databases were used to obtain miR-27a/b and LPL3 ’UTR sequence conservation in each species,miR-27a/b target predictions across many species,binding sites,stem loop structure and minimum free energy of miR-27a/b match LPL.2.Identification of LPL as a direct target of miR-27a/b by using luciferase reporter gene assay.3.Real-time PCR(q RT-PCR),western blot and kits were conducted respectively for the detection of the m RNA and protein expression and activity of LPL in THP-1 and RAW 264.7 macrophages transfected with miR-27a/b mimic/inhibitor.4.LPL expressions were detected by real-time quantitative PCR and western blot in tissue homogenate of isolated aortic roots and MPM in apo E KO mice treated with miR-27a/b agomir,antagomir or their respective scrambled controls.5.Immunofluorescence double labeling was used to detect colocalization of LPL and macrophages in lesion area.6.LPL expression was used silenced with si RNA or exogenous elevated by adding bovine LPL after macrophages transfection with miR-27a/b mimic or miR-27a/b inhibitor.Effect of miR-27a/b targeting LPL on inflammatory cytokines secretion were detected by ELISA,the content of FC,CE and TC were examined by HPLC 7.The level of NF-κB expression and phosphorylation were examined using western blot analysis in macrophages and MPM of apo E-/-mice to explore the anti-inflammatory mechanism of miR-27.8.Real-time quantitative PCR and western blot method was used to analyze the m RNA and protein expression,the key scavenger receptors of CD36,SR-A1,LOX-1 and CXCL16,in macrophages and MPM of apo E-/-mice.Results: 1.MiR-27a/b,which differ only by one nucleotide outside the seed sequence,highly conserved throughout evolution among vertebrates,has biological basis to regulate targetly the LPL gene with bioinformatics analysis.2.After HEK 293 T cells transiently co-transfected with wild type p-LPL UTR luciferase reporter vector and miR-27a/b mimic,activity of LPL3’UTR was reduced.Conversely,co-transfection of miR-27a/b inhibitorsignificantly increased its activity.The mutated LPL 3’UTR vector was co-transfected with the miR-27a/b mimic into HEK 293 T cells.As anticipated,miR-27a/b mimic markedly repressed the luciferase activity of the WT LPL3’UTR reporter construct,but the suppression was reversed in the group with mutation.Interestingly,the mutations of these two predicted binding sites had different responses to miR-27a/b mimic.Site-directed mutation(SDM)of Site2(SDM2)abolished the miR-27a/b repression of LPL 3’UTR activity,but the mutation of only Site 1 had no effect.3.Transfection of macrophages with miR-27a/b mimic significantly reduced the m RNA and protein expression and activity of LPL in both concentration-and time-dependent manners,compared with transfection with nonspecific micro RNA,whereas transfection of macrophages with miR-27a/b mimic had the opposite effects.4.Compared with AG-NC group,LPL m RNA and protein expression in aorta tissue and MPM of apo E-/-mice were decreased in AGa and AGb group,whereas these were increased in ANa and ANb group compared with AN-NC group.5.The results displayed positive area of LPL expression is consistent with rich area of macrophages in aortic sinus lesions by immunofluorescence double labeling assay.Macrophage LPL expression of AGa and AGb group was down-regulated significantly in aortic sinus compared with these of AG-NC group.The macrophage LPL expression was up-regulated significantly in ANa and ANb group.6.The increase effects of miR-27a/b inhibitor on inflammatory response and lipid accumulation in macrophages have been reversed as soon as those Macrophages transfected with LPL si RNA.7.Phosphorylation of NF-κB was decreased in response to miR-27a/b mimic treatment.On the contrary,its phosphorylation was increased by treatment of cells with miR-27a/b inhibitor.Macrophages transfected with miR-27a/b inhibitor could reverse the increase effect of miR-27 inhibitor on NF-κB phosphorylation after treated with LPL si RNA in macrophage.Phosphorylation of NF-κB was reduced in MPM of AGa and AGb groupcompared with these of AG-NC group,whereas ANa and ANb group had the opposite effects.8.Addition of LPL could increase obviously the contents