| BackgroundEpidemiological data showed that cardiovascular disease(CVD)was still the main cause of death worldwide.In 2021,the number of deaths from ischemic heart disease(IHD)was 9.44 million,and the disability adjusted life year(DALY)was 185 million person years.The most common cause of ischemic heart disease is atherosclerosis(AS).AS is a chronic inflammatory disease.A variety of cells are involved in the formation of atherosclerotic plaques,including endothelial cells,smooth muscle cells,macrophages,etc.Numerous studies have shown that macrophages can promote the formation of atherosclerotic plaques by secreting inflammatory factors interleukin-6(IL-6)and tumor necrosis factor-α(TNF-α).Both the CANTOS trial and COLCOT trial have confirmed that anti-inflammatory drugs reduced the occurrence of ischemic cardiovascular events.Therefore,exploring the regulatory mechanism of inflammatory response of macrophages in plaque has important scientific research value and clinical significance.N6-Methyladenosine(m6A)is the most common form of RNA modification.The modification process involves methylation of the 6th position N of adenylate in RNA under the action of methylases.The m6A modification affects the splicing,decay,translation and transport of targeted RNA.Therefore,m6A modification regulates life activities such as embryonic development,stem cell proliferation and differentiation,and tumorigenesis.The m6A modification process involves multiple enzymes,among which methyltransferase-like 3(METTL3)is capable of binding to methyl donors and catalyzing methyl transfer.In recent years,research on METTL3 inhibitors has become a hot topic and brings hope for the treatment of many diseases.Many studies confirmed that METTL3-dependent m6A modification was related to cardiovascular diseases,including myocardial hypertrophy,heart failure,and aortic aneurysm.However,the effect of METTL3-dependent m6A modification in macrophages on formation of atherosclerotic plaques needs further exploration.Mitogen activated protein kinases(MAPKs)are a class of serine/Threonine protein kinases that widely exist in mammalian cells.MAPKs regulates various cellular activities by transducing extracellular signals into cells.The activation of MAPK signaling pathway induced by oxidized low-density lipoprotein(ox-LDL)affects cell proliferation,migration,adhesion and inflammatory reaction to promote the formation of atherosclerotic plaques.Therefore,blocking the activation of MAPK signaling pathway induced by ox-LDL is one of the effective ways to control the occurrence and development of AS.METTL3 regulates multiple signaling pathways by targeting specific RNA.However,it is still unclear whether METTL3 in macrophages regulates ox-LDL induced MAPK signaling pathway activation.Therefore,we constructed macrophage-specific METTL3 knockout mice in the Apoe-/-background,and explored whether the deletion of METTL3 in macrophages affected the formation of atherosclerotic plaque in vivo.We investigated the effects of METTL3 on ox-LDL-induced macrophage inflammatory response and MAPK signaling pathway activation in vitro.Our research provided a new perspective for the formation of atherosclerotic plaques and found a new intervention target for the treatment of the disease.Objectives1.To investigate the effect of METTL3 in macrophages on atherosclerotic plaque formation.2.To explore the effect of METTL3 on ox-LDL-induced macrophage inflammatory response.3.To explore the effect of METTL3 in macrophages on ox-LDL-induced MAPK signaling pathway activation.4.To explore the molecular mechanism of METTL3 regulating MAPK signaling pathway.Methods1.To explore expression levels of macrophage METTL3 during atherosclerotic plaque formation1.1 Expression levels of macrophage METTL3 in atherosclerotic plaquesWe collected data from the database and analyzed the expression changes of Mettl3 in macrophage.in AS plaques.Apoe-/-mice(8 weeks)were divided irto three groups and fed a high-fat diet for 0,8,and 16 weeks.The vascular tissue of the aortic roots was fixed with paraformaldehyde,dehydrated,embedded,and cut into slices.Expression levels of macrophage METTL3 in plaques were measured by immunofluorescence