| Atherosclerosis(AS)is a chronic cardiovascular inflammatory disease that tends to occur in large and medium arteries caused by high-calorie and high-fat diet(HFD),mainly caused by infiltration of macrophages into the arterial wall.It is a major cause of high morbidity and mortality worldwide and is characterized by the formation of arterial plaques.In addition,atherosclerosis contributes to most cardiovascular diseases,including acute myocardial infarction,and increases cardiovascular mortality.It has been reported that increased lipid accumulation plays a critical role in the progression of atherosclerosis.Oxidized low-density lipoprotein(ox-LDL)has been shown to induce inflammatory responses during the formation and development of atherosclerosis.At present,despite significant advances in the treatment of atherosclerosis,the disease remains one of the leading causes of high morbidity and mortality worldwide.Therefore,it is urgent to explore the molecular mechanism of atherosclerosis,and to find new therapeutic targets and develop new therapeutic methods have become urgent problems for patients with atherosclerosis.MicroRNAs(miRNAs)are a class of small endogenous non-coding RNAs with 21-24 nucleotides in length,which bind to the 3’ untranslated region(3’-UTR)of target m RNAs and are widely involved in the regulation of many biological processes such as cell differentiation,proliferation,homeostasis,angiogenesis and tumorigenesis.In addition,miRNAs have been shown to play an important role in the development of atherosclerosis.For example,Schober et al.showed that miR-126-5p prevents the formation of atherosclerotic lesions by regulating EC conversion.Meanwhile,some scholars have also found that miR-15 a is an anti-inflammatory factor in the formation of atherosclerotic foam cells.Yebenes et al.showed that inhibition of miR-217 in blood vessels partially protects vascular function and alleviates atherosclerosis.In addition,Zhang et al.found that targeting miR-155 in combination with existing conventional therapies may be an effective approach to treat atherosclerosis.Zhang et al.also found that antisense oligonucleotide treatment of miR-144 may enhance reverse cholesterol transport and oxidative sterol metabolism in patients with cardiovascular disease.In addition,Wang et al.found that miR-296 plays an important role in the regulation of atherosclerosis.Earlier studies have shown that miR-185-5p is expressed as a tumor suppressor in many human cancers.However,the molecular mechanism of miR-185-5p in atherosclerotic disease remains unclear.Sterol Response Element-binding protein 2(SREBP2),a member of SREBP family,is an important regulatory factor in lipid metabolism.It is widely distributed in various tissues in the body and mainly participates in cholesterol metabolism.Cholesterol biosynthesis and uptake are negatively regulated by SREBP2 at the transcriptional level.In recent years,more and more studies have proved that miRNAs are involved in regulating genes related to lipid metabolism.Firstly,bioinformatics analysis showed that SREBP2 is involved in the pathogenesis of atherosclerosis.Biological predictive analysis showed that miR-185-5p could bind to SREBP2,and there was a targeting relationship between miR-185-5p and SREBP2.GO annotation and KEGG pathway analysis of GSE132651 data set showed that DEGs was enriched during lipid metabolism.This conclusion hypothesized that the effect of miR-185-5p and its target gene SREBP2 on atherosclerosis may be realized through lipid metabolism signaling pathway.Next,we investigated the effects of miR-185-5p on atherosclerosis and lipid accumulation in HFD-fed APOE-/-mice and ox-LDL-stimulated macrophages.Our results suggest that miR-185-5p reduces the formation of descending artery plaque and the lesion area of aortic root tissue.At the same time,at the molecular level,miR-185-5p reduced the expression of genes related to lipid metabolism by inhibiting the activation of SREBP2,thus confirming the key role of mi-185-5p as a lipid regulator in the clinical treatment of atherosclerosis and related lipid metabolism diseases.This study will explore the hypothesized role of miR-185-5p in regulating lipid metabolism in atherosclerosis