HDAC9 Mechanism Of Regulating Autophagy In Vascular Endothelial Cells

Background:Stroke is mainly a neurological dysfunction in the damaged site caused by the sudden rupture or death of brain cells or occlusion of the blood vessels that supply brain tissue,which has a very high rate of death and disability.The Genome Full Association Study(GWAS)found that single nucleotide polymorphism(SNP)of the histone deacetylase 9(HDAC9)gene was associated with major atherosclerotic type cerebral infarction(LAA)and identified the HDAC9 gene as a major risk source for large vascular stroke and coronary atherosclerotic disease.However,it is difficult to explore the specific variation site of HDAC9 gene SNP increasing vascular risk,and it is very difficult to understand the mechanism of HDAC9 on the epigenetic regulation of stroke.Therefore,some studies have found that HDAC9 can regulate the function of various cells through autophagy,but also the vascular endothelial cells(VECs)function,which provides the possibility to explore the relationship between HDAC9 and stroke.Since the molecular mechanism of HDAC9 regulating VECs autophagy is not yet clear,the mechanism of HDAC9-mediated autophagy in vascular endothelial cells still needs to be further studied.Objective:1.To investigate the effect of HDAC9-regulated cell autophagy on VECs function change;2.elucidates HDAC9 molecular mechanism mediated autophagy of VECs.Methods:1.Using VECs model and control cell model of EA.hy926 cells(gbd-NC,shRNA and NC,respectively,the transdye efficiency was observed under inverted fluorescence microscope after 48 hours.2.After successful transdyeing,HDAC9 overexpression and knockout VECs model were verified by Western blot(WB)and fluorescence quantitative PCR.3.The role of autophagy in the process was evaluated by detecting transwell,scratch tests to detect overexpressing and descending HDAC9,combined with an autophagy inhibitor(3-MA),and then detecting changes in VECs migration function.4.Electric microscopy detected changes in autophagy in the VECs model of overexpression and descending HDAC9.5.Immunofluorescence(TFI)detected aggregation changes of LC3 spots(red fluorescence)in VECs models that overexpressed and dropped HDAC9.6.expression levels of autophagage markers(LC3 Ⅰ/Ⅱ)were detected by Western blot.7.Western blot detected P62 protein levels during autophagy degradation,Atg7,Atg5,Beclin-1-related protein levels during autophagy formation,and associated channel protein levels regulating ULK 1,AMPK 1/2,mTOR,PI3K-1,MAPK,and identified candidate autophagy key protein molecules regulated by HDAC9.8.Real-time quantitative PCR technology screened candidate autophagy key signal paths regulated by HDAC 9.Results:1.The efficiency of slow virus transfection was observed under inverted fluorescence microscope and the protein and mRNA expression level of HDAC9 in cells by Western blot and qPCR.2.The transwell,scratch test found that,HDAC9 can affect VECs migration function by regulating autophagage,enhanced VECs migration ability in knockout group,decreased VECs migration ability in overexpression group,and the addition of autophagage inhibitor 3-MA,reduces the inhibition of cell migration ability in overexpression group.3.Higher LC3 Ⅱ expression levels in transmission radio scopy,immunofluorescence(IFT)and WB were detected in the overexpression group and decreased in the knockout group.4.Regulatory autoophage-related channel molecules were detected by Western blot,increased Atg7 protein expression,P62,mTOR and PI3 K-1 expression,Atg7,P62 protein expression in VECs,and mTOR and PI3K-1 expression in VECs.5.Through qPCR validation of translation molecules associated with autophagy,increased mRNA expression of Atg7 in VECs decreased,mRNA expression in shRNA group and mRNA expression of mTOR in VECs.There were no significant differences in the LC3 and P62 mRNA expressions.Conclusion:1.HDAC9 can inhibit the migration function of VECs by promoting autophagy of VECs.2.HDAC9 can regulate the autophagage formation stage and may have no effect on the autophagage degradation phase.3.HDAC9 promotes the occurrence of VECs autophagy by inhibiting the expression of molecules in the PI3K/AKt/mTOR signal transfer pathway.