The Mechanism Of WTAP Drives Macrophage Apoptosis And Accelerating Atherosclerosis

Background:Atherosclerosis is the common pathological basis of coronary artery disease,cerebrovascular disease and peripheral artery disease.It is characterized by vascular intima damage,inflammatory cell infiltration,macrophage foam and atherosclerotic plaque formation.Myocardial infarction and stroke caused by atherosclerotic plaque rupture are the leading causes of death worldwide.Wilms tumor 1-associating protein（WTAP）is a core N6-methyladenosine（m⁶A）methylation writer,although m⁶A methyl modification plays an important role in a variety of basic biological and disease processes,no clear studies have reported the relationship between WTAP and the development of atherosclerosis.Hence,this study investigated the precise role of macrophage WTAP in atherogenesis.Methods:Firstly,m⁶A modification levels was detected atherosclerotic aortic plaque of Apo E^-/-mice and peripheral blood monocytes of coronary artery disease（CAD）by m⁶A methylation quantitatively.Next,we constructed a stable LPS-induced inflammatory microenvironment in macrophages and evaluated the expression of m⁶A modified-associated proteins to identify the major m⁶A modified-associated protein WTAP involved in this process by q RT-PCR and Western blot.Meanwhile,the expression of WTAP in macrophages in human carotid plaque and Apo E^-/-mouse atherosclerotic plaque tissue was detected by immunofluorescence and Western blot.Furthermore,we detected WTAP m RNA and protein levels in peripheral blood monocytes collected from atherosclerotic CAD patients by q RT-PCR and Western blot.Next,we demonstrated that WTAP regulates macrophage apoptosis rather than inflammation by knocked down the expression of WTAP.To study its function in regulating macrophage apoptosis and the development of atherosclerosis,we used AAV8（in vivo）to generate macrophage-specific WTAP knockdown mice to determine the effect of WTAP on atherosclerosis in Apo E^-/-mice.Furthermore,we demonstrated that WTAP can regulate macrophage apoptosis by regulating the expression of myosin heavy chain 11（MYH11）.However,it was indirectly or directly demonstrated that WTAP regulation of MYH11 expression was independent of WTAP-mediated m⁶A modification by testing m RNA stability,protein stability and RNA pull-down experiments.Next,we demonstrated that WTAP regulates the expression of MYH11 by miR-124-3p sequestration through bioinformatics prediction,protein immunofluorescence and dual-luciferase reporter gene.Finally,we confirmed that the expression of WTAP/miR-124-3p/MYH11 axis regulates macrophages apoptosis in vitro.Results:We first demonstrated that the level of m⁶A modification was significantly upregulated in atherosclerotic aortic plaque of Apo E^-/-mice and peripheral blood monocytes of CAD patients.We further found that WTAP is highly expressed in atherosclerotic lesions of Apo E^-/-mice and peripheral blood monocytes of human CAD patients.At the same time,In vitro experiments demonstrated that WTAP regulates macrophage apoptosis rather than inflammation.macrophage-specific WTAP knockout alleviates macrophage apoptosis and atherosclerosis development in Apo E^-/-mice.Mechanically,we confirmed that MYH11 mediated macrophage apoptosis regulated by WTAP.However,WTAP upregulates MYH11expression in macrophages with m⁶A independent modification.Next,we reported that WTAP m RNA promotes MYH11 expression by miR-124-3p sequestration.Finally,the WTAP/miR-124-3p/MYH11 axis was shown to regulate macrophage apoptosis.Conclusions:WTAP increases MYH11 expression by miR-124-3p sequestration,thereby promoting macrophage apoptosis and atherosclerosis,indicating that WTAP may be a potential therapeutic target against atherosclerosis.