of TC、FC and CE,whereas silence of LPL could decrease the contents of those in macrophages.Macrophages transfected with LPL si RNA could reverse the increase effect of miR-27a/b inhibitor on the contents of cholesterol in macrophage.9.miR-27a/b mimic significantly repressed the expressions of SR-A1,LOX-1,CD36 and CXCL16 both at the m RNA and protein levels,whereas miR-27a/b inhibitor treatment had the opposite effects.Silence of LPL in miR-27a/b inhibitor group could reverse the trend increased effect of miR-27a/b inhibitor on the expression of associated scavenger receptors in ox-LDL-stimulated THP-1 macrophages.10.q RT-PCR and Western blotting assays results showed that the m RNA and protein expression of CD36 、SR-A1、LOX-1、CXCL16 reduced obviously in MPM of AGa and AGb group compared with these of AG-NC group,whereas ANa and ANb group had the opposite effects.Conclusion: 1.MiR-27 directly targets LPL gene through their 3’UTR sequence to inhibit m RNA and protein expression of LPL in macrophages.2.miR-27,a novel antiatherogenic factor,attenuates inflammatory response and lipid accumulation via downregulation gene expression of LPL,closely related to negative regulation scavenger receptors CD36、SR-A1、LOX-1、CXCL16expressions and inflammatory transcription factor NF-κB phosphorylation,suggesting a potential strategy to the diagnosis and treatment of atherosclerosis.Part Ⅳ: Kruppel-Like Factor 14 Sufficiency in Macrophages Decelerate Atherosclerosis via Down-regulation of Lipoprotein Lipase Expression Dependented on miR-27aObjective: To explore antiatherogenic effect of gypenosides(GP)mediated KLF14 Sufficiency via miR-27 a downregulating LPL expression and inhibition of inflammatory response and lipid accumulation by detecting miR-27 and LPL expression in THP-1 macrophage,and atherosclerotic lesion,plasma inflammatory cytokines,lipid level in apo E-/-mice.Methods: 1.To investigate the cytotoxic effects of GP on macrophages via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)and lactate dehydrogenase(LDH)analyses.2.THP-1 macrophages incubated with different GP concentrations(0,10,25,50,100 μg/m L)and time(0、6、12、24、48 h),were detected the effects of GP on to LPL expressions by q RT-PCR and western blot.3.The macrophages was infected lentiviral vector LV-h LPL to overexpress endogenous LPL or bovine LPL to add exogenous LPL after incubated with 50μg/ml GP.The effect of GP on inflammatory cytokines secretion were detected by ELISA,the content of FC,CE and TC were examined by HPLC,and lipid accumulation were observed by Oil red O staining.4.miR-27a/b expressions were detected by q RT-PCR when the macrophages incubated with GP for 24 h.5.THP-1 macrophages were transfected with miR-27 a inhibitor after incubated with 50μg/ml GP,q RT-q PCR and western blot were used to detect the m RNA and protein expressions of LPL,ELISA to analyze inflammatory cytokines secretion,HPLC to examine the content of FC,CE and TC,and lipid accumulation were observed by Oil red O staining.6.The effect of GP on CCAAT/enhancer binding protein(KLF14)and pre-miR-27 a expressions were detected by q RT-PCR or Western blot.7.The THP-1 macrophages was transfected with si RNA to silence KLF14 expression or infected lentiviral vector LV-h KLF14 to overexpress KLF14,q RT-PCR was used to detect the expression of pre-miR-27 a.8.THP-1 macrophages were infected with LV-h KLF14 for 24 hours and incubated in medium containing actinomycin D for another 24 hours,q RT-PCR was conducted for the detection of the expression of pre-miR-27 a.9.The bioinformatic tools were used to predicte the the potential probability of binding KLF14 to miR-27 a promoter sites.10.Identification of miR-27 a gene promoter as a direct target of KLF14 by and Chromatin Immunoprecipitation(Ch IP)and Luciferase Reporter Gene Assay.11.Eight-week-old male apo E-/-mice,fed high-fat diet were randomly allocated to 4 groups(n=10 mice per group),GP group was fed with 200mg/kg/d GP in high-fat diet,GP+ANa or GP+LV-h LPL group fed with GP were given a tail vein injection with 80 mg/kg miR-27 a antagomir or 6×107 TU/200 ul human LPL lentiviral vector every 4 weeks,respectively.After 8 weeks,the mice were bled