staining.1.2 METTL3 expression levels in ox-LDL-induced macrophagesPeritoneal macrophages and RAW264.7 cells were stimulated with ox-LDL for different time periods.Western blot was performed to detect the expression levels of METTL3 protein in cells.In addition,we used immunofluorescence staining to detect METTL3 protein levels in ox-LDL-induced macrophages.2.Construction and identification of macrophage-specific METTL3 knockout miceMettl3-Flox Lyz2-Cre(Mettl3fl/fl Lyz2Cre)mice were obtained by crossing Mettl3-Flox(Mettl3fl/fl)mice with Lyz2-Cre(Lyz2Cre)mice.The polymerase chain reaction(PCR)was used to identify mouse genotypes.Peritoneal macrophages were extracted from Mettl3fl/fl Lyz2Cre mice and littermate control Mettl3fl/fl mice.Mettl3 mRNA levels in macrophages were detected by real-time PCR(RT-PCR)and METTL3 protein levels were detected by Western blot.3.To explore the effect of macrophage-specific METTL3 knockout on atherosclerotic plaque formation3.1 Effect of macrophage-specific METTL3 knockout on body weight,blood cell count,and blood lipids in miceMettl3fl/fl Lyz2Cre Apoe-/-mice and its littermate control Mettl3fl/fl Apoe-/-mice were obtained by crossing Mettl3fl/fl Lyz2Cre mice with Apoe-/-mice.Mice were fed a high-fat diet for 14 weeks,starting at 8 weeks of age.Mice were euthanized,weighed,and blood drawn.Blood routine test and blood lipid measurement were performed.3.2 Effect of macrophage-specific METTL3 knockout on plaque size,lipid infiltration,the content of collagen and macrophagesThe plaques in the aortic arch and branches were photographed during tissue isolation.Then,we obtained the heart and vascular tissues of mice.The whole aortas of mice were stained with oil red O.The vascular tissue of the aortic roots was fixed with paraformaldehyde,dehydrated,embedded,and cut into slices.Hematoxylin-eosin(HE)staining,oil red O staining,masson staining,and immunofluorescence staining of slices were performed.3.3 Effect of macrophage-specific METTL3 knockout on the inflammatory response in aortic plaquesIL-1β,IL-6,TNF-α,IL-4,IL-10,and IL-13 protein levels in atherosclerotic plaque sections were detected by histochemical staining.4.To investigate the effect of METTL3 on the inflammatory response of ox-LDLinduced macrophages4.1 Effect of METTL3 knockout on inflammatory response of ox-LDL-induced macrophagesPeritoneal macrophages were extracted from Mettl3fl/fl Lyz2Cre mice and littermate control Mettl3fl/fl mice.Macrophages were stimulated for different time periods by oxLDL.The inflammatory factors Il-1β,Il-6,Tnf-α,Mcp-1,Il-4,11-10,and Il-13 mRNA levels were measured by RT-PCR.Two groups of macrophages were stimulated with ox-LDL for different time periods and then the supernatant was collected.IL-1β,IL-6,and TNF-α protein levels of the supernatant were detected using enzyme-linked immunosorbent assay(ELISA).4.2 Effect of METTL3 knockdown on the inflammatory response of ox-LDLinduced macrophagesEfficient knockdown of Mettl3 using a small interfering RNA(siRNA)was validated by RT-PCR and Western blot.Macrophages were stimulated for different time periods by ox-LDL.The inflammatory factors Il-1β,Il-6,Tnf-α,and Mcp-1 mRNA levels were measured by RT-PCR.Macrophages were stimulated with ox-LDL for 0,6 hours and then the supernatant was collected.IL-1β,IL-6,and TNF-α protein levels of the supernatant were measured using ELISA.4.3 Effect of METTL3 overexpression on ox-LDL-induced macrophage inflammatory responseMacrophages were infected by Mettl3 adenovirus(Ad-Mettl3)and control green fluorescent protein(GFP)adenovirus(Ad-GFP)for 24 hours and then stimulated by ox-LDL for 0,2 hours.Expression levels of Il-1β,Il-6,Tnf-α and Mcp-1 mRNA were detected by RT-PCR.5.To explore the influence of METTL3 in macrophages on the MAPK signaling pathway and its targets5.1 Effect of METTL3 in macrophages on the phosphorylation levels of ERK,JNK,and P38Firstly,peritoneal macrophages were extracted from Mettl3fl/fl Lyz2Cre mice and littermate control Mettt3fl/fl mice.Western blot measured ERK,JNK,and P38 phosphorylation levels in peritoneal macrophages stimulated with ox-LDL for different time periods.Secondly,METTL3 was knocked down using siMettl3.ERK,JNK,and P38 