| Objective: Atherosclerosis(AS)is a chronic cardiovascular disease caused by inflammation and lipid accumulation,which seriously endangers human life and health and is the main cause of coronary artery disease,peripheral arterial disease,cerebral infarction and stroke.There are many theories that contribute to the pathogenesis of AS,such as lipid theory,endothelial injury theory,inflammatory reaction theory,and oxidation theory.It is now generally accepted that AS is a chronic inflammatory disease that is a process of vascular injury caused by the interaction of smooth muscle,endothelium and macrophages under the action of cytokines.Under normal physiological conditions,macrophages are less present in the blood.Only after endothelial injury,monocytes enter the endometrium through the injured endothelium and transform into macrophages.Macrophages phagocytose oxidized low-density lipoprotein through the scavenger receptor on the cell surface to form a large number of foam cells,and foam cells accumulate to form lipid streaks to further form atheromatous plaques.The inflammatory response is a complex response of the body's immune system to infection and tissue damage.Macrophages are the main effector cells of the inflammatory response,and cytokines and chemokines can be produced in response to external stimuli to limit infection.Disordered inflammation can cause a variety of pathological conditions,including autoimmunity,septic shock,atherosclerosis,etc.,so the inflammatory response must be strictly regulated.MicroRNAs(miRNAs)are endogenous,non-coding,single-stranded RNA molecules of approximately 18-25 nt in length,and studies have shown that miRNAs can regulate gene expression.The miRNA regulates gene expression at the post-transcriptional level by degrading or inhibiting translation in the 3'-UTR region of the target gene mRNA,thereby the miRNA exerts a functional influence through the target gene.Studies have shown that a variety of miRNAs are involved in the regulation of AS and inflammatory responses.It is known from the literature that miR-520c-3p is widely studied in many types of tumors,and that by luciferase assay,RELA is a target gene of miR-520c-3p,then whether the correlation in the tumor also correlated in the AS inflammatory response has not been reported.Based on the above research background,this study focused on the stimulation of THP-1 macrophages by lipopolysaccharide(LPS)to establish an inflammatory model,and explored the role of miR-520c-3p in THP-1 macrophage inflammatory response and oxidative stress.The purpose is: miR-520c-3p and RELA regulates the release of inflammatory factors(IL-1?,TNF-?,IL-6),phagocytizing fluorescently labeled oxidized low density lipoprotein(Dil-Labeled oxidized low density,Dil-ox-LDL),regulates the expression of Malondialdehyde(MDA)and Reactive Oxygen Species(ROS)in THP-1 macrophages.Method:(1)Quantitative Real-time Polymerase Chain Reaction(qPCR)was used to detect the expression of miR-520c-3p in THP-1 macrophages under different concentrations of LPS.(2)Transfection of miR-520c-3p mimic(Pre-miR-520c)and inhibitor(Anti-miR-520c)in THP-1 macrophages,qPCR was used to detect the expression of miR-520c-3p.(3)Using ELISA(Enzyme linked immunosorbent assay)and qPCR method to detect the expression of cellular inflammatory factors IL-1?,TNF-? and IL-6 in both mRNA and protein after overexpression and inhibition of miR-520c-3p.(4)Immunofluorescence technique was used to analyze the effect of miR-520c-3p on the uptake of oxidized low density lipoprotein by THP-1 macrophages.(5)The effect of miR-520c-3p on the expression of oxidative stress markers MDA and ROS in THP-1 macrophages was detected by multi-function fluorescent microplate reader.(6)Using qPCR and Western blot to detect the correlation between RELA and miR-520c-3p,and verify the transfection efficiency of three siRELA at mRNA and protein levels.(7)ELISA and qPCR methods were used to detect the expression of cellular inflammatory factors IL-1?,TNF-?,IL-6 at mRNA and protein levels after transfection of siRELA.(8)The effect of RELA on the uptake of Dil-ox-LDL by THP-1 macrophages was analyzed by immunofluorescence technique.(9)The effect of RELA on the expression of oxidative stress marker MDA and ROS activity in THP-1 macrophage was detected by multi-function fluorescence microplate reader.Results:(1)The expression of miR-520c-3p was down-regulated in different concentrations of LPS-stimulated THP-1 macrophages.(2)Pre-miR-520 c increased the expression of miR-520c-3p,Anti-miR-520 c inhibited the expression of miR-520c-3p,and the transfection efficiency could be used for experiments.(3)Pre-miR-520 c inhibits the expression of cellular inflammatory factors IL-1?,TNF-? and IL-6,and Anti-miR-520 c promotes the expression of cellular inflammatory factors IL-1?,TNF-? and IL-6.(4)Pre-miR-520 c can inhibit the uptake of Dil-ox-LDL by THP-1 macrophages,Anti-miR-520 c can promote the uptake of Dil-ox-LDL by THP-1 macrophages.(5)Pre-miR-520 c down-regulated the expression of MDA and ROS in THP-1 macrophage oxidative stress markers,and Anti-miR-520 c had the opposite effect.(6)RELA expression was up-regulated in different concentrations of LPS-stimulated THP-1 macrophages,and RELA mRNA and protein expression levels showed a negative correlation with miR-520c-3p.(7)siRELA can inhibits the expression levels of inflammatory factors IL-1?,TNF-?,IL-6.(8)siRELA can inhibit THP-1 macrophage phagocytosis of Dil-ox-LDL.(9)siRELA inhibits the expression of THP-1 macrophage lipid oxidation markers MDA and ROS.Conclusion:(1)miR-520c-3p is down-regulated in different concentrations of LPS-stimulated THP-1 macrophages,inhibits the expression of IL-1?,TNF-?,IL-6,and inhibits THP-1 phagocytosis of Dil-ox-LDL and the expression of oxidative stress markers MDA and ROS.(2)RELA was up-regulated in different concentrations of LPS-stimulated THP-1 macrophages and negatively correlated with miR-520c-3p expression.(3)In THP-1 macrophages,siRELA inhibits the expression of IL-1?,TNF-?,and IL-6,inhibits THP-1 phagocytosis of Dil-ox-LDL and the expression of oxidative stress markers MDA and ROS. |
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