Article One:RNA Heiicase DDX5 Participates In OxLDL-induced Macrophage Scavenger Receptor 1 Expression By Suppressing MRNA Degradation Article Two:lentivirus-mediated Overexpression Of CD97/ADGRE5 Protein Reverses High Glucose-induced Endothelial Cell Mig

Article one RNA helicase DDX5 participates in oxLDL-induced macrophage scavenger receptor 1 expression by suppressing mRNA degradationBackground and Aim:The hallmark feature of atherosclerosis is that monocyte-derived macrophages enter into the endothelium of the blood vessels and phagocyte lipids into foam cells.In the initial stage of plaque formation,the phagocytosis of macrophages helps to eliminate oxidized low-density lipoprotein(oxLDL)and apoptotic cells in the blood vessel wall,but in the later stage,that macrophages f’orm toam cells,and plaque grows and ruptures eventually leads to throlbosis.Therefore,it is very necessary to study and analyze the lipid phagocytosis process of macrophages.DDX5(the DEAD box protein 5)is an ATP-dependent RNA helicase,which plays an important regulatory role in various solid tumor diseases through transcriptional regulation.However,little is known about the role ot DDX5 in cardiovascular diseases,so here we mainly explore whether DDX5 is involved in the lipid phacgocytosis process of macrophages and the specific mechanism by which DDX5 regulates this process.Methods:In this study,we first tested the overall expression of DDX5 by giving oxLDL stimulation of time gradient and concentration gradient to macrophages to observe whether the distribution of DDX5 in cytoplasm and nucleus was different.Secondly,siRNA was constructed to inhibit the expression of DDX5 in macrophages,and the influence of DDX5 on lipid phagocytosis in macrophages was studied to explore its potential mechanism,that is,whether DDX5 plays a role in lipid phagocytosis by regulating the expression of receptors.Finally,the specific mechanism of DDX5 affecting the expression of phagocytic receptors was explored by double luciferase reporter gene method,mass spectrometry,protein immunoprecipitation and RNA-protein immunoprecipitation.Results:In macrophages,after oxLDL stimulation of time gradient and concentration gradient,the expression of DDX5 in mRNA and protein levels are increased,which does not depend on the MAPK and NF-kB signaling pathways.After DDX5 expression was inhibited by small interfering RNA,the lipid phagocytic function of macrophages decreased significantly,and the decrease was related to the effect of DDX5 on the expression of scavenger receptor 1(MSR1)on the surface of macrophages.Through dual luciferase reporter assay,it was found that DDX5 promoted MSR1 expression not by enhancing its transcriptional activity,but by inhibiting MSRlmRNA degradation.The interaction between DDX5 and METTL3 was found by mass spectrometry,and the latter would reduce the expression of MSRI.Finally,through protein immunoprecipitation method,we learned that METTL3 binds to MSRlmRNA to promote its m6A methylation and the degradation of MSRlmRNA.By binding to METTL3,DDX5 partially inhibited the binding of the former to MSRlmRNA,thereby inhibiting m6A nethylation and degradation of MSRlmRNA.Conclusions:DDX5 is involved in the regulation of lipid phagocytosis in macrophages.First,the increased expression of DDX5 1s induced when macrophages are phagocytic,and the increased DDX5,by binding to METTL3 protein,inhibits the m6A methylation of MSRlmRNA and reduces the degradation oi-MSRlmRNA,thereby promoting the increase of MSR1 expression and contributing to the improvement of macrophages’lipid phagocytosis.Therclore,DDX5 can be used as a new target tor the prevention and treatment of atherosclerosis.Article two Lentivirus-mediated overexpression of CD97/ADGRE5 protein reverses high glucose-induced endothelial cell migration damageBackground and Aim:As a systemic metabolic disease,diabetes is a serious threat to human health.Among them.a variety of diabetic vascular complications 1s associated with the impairment of endothelial function、including visual loss caused by retinal vascular diseases,chronic unhealed ulcers,and cardiovascular and cerebrovascular diseases caused by diabetes.Impaired endothelial l’unction caused by diabetes includes abnorrnal endothelial cell contraction and relaxation,migration and adhesion disorders.Cluster of differentiation(CD)97/adhesion G protein coupled receptor 5(ADGRH5),a member of the G protein coupled rceceptor family,is expressed on a variety ol cell surfaces,including hematopoietic cells,epithelial cells and muscle cells,etc.,and is also involved in several physiological development processes and tumor migration and proliferation.Therefore,we hypothesized whether CD97 could be used to treat impaired endothelial migration induced by diabetes.Methods:In this study、to investigate the role of CD97 in impaired endothelial migration in diabetes,we first examined the expression of CD97 in HUVECs cells which were in a high-glucose state and in aortic endothelium of diabetic mice.Then CD97 overexpressed endothelial cell lines were constructed by using lentiviral vectors to observe the wound healing assays between different treatment groups.CD97 knockout endothelial cell lines were constructed by short palindromic repeat sequence(CRISPR)/crisPr-associated protein 9(Cas9),and the expression changes of various factors related to cell migration were detected together with CD97 overexpressed cell lines.In addition,we studied the eftects of CD97 on cytoskeleton by cytoskeleton staining.Finally、the potential mechanism of high glucose affecting CD97 expression was investigated by dual luciferase reporter gene assay and ChlP assay.Results:(l)the expression of CD97/ADGRE5 was significantly decreased in high-glucose induced human umbilical vein endothelial cells(HUVECs)and diabetic mice.(2)the overexpression of CD97(EGFK2,5)in HUVECs can alleviate the dysfunction of migration induced by high glucose.(3)CD97 pronotes actin enrichment and reorganization iii endothelial cells through a CDC42-ARP2-dependent pathway;(4)the high glucose inhibits the transcription of CD97 by regulating STAT1.Conclusions:The expression of CD97 was inhibited,and endothelial migration was impaired in both diabetic mice and high glucose-treated HUVECs.In addition,overexpression of CD97 in HUVECs can effectively alleviate the damage of endothelial migration,and this process is related to the CDC42-APR2 dependent pathway.That is,through this pathway,CD97 promotes actin enrichment and recombination in endothelial cells、and then improves the function of endothelial migration.Finally,we also found that the inhibitory effect of high glucose on CD97 expression was achieved by regulating STAT1.