phosphorylation levels in peritoneal macrophages stimulated with ox-LDL for different time periods were measured by Western blot.Thirdly,METTL3 protein was overexpressed in macrophages extracted from C57BL/6L mice using Ad-Mettl3 or METTL3 truncation virus(Ad-Mettl3-AMTD),and the phosphorylation levels of ERK were detected using Western blot.5.2 To explore effect of METTL3 in macrophages on the expression levels of upstream molecules of ERK pathwayPeritoneal macrophages were extracted from Mettl3fl/fl Lyz2Cre mice and littermate control Mettl3fl/fl mice and stimulated by ox-LDL for different time periods.Expression levels of Mekl,Mek2,Araf,Braf,Craf,Nras,Kras,and Hras mRNA were measured by RT-PCR.Western blot was used to detect the protein expression levels of MEK1,MEK2,p-MEK1/2,ARAF,BRAF,CRAF,NRAS,KRAS,and HRAS in macrophages.METTL3 in macrophages from C57BL/6J was knocked down using siMettl3.Macrophages were stimulated by ox-LDL for different time periods Then,conduct the same experiment as above.In addition,after overexpressing METTL3 protein in macrophages from C57BL/6J mice using Ad-Mettl3 or Ad-Mettl3-ΔMTD,Western blot was used to detect the expression levels of BRAF protein.Similarly,after overexpressing METTL3 protein in macrophages from Mettl3fl/fl Lyz2Cre mice using AdMettl3 or Ad-Mettl3-AMTD,Western blot was used to detect the expression levels of BRAF protein.5.3 To investigate effect of macrophage-specific METTL3 knockout on BRAF protein expression levels in macrophages of atherosclerotic plaquesBRAF expression in macrophages of plaques from Mettl3fl/fl Lyz2Cre mice and littermate control Mettl3fl/fl mice was measured by immunofluorescence staining.5.4 To investigate effect of BRAF overexpression on the oz-LDL-induced macrophage inflammatory responseWe constructed Brafwild type adenovirus(Ad-Braf-WT)and analyzed its expression levels by RT-PCR.We overexpressed BRAF protein in macrophages from Mettl3fl/fl Lyz2Cee mice with Ad-Braf-WT adenovirus and then stimulated macrophages with oxLDL for different time periods.Expression levels of Il-1β,Il-6,Tnf a,and Mcp-1 were detected by RT-PCR.6.To explore specific adenosine on Braf mRNA modified by m6A modification and its biological function6.1 To clarify whether Braf mRNA is modified by m6AMacrophages were obtained from C57BL/6J mice,stimulated by ox-LDL and then extracted RNA for m6A-methylated RNA immunoprecipitation sequencing(MeRIPseq)analysis.At the same time,we knocked out METTL3 or knocked out METTL3,extracted RNA from macrophages,and then performed MeRIP-qPCR.6.2 To explore specific adenosine on Braf mRNABased on the MeRIP-seq data and the base sequence characteristics of the m6A modification,we speculated the m6A modification site(39725126 in chromosome 6)and replaced the adenine on the RNA with guanine(Braf MUT1).In addition,we replaced adenine(39725174 in chromosome 6)with guanine(Braf-MUT2).We constructed plasmids of GFP-Braf-MUT1 and GFP-Braf MUT2 and transfected them into HEK293T cells along with the Flag-Mettl3 plasmid.6.3 To explore effect of specific adenosine on Braf mRNA on ox-LDL-induced inflammatory responseAd-Braf-MUT1 adenovirus was constructed.Macrophages were infected with AdBraf-WT adenovirus and Ad-Braf-MUT1 adenovirus for 24 hours and stimulated by ox-LDL for different time periods.We extracted RNA from cells and detected Il-1β,Il6,Tnf-α,and Mcp-1 mRNA levels in cells using RT-PCR.7.To investigate the mechanism by which METTL3 affected expression levels of BRAF proteinMacrophages were extracted from Mettl3fl/fl Lyz2Cre mice and its littermate control Mettl3fl/fl mice.Firstly,RNA in the cytoplasm and the nucleus was separately extracted,and then Braf mRNA was detected by RT-PCR.Secondly,peritoneal macrophages were stimulated with actinomycin D(10 μg/ml)for different time periods,and then the levels of Braf mRNA were measured by RT-PCR.Thirdly,peritoneal macrophages were stimulated with cycloheximide(CHX)for different time periods,and then the levels of BRAF protein were measured by Western blot.8.To explore reading proteins bound to Braf mRNAYTHDF1,YTHDF2,and YTHDF3 were knocked down in macrophages by transfection with siRNA.RT-PCR was used to detect the expression levels