to death using 20% urethane anesthetized,blood was obtained,and tissues were collected for further analysis.12.The stereoscopic microscope for en face analysis,was used to observe the whole aorta excised from the aortic arch to the common iliac artery.13.The atherosclerotic lesions in the aortic sinus,frozen sections of the aortic root were prepared to analyze by HE and oil red O stain assay.14.Collagen contents were evaluated by Masson’s staining.15.ELISA was applied to detect the level of plasma inflammatory cytokines,including IL-1β、 IL-6、MCP-1 and TNF-α.16.TG,TC,HDL-C and LDL-C were detected by the automatic biochemistry analyzer.Results: 1.THP-1 and RAW 264.7 macrophages treated with GP for 24 h did not affect the cell viability and the LDH release from macrophages,indicating GP exhibited a low cytotoxicity to the cells was used for further experiments.2.The both macrophages treated with GP had a significant down-regulation to the m RNA and protein expression of LPL in both concentration-and time-dependent manners.3.Infection lentiviral vector LV-h LPL or addition bovine LPL after incubated with GP could reverse thereduction effects of GP on the inflammatory cytokines secretion and the cellular lipid accumulatioin in macrophages.4.GP could up-regulate significantly the expressions of miR-27 a in macrophages treated with ox-LDL,whereas miR-27 a inhibitor treatment had weaken effect of GP on inhibition LPL expression and reversed the reduction effects of GP on the inflammatory cytokines secretion and the lipid accumulation in macrophages.5.The correlated positive expression of KLF14 with pre-miR-27 a had a significant up-regulation in time-dependent manners.6.Overexpression of KLF14 in macrophages infected with lentiviral vector LV-KLF14 could up-regulate significantly the expression of pre-miR-27 a,whereas silence of KLF14 in macrophages treated with GP could reverse the trend increased effect of GP on the expression of miR-27 a.7.KLF14 induced pre-miR-27 a upregulation was blocked by actinomycin D,a transcriptional inhibitor,suggesting that KLF14 regulates miR-27 a at the transcriptional level.8.Bioinformatic sequence analysis tools was performed to predicte and revealed a putative CATGGTGAAAC site in the promoter region of human miR-27 a.9.Ch IP and Luciferase Reporter Gene Assay validated promoter binding and showed that the-1521~-1508 site had KLF14 occupancy in GP-treated THP-1macrophages.10.The stereoscopic microscope and Oil red O staining results showed that GP reduced the number and size of lesion in the aorta region,but given an additional tail vein injection with miR-27 a antagomir(ANa)or human LPL lentiviral vector(LV-h LPL)had more severe effects.11.The assessment of plaque in the HE stained cross-sections of the aortic root revealed that the lesion area in GP-treated mice were less than that those in control mice,the aortic root of ANa and LV-h LPL treated additionally mice contained more severe lesion than GP treated mice.12.Furthermore,severity of lipid deposition in the Oil red O stained cross-sections of the aortic root was alleviated in mice treated additionally with GP,but enhanced in mice treated with ANa and LV-h LPL.13.Masson trichromatic staining results showed thatfiber content in the aortic sinus collagen was decreased in GP groups,compared with CON group,whereas the increased significantly in GP+ANa and GP+LV-h LPL group compared with the GP group.14.Treatment of apo E-/-mice with GP significantly decreased the plasma levels of inflammatory factors compared with those in control mice,but addition GP+ANa and GP+LV-h LPL increased significantly those compared with the GP group.15.GP significantly increased plasma HDL-C levels,but decreased plasma TC and LDL-C levels compared with control mice.On the other hand,addition ANa and LV-h LPL decreased plasma TG and HDL-C levels,increased significantly TC and LDL-C levels compared with the GP group.Conclusion: Antiatherogenic effect of Gypenosides via down-regulation of lipoprotein lipase expression and then inhibition of inflammatory response and lipid accumulation dependented KLF14 and MiR-27 a Pathway. |
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