The Role And Mechanism Of METTL3 In Vascular Endothelial Cells-Mediated Coagulation

Background and objective:After blood vessels are damaged,the coagulation system is activated immediately.Thrombin acts on fibrinogen to generate fibrin through the endogenous or exogenous coagulation cascade reaction,which changes blood from flow state to gel state,thus achieving the effect of hemostasis.The whole process involves coagulation,anticoagulation and fibrinolysis systems.Physiologically,these three systems of the body include platelets,red blood cells,white blood cells,vascular endothelial cells and other cells,which are regulated by a variety of coagulation factors and coagulation related proteins to maintain dynamic balance^[1].Disorder in any portion of the coagulation can result in abnormal bleeding or thrombolic disease,as shown in 20%to 50%of patients with sepsis who have a marked Disseminated intravascular coagulation(DIC)^[2].In addition,severe trauma^[3],liver dysfunction^[4],malignant blood disease^[5]were accompanied by a certain degree of coagulation abnormalities.Therefore,the depth study of the mechanism in the process of coagulation will undoubtedly help to provide new targets for coagulation disorders.Vascular endothelial cells,which cover the surface of all blood vessels,provide an important barrier to blood circulation,cellular and acellular components of the interstitium,and play a role in regulating tissue perfusion,supplying oxygen and transporting nutrients,and controlling blood pressure.In addition,endothelial cells,as a bridge connecting coagulation,anticoagulation and fibrinolysis,also play an extremely important role in the procoagulation,anticoagulation and fibrinolysis process of coagulation^[1].When blood vessels are damaged,endothelial cells can activate exogenous coagulation pathway and activate platelets through high expression of Tissue factor(TF)and von Willebrand factor(v WF),respectively^[6].Endothelial cells also secrete Antithrombin(AT),Thrombomodulin(TM)and protein C receptor to participate in the anticoagulation process.In addition,endothelial cells also participate in the regulation of thrombus lysis by expression of Tissue/Urokinase plasminogen activator(t-PA/u-PA)and plasminogen activator inhibitor(PAI-1)^[7,8].Previous studies have shown that vascular endothelial cells participate in coagulant balance and are regulated by transcriptional and post-transcriptional mechanisms such as transcription factors and micro RNAs^[9,10].In recent years,post-transcriptional modification of mRNA has been reported to play an important role in many fields.N⁶-methyladenosine(m⁶A)is the most common post-transcriptional modification of eukaryotic mRNA.N⁶-methyladenosine(m⁶A)is the most common post-transcriptional modification of eukaryotic mRNA.m⁶A modification is catalyzed by Methyltransferase complex,of which methyltransferase-like protein 3(METTL3)is the core subunit of the complex^[11].Studies have shown that METTL3 affects endothelial cells formation in embryonic hematopoietic endothelial cells^[12].In hepatocellular carcinoma and prostate cancer,METTL3 affects the coagulation pathway^[13].Since METTL3 is highly enriched in endothelial cells,and endothelial cells are the main participants in the regulation of coagulation,it is worth further study whether METTL3 is involved in the regulation of endothelial anticoagulation process.To explore the role of METTL3 in endothelial cells-mediated anticoagulation,we first constructed a Human umbilical vein endothelial cells(HUVEC)model with METTL3 gene silencing,further explored whether METTL3 regulates endothelial cells-mediated coagulation process by transcriptome sequencing.We found that METTL3 was closely related to coagulation.METTL3 promotes the expression of PAI-1,a key molecule inhibiting fibrinolysis.Preliminary functional verification showed that METTL3 inhibited fibrinolysis.Then,the molecular mechanism of METTL3 regulating PAI-1 was explored by constructing HUVEC cell model with gene silencing,overexpression and complement.Finally,the regulatory effect of METTL3 on PAI-1 was studied in vivo by using sepsis mouse model.The main research results are as follows:Part ?:METTL3 upregulates the expression of PAI-1 in endothelial cells and inhibits fibrinolysis.1.METTL3 is involved in the regulation of endothelial cells-mediated coagulation process.HUVEC was infected with Short hairpin RNA(shRNA)mediated by lentivirus.Real-time fluorescent quantitative PCR(qPCR)and Western blot(WB)were used to detect the knockdown efficiency of METTL3.Subsequently,RNA of HUVEC cells silenced by METTL3was extracted for high-throughput RNA sequencing(RNA-seq)and bioinformatics analysis.The results of differential gene cluster analysis indicated that many biological processes,such as coagulation and cell migration,were significantly affected after silencing METTL3.The role of METTL3 in cell migration is shown in the appendix.This study focuses on the role of METTL3 in endothelial cell-mediated coagulation.Further differential genes heat map analysis revealed that the expression level of PAI-1,a key molecule inhibiting fibrinolysis,was significantly down-regulated.2.METTL3 up-regulates the expression of PAI-1To further verify the bioinformatics results,qPCR and WB were used to detect the expression level of PAI-1 in HUVEC with silenced METTL3,and it was found that the expression level of PAI-1 