| Background:Cardiovascular and cerebrovascular diseases has become the primary reason threatening the health of residents.Vascular stenosis and occlusion caused by atherosclerosis as well as vascular embolism caused by plaque shedding constituted the most important components of cardiovascular and cerebrovascular diseases.Lipid metabolism disorder is an important risk factor for atherosclerotic cardiovascular disease.Elevated circulating LDL-C directly contributes to the onset and development of atherosclerosis.Foam cells are the pathological basis of atherosclerosis,which formed from the macrophages by phagocytizing ox-LDL.Circulating LDL-C level has become the most important indicator for clinical evaluation of lipid-lowering drugs.Many miRNAs are involved in the regulation of lipid metabolism.Circulating miRNAs have been proved to be useful in the diagnosis of diseases as well as the evaluation of therapeutic effects and prognosis.Screening differentially expressed miRNAs from clinical samples(serum or pathological tissues)has become an important direction to study the relationship between miRNAs and diseases.Object:To explore the miRNAs associated with lipid metabolism disorders from clinical samples,and to provide new ideas for the early diagnosis and treatment of ASCVD.Method:1.Subjects,including healthy volunteers,outpatients,and in-patients,were recruited from October 2017 to December 2018 at Affiliated Hospital of Yangzhou University.According to the blood lipid level(mainly the serum LDL-C level)of patients,the subjects were divided into hyperlipidemia group and healthy control group.Five serum samples were taken from the hyperlipidemia group and the healthy control group respectively according to the completely random principle for miRNA serum expression profiling.Target Scan,PITA and microRNAorg were used to predict the target genes of these differentially expressed miRNAs,GO and KEGG databases were used to analyze the functions and pathways of target genes.2.Expanded the number of experimental samples.The basic clinical information of the two groups was analyzed,and the difference between the two groups was compared by using two independent sample T test.The expressions of miR-933,miR-191-3p and miR-425-5p were detected and verified by qRT-PCR(n=20).The expression of miR-933 was further detected and compared between hyperlipidemia group(n=122)and healthy control group(n=168).Pearson correlation analysis was used to evaluate the correlation between serum levels of miR-933 and LDL-C.Logistic regression analysis was used to evaluate the accuracy of miR-933 as an influencing factor of circulating LDL-C.ROC curve was used to evaluate the predictive value of miR-933 for hyper-LDL-C.3.Construct ADCY9 S'UTR-PmirGLO plasmid,synthesize miR-933 mimics and NC by chemical method.Query the binding site between miR-933 and ADCY9 through TargetScan database to mutate the binding site.Using Dual Luciferase Reporter assly Kit in 293T cells to detect the transcriptional regulation relationship between miR-933 and ADCY9.Results:1.miRNA microarray analysis showed that there were 22 differentially expressed miRNAs,of which 12 miRNAs showed up-regulated,including:hsa-miR-320b,hsa-miR-6812-5p,hsa-miRNA-4442 and 10 miRNAs showed down-regulated,including:hsa-miR-425-3p,hsa-miR-933,hsa-miR-191-3p.TargetScan,PITA,and microRNAorg databases were used to predict target genes for these differentially expressed miRNAs,the results were cross-matched to obtain 616 predicted targets.GO analysis showed that these target genes were mainly enriched in protein binding,histone deacetylase binding,phosphoprotein binding,transcriptional activator activity and other molecular functions.Pathway analysis was conducted using the KEGG database,the result revealed the top 20 signal pathways with the highest target gene enrichment,including Hippo signaling pathway,Focal adhesion,PI3K-Akt signaling pathway and other pathways.2.qRT-PCR was used to verify the expression of three differentially expressed miRNAs selected from the chip results.The result showed the expression of these three miRNAs was mostly consistent with the results reported by the chip.Large sample qRT-PCR further verified the expression trend of miR-933.Pearson correlation analysis showed a significant negative correlation between serum miR-933 and LDL-C(R=-0.691);multivariate logistic regression analysis showed decreased level of serum miR-933(OR 0.339,95%Cl 0.138-0.829,P=0.018)was an independent risk factor for hyper-LDL-C.The area under the ROC curve(AUC)is 0.7388(95%Cl,0.6822-0.7995),suggesting that circulating miR-933 possesses certain predictive value for LDL-C metabolic disorder3.The relationship between miR-933 and ADCY9 was confirmed by the dual luciferase reporter gene assay in 293T cells.The results showed that ADCY9-3'UTR can be negatively regulated by miR-933,while in NC group,miR-933 could not reduce the luciferase activity of ADCY9 3'UTR,and after mutating the binding site,miR-933 could not reduce the luciferase activity of ADCY93'UTR-mut,suggesting that ADCY9 was a direct target gene of miR-933.Conclusion:Several miRNAs possibly involved in the regulation of circulating LDL-C were found,of which miR-933 was closely related to LDL-C regulation.MiR-933 probably participated in the regulation of lipid metabolism through ADCY9. |
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