of Braf mRNA and Western blot was used to detect the expression levels of BRAF protein.RNA immunoprecipitation(RIP)assay was used to explore whether YTHDF1 bound directly to Braf mRNA.ERK phosphorylation levels induced by ox-LDL in macrophages transfected by siYthdfl were detected by Western blot.9.Statistics analysisGraphPad Prism 9(GraphPad,CA,USA)was used for all analyses.Data are presented as mean ± SD.To compare the two groups,the Mann-Whitney U test or twotailed unpaired t test was used to determine significant differences.To compare multiple groups,the Kruskal-Wallis test followed by a Dunn test was used to determine the significance.P<0.05 was considered significant.Results1.METTL3 was upregulated in macrophages during atherosclerotic progressionThe reanalysis of scRNA-seq data from mouse plaques showed that as the plaques progressed,the number and proportion of Mettl3+ macrophages in the plaques gradually increased.Immunofluorescence staining of plaques showed that the METTL3 protein levels were significantly upregulated in macrophages as the plaques progressed.Western blot showed that METTL3 protein levels increased in a time-dependent manner in peritoneal macrophages and RAW264.7 cells.Similarly,immunofluorescence staining showed that the expression levels of METTL3 protein in macrophages gradually increased in a time-dependent manner.2.Construction of macrophage-specific METTL3 knockout miceThe absence of METTL3 expression in macrophages from Mettl3fl/fl Lyz2Cre mice was confirmed by RT-PCR and Western blot.3.Macrophage-specific METTL3 knockout attenuated the formation of atherosclerotic plaques3.1 Body weight,blood routine test,and blood lipid testThere were no significant differences in body weight.Circulating white blood cell(WBC)populations,the proportion of monocyte(Mon%)and the proportion of neutrophils(Neut%)did not differ between the two groups.There were no differences in serum levels of triglyceride(TG),cholesterol(CHO),high-density lipoprotein(HDL),and low-density lipoprotein(LDL).3.2 Plaque size,lipid infiltration,collagen and macrophage contentMettl3fl/fl Lyz2Cre Apoe-/-mice exhibited fewer atherosclerotic plaques than Mettl3fl/fl Apoe-/-mice in aortic arches and its branches.Hematoxylin and eosin(HE)staining revealed more minor lesions in the aortic roots from Mettl3fl/fl Lyz2Cre Apoe-/-mice than in Mettl3fl/fl Apoe-/-mice.Oil red O staining showed METTL3 deletion reduced lipid infiltration of plaques.Similarly,we found significantly reduced atherosclerosis progression in the whole aortas of Lyz2Cre Apoe-/-mice.Masson staining showed METTL3 deletion increased the content of collagen and reduced the ratio of the necrotic area.Immunofluorescence staining showed METTL3 deletion reduced the content of macrophages in plaques.3.3 Macrophage-specific METTL3 knockout reduced the inflammatory response in atherosclerotic plaquesImmunohistochemical staining showed METTL3 deletion in macrophages decreased the protein expression levels of IL-1β,IL-6,and TNF-α in plaques.However,there were no statistical differences in the protein expression levels of IL-4,IL-10 and IL-13 in the two groups.4.METTL3 promoted ox-LDL-induced macrophage inflammatory response4.1 METTL3 knockout inhibited ox-LDL-induced macrophage inflammatory responseIl-1β,Il-6,Tnf-α,and Mcp-1 mRNA levels were reduced in peritoneal macrophages from Mettl3fl/fl Lyz2Cre mice,while Il-4,Il-10,and Il-13 mRNA levels were unchanged.ELISA showed that the secretion of IL-1β,IL-6,and TNF-α proteins was significantly decreased in peritoneal macrophages from Mettl3fl/fl Lyz2Cre mice.4.2 METTL3 knockdown inhibited ox-LDL-induced macrophage inflammatory responseRT-PCR showed that METTL3 knockdown decreased the expression levels of inflammatory factors Il-1β,Il-6,Tnf-α,and Mcp-1 mRNA in macrophages.ELISA showed that METTL3 knockdown in macrophages reduced the protein levels of IL-1β,IL-6 and TNF-α.4.3 METTL3 overexpression promoted inflammatory response in ox-LDLinduced macrophagesRT-PCR showed METTL3 overexpression via Ad-Mettl3 elevated Il-1β,Il-6,Tnf-α,and Mcp-1 mRNA levels in peritoneal macrophages.5.METTL3 affected the ERK signaling pathway by regulating the expression of BRAF protein5.1 