and its possible mechanism of action,providing new strategies and methods for the treatment of atherosclerotic diseases.Part One Study on SREBP2 expression in peripheral blood of patients with coronary atherosclerotic heart diseaseObjective:The serum level of SREBP2 in peripheral blood of normal control group and clinical patients with coronary heart disease was detected by ELISA to describe the expression and changes of SREBP2 in peripheral blood of patients with coronary heart disease.Methods:1.Select subjects.A total of 30 patients diagnosed with coronary atherosclerotic heart disease(CAD)who were admitted to the Department of Cardiology of the First Hospital of Hebei Medical University from October2020 to January 2021 were selected.All patients were examined by percutaneous selective coronary angiography(CAG).The control group involving of 30 patients,all of whom were from the Physical Examination Center of the First Hospital of Hebei Medical University.Peripheral blood samples were collected from patients before they were admitted to hospital during the acute episode and before any treatment was initiated.2.ELISA was used to detect the serum level of SREBP2 in peripheral blood of patients in normal control group and CAD group.3.All the experimental data were analyzed by SPSS22.0 software,and the measurement data were expressedx±s.The t test was used for both groups of data,which was in line with P<0.05 was considered statistically significant.Results: The results showed that the expression level of SREBP2 in the serum of patients with coronary heart disease was significantly higher than that of the control group.Summary: By analyzing the peripheral blood serum of patients with coronary atherosclerotic heart disease,it was found that the expression level of SREBP2 increased compared with the normal control group.Next,we further study the mechanism of lipid regulation in atherosclerotic diseases,so as to find new ideas for the treatment of atherosclerotic diseases.Part Two miR-185-5p alleviates atherosclerosis and down-regulates the expression of lipid metabolism genes and inflammatory factors by inhibiting SREBP2 activation in APOE-/-miceObjective: Bioinformatics analysis indicated that SREBP2 was involved in the pathogenesis of atherosclerosis.We investigated the effect of miR-185-5p on atherosclerosis and lipid accumulation in HFD-fed APOE-/-mice,and proposed the hypothesized role of miR-185-5p in regulating lipid metabolism in atherosclerosis in mice and its possible mechanism,providing a new strategy for the treatment of atherosclerosis.Methods:1.Gene chip data were retrieved and downloaded to screen differentially expressed genes.The gene expression profiles of GSE34812,GSE132651 and GSE28829 were downloaded from GEO database.Differentially expressed genes(DEGs)to | logFC | & gt;2 and P < 0.05 for difference analysis.Then,the overlapping DEGs obtained from microarray analysis were described as Venn diagrams using an online Venn diagram tool and key genes were identified.Hypothesized miRNAs for DEG were predicted using targescan,miRDB,miRWalk and DIANA tools.Degs between datasets GSE34812,GSE132651 and GSE28829 are identified by intersection functions in R packets.2.Select experimental animals and groups.Male APOE-/-C57BL/6 mice aged 6-8 weeks were selected for this experiment.Mice were fed a high-fat diet(18% fat and 1.25% cholesterol)for 3 months to induce atherosclerosis.Subsequently,the mice were randomly divided into two groups,miR-185-5P intervention group and miR-185-5P negative control group.Intervention results were tested after 12 weeks.Finally,the mice were euthanized by intraperitoneal injection of pentobarbital sodium(50 mg/kg)and sampled.3.Bioinformatics analysis of SREBP2 involved in the pathogenesis of atherosclerosis.4.Oil red O staining was used to detect the effects of miR-185-5p on serum and liver cholesterol levels in HFD-fed APOE-/-mice,and whether miR-185-5p played a role in the progression of atherosclerosis in mice.5.Immunohistochemical staining,Western blotting and Q-PCR were used to detect various protein levels in HFD-fed apoE-/-mice aorta sections to prove that miR-185-5p down-regulated the expression of lipid metabolism genes and