Long Noncoding RNA MALAT1 Promotes High Glucose-induced Human Epithelial Cell Pyroptosis By Affecting NLRP3 Expression Through Competitively Binding MiR-22

Background:Atherosclerosis is the leading cause of morbidity and mortality worldwide.Many studies have shown that cell death and inflammation play important roles in different stages of atherosclerosis.Increasing evidence indicates that the pathogenesis of atherosclerosis in patients with diabetes mellitus involves persistent chronic inflammation induced through the excessive activation of inflammation by pathogen-associated molecular patterns(PAMPs)and danger-associated molecular patterns(DAMPs).A role of lncRNA MALAT1 has been reported in various disease,such as diabetic nephropathy,diabetic retinopathy,coronary atherosclerotic heart disease,and others.A role for NLRP3 has been reported in diabetes mellitus,atherosclerosis,and others.Although liminited information is available concerning the role of lncRNA MALAT1 and NLRP3 in patients with type 2 diabetes mellitus(T2DM)and carotid atherosclerosis(CAS).Therefore,this cross-sectional study investigated these in patients with T2DM complicated with carotid atherosclerosis(T2DM+CAS).Pyroptosis,a novel proinflammatory programmed cell death process,participates in atherosclerosis pathogenesis.Recently,independent studies have revealed that lncRNAs are involved in the regulation of pyroptosis.LncRNA MALAT1 was identified as a pyroptosis-related long noncoding RNA(lncRNA).Here,we investigated the potential role and underlying mechanism of lncRNA MALAT1 in endothelial cell pyroptosis.Methods:A total of 87 inpatients were included,including 61 T2DM+CAS patients and 26 T2DM patients.Patients with T2DM or T2DM+CAS were recruited to compare the expression levels of NLRP3 mRNA and lncRNA MALAT1 and to analyze the correlation between them.In the T2DM+CAS group,patients with thickened intima media thickness and those with plaques were compared.EA.hy926 human endothelial cells(EA.hy926 cells)were treated with different concentrations of glucose.The distribution of lncRNA MALAT1 expression was detected in EA.hy926 cells by FISH.LncRNA MALAT1 and miR-22 expression was detected by qRT-PCR.Pyroptosis was evaluated by immunofluorescence staining and LDH release.The mRNA and protein levels of pyroptosis-related molecules were analyzed by qRT-PCR and western blotting.EA.hy926 cells were transfected with a MALAT1-targeting antisense oligonucleotide(ASO)to inhibit MALAT1 expression.MALAT1 target genes were explored by bioinformatics analysis and luciferase reporter assays.Western blotting and Hoechst/PI double fluorescence staining were performed on endothelial cells transfected with or without the MALAT1-targeting ASO and miR-22 mimic.Results:The expression of lncRNA MALAT1 and NLRP3 mRNA were significantly higher in patients with T2DM+CAS compared to T2DM patients.The expression levels of lncRNA MALAT1 and NLRP3 were increased in patients with T2DM and plaque compared to those with T2DM.The expression level of lncRNA MALAT1 in patients with type 2 diabetes mellitus complicated with carotid atherosclerotic plaque was positively correlated with the expression level of NLRP3 gene.LncRNA MALAT1 expression was upregulated in high glucose-treated EA.hy926 cells.LncRNA MALAT1 silence significantly inhibited high glucose-induced EA.hy926 cell pyroptosis,which may critically impact atherosclerosis.MiR-22 was a target of lncRNA MALAT1.NLRP3 expression was significantly suppressed by transfection with the MALAT1-targeting ASO.MiR-22 overexpression abrogated the effect of MALAT1 on EA.hy926 cell pyroptosis.Conclusions:The expression levels of IncRNA MALATland NLRP3 mRNA are significantly increased in patients with T2DM and carotid plaque formation.LncRNA MALAT1 promotes high glucose-induced EA.hy926 cell pyroptosis partly by affecting NLRP3 through competitively binding miR-22.Our findings indicate a new regulatory mechanism for EA.hy926 cell pyroptosis under high-glucose stress,providing a novel therapeutic target for atherosclerosis.