The Anti-atherosclerosis Mechanism Of MicroRNA-302c-3p Inhibiting Endothelial Cell Pyroptosis Via Targeting NLRP3

Objective: To explore the function and mechanism of miRNA targeting NLRP3 in endothelial cells and provide a new strategy for the prevention and treatment of atherosclerosis.Methods:(1)Four online bioinformatics websites(Target Scan,miRanda,miRWalk,miRMAP)were used to screen out four potential miRNAs(miR-302C-3p,miR-490-5p,miR-421 and miR-876-5p)targeting NLRP3 through comprehensive evaluation of site prediction score,miRNA conservatism,and association with cardiovascular inflammation.(2)Real-time quantitative reverse transcription polymerase reaction(qRT-PCR)and fluorescence in situ hybridization(FISH)were used to detect the differential expression of candidate miRNAs in human normal vascular tissues and atherosclerotic vascular tissues.(3)Human umbilical vein endothelial cells(HUVECs)treated with oxidized low density lipoprotein(ox-LDL)were used as cell model in vitro.Mi R-302C-3p was overexpressed and knocked down,respectively.qRT-PCR and western blot were used to detect the expression levels of pyrotosis-related proteins(NLRP3,cleaved caspase-1,cleaved IL-1β,and GSDMD).(4)Lactate dehydrogenase(LDH)release assay and Hoechst 33342/PI staining were used to detect the occurrence of pyroptosis of cells.(5)Magnetic bead RNA pull down assay and Dual luciferase reporter gene assay were used to verify the direct targeting relationship between miR-302C-3p and NLRP3.(6)The expression of NLRP3 in HUVECs was silenced by si RNA,which were then transfected with miR-302C-3p mimic and inhibitor to investigate whether the silencing of NLRP3 affected the inhibitory effect of miR-302C-3p on cell pyrotosis.(7)A mouse model of atherosclerosis was established.miR-302C-3p agomir was injected into tail vein.The expression levels of pyroptosis-related proteins and miR-302C-3p were detected by oil red staining,immunofluorescence,immunohistochemistry,FISH,qRT-PCR and western blot.Results:(1)miR-302C-3p was down-regulated in human atherosclerotic plaque tissues and ox-LDL-treated HUVECs.Cytokines associated with NLRP3 inflammasome activation(NLRP3,cleaved caspase-1,cleaved IL-1β,and GSDMD)were negatively correlated(P<0.05).(2)Pull down assay and dual luciferase reporter gene assay confirmed that miR-302C-3p could target specific sites of NLRP3 and inhibit its transcriptional expression(P < 0.05).Further functional experiments showed that overexpression of miR-302C-3p inhibited the expression of NLRP3,cleaved caspase-1,cleaved IL-1β,and GSDMD,and reduced the degree of cell pyroptosis(P < 0.05);However,inhibition of endogenous miR-302C-3p expression up-regulated the expression of NLRP3,cleaved caspase-1,cleaved IL-1β,and GSDMD,and enhanced cell pyroptosis(P < 0.05).(3)Overexpression or knockdown of miR-302C-3p expression while silenting NLRP3 expression further down-regulated the expression of NLRP3,cleaved caspase-1,cleaved IL-1β,and GSDMD,and reduced the number of cell pyrotosis(P<0.05).(4)Animal studies showed decreased lipid areas,improved atherosclerotic lesions,and down-regulated expression of NLRP3,cleaved caspase-1,cleaved IL-1β,and GSDMD in the miR-302C-3p agomir treatment group compared with the atherosclerosis group(P<0.05).Conclusions: Experiments in vivo or in vitro demonstrated that miR-302C-3p inhibited NLRP3 activation by targeting specific sites of NLRP3,thereby inhibiting endothelial inflammation and cell pyrotosis.Overexpression of miR-302C-3p can inhibit endothelial cell pyrotosis and improve atherosclerosis,suggesting that miR-302C-3p can be used as a new target for prevention and treatment of atherosclerosis.