was significantly down-regulated(P<0.001).Subsequently,we constructed HUVEC cell model with METTL3 overexpression and complement,and detected the expression of PAI-1 by qPCR and WB.We found that the mRNA and protein expression levels of PAI-1 were significantly up-regulated(P<0.001).These results suggest that METTL3promotes the expression of PAI-1.3.METTL3 inhibits fibrinolysisPAI-1 is an important regulator of fibrinolysis.In order to define the METTL3 regulation function in the process of fibrinolysis,we adopt fibrinolysis experiment in METTL3 silence,METTL3 expression,METTL3 covering HUVEC cell observation fibrinolysis,respectively.We found that fibrinolysis was accelerated in METTL3 silenced cells,and significantly slowed down in METTL3 overexpressed cells,while fibrinolysis was accelerated after METTL3supplementation.These results suggest that METTL3 inhibit fibrinolysis.Part ?:The promotion of PAI-1 expression by METTL3 depends on the m⁶A-JUN-YTHDF1 pathway.1.m⁶A modification was rich in JUN mRNA.In order to explore the molecular mechanism of METTL3 regulating PAI-1,the m⁶A modified mRNA in HUVEC was enriched by m⁶A antibody,and then methylated RNA Immunoprecipitation sequencing(RIP-seq)was conducted.The results showed that PAI-1mRNA was not significantly methylated,while the transcription factor JUN mRNA was hypermethylated.2.METTL3 mediated the m⁶A modification of JUN mRNA and promoted its translation.We quantified the expression of JUN mRNA or protein by qPCR and WB in HUVEC with silenced or overexpressed METTL3.The results showed that silenced or overexpressed METTL3 had no significant effect on the expression of JUN mRNA,but significantly down-regulated and up-regulated the expression of JUN protein,respectively,suggesting that METTL3-mediated modification of JUN methylation affected the translation of JUN.3.JUN protein promoted PAI-1 gene transcription.In order to explore whether JUN regulates PAI-1 expression.Chromatin immunoprecipitation(Ch IP)was used to detect the binding of JUN to the PAI-1 promoter region.We found that JUN could bind to the PAI-1 promoter.Then,qPCR and WB were used to detect the expression level of PAI-1 in HUVEC with JUN silenced or overexpressed,and in HUVEC with JUN overexpressed after METTL3 silenced,and performed the fibrinolysis assay.The results showed that PAI-1 was significantly down-regulated in JUN-silenced cells(P<0.001),and up-regulated in JUN overexpression cells(P<0.01).Overexpression of JUN can remedy the low expression of PAI-1 in METTL3-silenced cells(P<0.001).The rate of fibrinolysis was decreased after silencing of JUN,but increased after overexpression of JUN.Overexpression of JUN could remedy the fibrinolytic impaired phenotype in silenced METTL3cells.These results suggested that JUN promoted the transcription of PAI-1 and inhibited fibrinolysis.And METTL3 promoted the expression of PAI-1 depend on JUN.4.JUN mediated up-regulation of PAI-1 expression depend on the recognition protein YTHDF1.m⁶A modified mRNAs are recognized by different m⁶A recognition proteins to regulate mRNA expression.Combined with the second result in this part,METTL3 promoted JUN translation,so we constructed a HUVEC model in which m⁶A modified recognition protein YTHDF1 was silenced.WB was used to detect JUN/PAI-1 expression levels.The results showed that the expression of JUN protein was significantly down-regulated.The expression levels of PAI-1 were significantly down-regulated(P<0.05).These results indicated that the m⁶A modification of JUN mRNA was recognized by YTHDF1,and the JUN promoted the expression of PAI-1 depend on YTHDF1.Part ?:METTL3/m⁶A/JUN/PAI-1 pathway may be involved in regulating fibrin deposition in sepsis mouse.1.Establishment of sepsis mouse model.In order to investigate the regulation effect of METTL3 on PAI-1 expression in vivo.C57/BL6 mice were intraperitoneally treated with 6mg/kg LPS to establish sepsis mouse model.After 4h,we collected lung and liver tissues and used immunohistochemistry and Hematoxylin-Eosin(HE)staining to detect fibrin deposition and thrombosis,respectively.There were obvious fibrin deposition and thrombosis in liver and lung tissue in sepsis mouse.2.METTL3/m⁶A/JUN/PAI-1 pathway was highly expressed in the blood vessels of sepsis mouse.We collected celiac vein and thoracic aorta from sepsis mouse and control group.m⁶A methylation assay kit was used to detect the m⁶A modification level.We used qPCR and WB to detect the expression of METTL3/JUN/PAI-1.The results showed that m⁶A modification level in the blood vessels of sepsis mouse was significantly increased(P<0.05),the expression levels of METTL3/JUN/PAI-1 were significantly up-regulated(P<0.01).Conclusion:Through this study,we can obtain the following conclusions:(1)METTL3 is involved in the regulation of endothelial cells-mediated coagulant function,inhibiting fibrinolysis by upregulating the expression of PAI-1.(2)Mechanismly,METTL3 promotes the expression of PAI-1 through the m⁶A-JUN-YTHDF1 pathway.(3)Fibrin deposition and microthrombosis in sepsis mouse may be related to the METTL3/m⁶A/JUN/PAI-1 regulatory pathway.However,further experimental is needed.This study reveals the mechanism of METTL3 regulation of fibrinolysis,which provides an experimental basis for the involvement of m⁶A modification in coagulation,and provides a possible new target for the clinical prevention and treatment of coagulation disorders.