inflammatory factors.6.The levels of liver homogenate and serum cholesterol were detected by ELISA.7.All the experimental data were analyzed by SPSS22.0 software,and the measurement data were expressedx±s.The t test was used for both groups of data,which was in line with P<0.05 was considered statistically significant.Results: Firstly,SREBP2 is involved in the pathogenesis of atherosclerosis.secondly,miR-185-5p down-regulated the expression of lipid metabolism genes and inflammatory factors in HFD-fed APOE-/-mice;Thirdly,miR-185-5p was confirmed to down-regulate the expression of lipid metabolism genes and inflammatory factors in HFD-fed APOE-/-mice.Summary: In this part of the experiment,bioinformatics analysis indicated that SREBP2 is involved in the pathogenesis of atherosclerosis.Subsequently,we investigated the effect of Mir-185-5p on atherosclerosis and lipid accumulation in HFD-fed APOE-/-mice,and proposed the hypothesized role of Mir-185-5p in regulating lipid metabolism in atherosclerosis in mice and its possible mechanism.Part Three miR-185-5p reduces protein levels and lipid accumulation of genes related to lipid metabolism by inhibiting SREBP2 act-ivation in macrophages in vitroObjective: From the perspective of in vitro,whether miR-185-5p can reduce the protein level and lipid accumulation of genes related to lipid metabolism by inhibiting the activation of SREBP2 in macrophages in vitro was analyzedMethods:1.Cell culture.RAW264.7 Macrophage recovery,cell passage and cell cryopreservation.2.Cell transfection.The lentiviral expression vector and control vector of miR-185-5P overexpressed and silenced were transfected into RAW264.7macrophages using Lipo 2000 TM transfection reagent.3.Western blot,oil red O staining and immunofluorescence staining were used to confirm the protein levels of genes related to lipid metabolism in ox-LDL-stimulated macrophages by miR-185-5p intervening SREBP2.4.The number of lipid droplets in macrophages was detected by oil red O staining.5.The content of microspheres in ox-LDL-stimulated macrophages was determined by microsphere phagocytosis assay,and the effect of Mir-185-5p on the phagocytosis ability of macrophages was studied.6.All the experimental data were analyzed by SPSS22.0 software,and the measurement data were expressed asx±s.The t test was used for both groups of data.Results:1.Western Blot and immunofluorescence staining confirmed that miR-185-5p inhibited the lipid metabolism pathway by inhibiting the activation of SREBP2.2.Oil red O staining and microsphere phagocytosis confirmed that the phagocytosis of ox-LDL by macrophages was impaired by miR-185-5p,which helped to inhibit lipid accumulation in ox-LDL-stimulated macrophages.Summary: Mir-185-5p inhibits lipid metabolism pathways by inhibiting SREBP2 activation,and Mir-185-5p inhibits lipid accumulation in macrophages.The inhibition of mir-185-5p in the progression of atherosclerosis is based on the down-regulation of SREBP2 to induce the inhibition of lipid metabolism signaling pathway.Conclusions:1.In this study,through the analysis of peripheral blood serum of patients with coronary atherosclerotic heart disease,it was found that the expression level of SREBP2 showed an increased trend compared with the normal control group,which also verified the close relationship between SREBP2 and atherosclerotic disease.2.It is concluded that SREBP2 is involved in the pathogenesis of atherosclerosis in vivo.Furthermore,miR-185-5p down-regulated the expression of lipid metabolism genes and inflammatory factors in HFD-fed APOE-/-mice;Third,miR-185-5p was confirmed to down-regulate the expression of lipid metabolism genes and inflammatory factors in HFD-fed APOE-/-mice.3.This study demonstrated in vitro that miR-185-5p inhibited lipid metabolism pathway by inhibiting SREBP2 activation,and miR-185-5p inhibited lipid accumulation in macrophages.The inhibition of mir-185-5p in the progression of atherosclerosis is based on the down-regulation of SREBP2 to induce the inhibition of lipid metabolism signaling pathway.These results suggest that miR-185-5p has important value as a novel therapeutic strategy for atherosclerosis. |
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