METTL3 promoted ox-LDL-induced ERK phosphorylation,but not JNK and P38 phosphorylationWestern blot showed that METTL3 knockout or knockdown suppressed ox-LDLinduced ERK phosphorylation,but not JNK and P38 phosphorylation.In contrast,Western blot showed that the phosphorylation levels of ERK protein were higher in peritoneal macrophages infected with Ad-Mettl3 than in peritoneal macrophages infected with Ad-GFP or Ad-Mettl3-AMTD.5.2 METTL3 promoted MEK1 and MEK2 phosphorylation and expression of BRAF proteinRT-PCR showed that METTL3 knockout or knockdown had no effect on Mekl,Mek2,Araf,Braf,Craf,Nras,Kras,and Hras mRNA expression.Western blot showed that METTL3 knockout or knockdown reduced MEK1 and MEK2 phosphorylation levels and BRAF protein expression levels,but had no effect on ARAF,CRAF,NRAS,KRAS,and HRAS protein expression levels.In contrast,MEK1 and MEK2 phosphorylation levels and BRAF protein levels were higher in peritoneal macrophages infected with Ad-Mettl3 than in cells infected with Ad-GFP or Ad-Mettl5-ΔMTD.BRAF protein levels in peritoneal macrophages from Mettl3fl/fl Lyz2Cre mice infected with Ad-Mettl3 were rescued.5.3 Macrophage-specific METTL3 knockout reduced BRAF expression of macrophages in atherosclerotic plaquesImmunofluorescence staining of aortic roots showed that METTL3 deletion decreased the BRAF protein expression in macrophages.5.4 BRAF overexpression restored ox-LDL-induced inflammatory response in macrophages from MettI3fl/fl Lyz2Cre mice.RT-PCR showed that BRAF overexpression via rescued the Il-1β,Il-6,Tnf-a,and Mcp-1 mRNA levels in peritoneal macrophages from Mettl3fl/fl Lyz2Cre mice.6.METTL3 mediated Site-specific adenine methylation of Braf mRNA and affected its biological function6.1 METTL3 promoted m6A modification of Braf mRNAMeRIP-seq data showed that m6A modification was present near the 5’ end(exon 1)of Braf mRNA.Ox-LDL stimulation promoted m6A modification of Braf mRNA in macrophages.MeRIP-qPCR showed that METTL3 knockdown or knockout in macrophages reduced the m6A modification ratio of Braf mRNA.6.2 METTL3 mediated Site-specific adenine methylation of Braf mRNAWestern blot showed that METTL3 overexpression did not affect GFP-BRAF-MUT1protein levels,whereas it promoted GFP-BRAF-WT and GFP-BRAF-MUT2 protein expression.Thus,the adenine at 39725126(chromosome 6)of the Braf mRNA was identified as the specific modification site.6.3 METTL3 regulated inflammation of macrophages by mediating themethylation of adenine(39725126)of Braf mRNA RT-PCR showed Il-1β,Il-6,Tnf-α,and Mcp-1 mRNA levels were lower in peritonealmacrophages infected by Ad-Braf-MUT1 adenovirus than in those infected by Ad-BrafWT adenovirus.7.METTL3 promoted Braf mRNA translationRT-PCR showed METTL3 knockout did not affect the Braf mRNA distribution inthe cytoplasm and nucleus in peritoneal macrophages.METTL3 did not affect the Braf mRNA decay rate in peritoneal macrophages.Western blot showed METTL3 deletion did not affect the degradation rate of BRAF protein in peritoneal macrophages.8.YTHDF1 bound to Braf mRNA and promoted its translationEfficient knockdown of Ythdf1,Ythdf2,and Ythdf3 was validated by RT-PCR.RTPCR showed that Ythdfl,Ythdf2,and Ythdf3 deficiency in peritoneal macrophages did not reduce the Braf mRNA levels.Western blots showed that BRAF protein levels were lower in siYthdf1-transfected cells than in siNC-transfected cells.Ythdf2 and Ythdf3 deficiency did not reduce BRAF protein levels.Importantly,RIP assay demonstrated that YTHDF1 bound to Braf mRNA.Furthermore,ERK phosphorylation levels were lower in siYthdf1-transfected cells than in siNC-transfected cells.Conclusions1.METTL3 expression levels were increased in macrophages during AS progress.2.Macrophage-specific METTL3 knockout inhibited hyperlipidemia-induced atherosclerotic plaque formation and reduced atherosclerotic inflammation in mice.3.METTL3 promoted the expression of BRAF protein by regulating the m6A methylation modification of Braf mRNA,which affects the activation of the ERK pathway and inflammatory response in ox-LDL-induced macrophages.4.YTHDF1 recognized m6A-modified Braf mRNA and promoted its translation. |
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