Screening And Validation Study Of LncRNA For The Difference Of Large Artery Atherosclerosis Cerebral Infarction

Objective: 1)This study screened differentially expressed genes and their associated LncRNAs and mRNAs in atherosclerotic cerebral infarction through a case-control LncRNA expression profiling of atherosclerotic cerebral infarction,using bioinformatics methods to predict and analyze differences.The co-expression of LncRNA,the biological function clustering of LncRNA and the involved signaling pathways provide insights into the biological value of LncRNA in atherosclerotic cerebral infarction;2)In the case control group that further expanded the sample,the differential expression of LncRNA(MALAT1)screened in this study was repeated,and the expression level of lncRNA(MEG3,SENCR)related to vascular biological processes selected based on the literature was compared.To explore the expression pattern of LncRNA in atherosclerotic cerebral infarction,and to analyze the relationship between LncRNA expression and risk factors related to cerebral infarction,in order to screen for LncRNA biomarkers for diagnosis of atherosclerotic cerebral infarction;3)In the atherosclerotic cell model constructed by ox-LDL-stimulated human umbilical vein endothelial cells,the expression levels of representative differentially expressed LncRNA,mRNA and predicted co-expressed target miRNAs were screened in this study.The effect of ox-LDL on the expression of LncRNA,mRNA and miRNA in human umbilical vein endothelial cells.Methods: 1)patients with atherosclerotic cerebral infarction and 3 patients with small occlusion cerebral infarction and 3 healthy controls were enrolled in the study.RNA was extracted from the blood samples of the subjects and processed by transcript sequencing technology.Expression profiling of LncRNA and mRNA at the genome level.GO(gene ontology)functional enrichment analysis and KEGG signaling pathway enrichment analysis were used to explore the potential biofunctional association between differentially expressed LncRNA and differentially expressed mRNA.A co-expression network was constructed by calculating the correlation between LncRNA and mRNA in the same sample to find mRNA closely related to LncRNA.Using the Target Scan database and the mi Randa database,predicted target miRNAs were obtained,predicting the differentially expressed1 ncRNAs,mRNA,and biological functions that target miRNAs may participate in;2)Based on differential multiples,P values,predicted target genes,and screening criteria such as LAAS or vascular endothelial susceptibility loci,combined with literature reading,selected 3 LncRNAs,using real-time fluorescent quantitative PCR,in 16 cases of large atherosclerotic brain Repeated validation was performed in patients with infarction and 17 patients with small occlusion cerebral infarction and 17 healthy controls.Using ROC curve to investigate the efficacy of LncRNA in the diagnosis of atherosclerotic cerebral infarction;3)The human umbilical vein endothelial cells(HUVECs)induced by ox-LDL were used to construct an atherosclerotic cell model,and the differential LncRNA was repeatedly verified in atherosclerotic cell model and normal endothelial cells by real-time fluorescent quantitative PCR and western blot.And mRNA expression,at the same time,observed ox-LDL induced HUCECs vascular endothelial factor,differential expression of LncRNA,mRNA,miRNA expression changes.Results: 1)There were 454 genes differentially expressed in the peripheral blood of patients with aortic atherosclerotic cerebral infarction.A total of 454 genes were differentially expressed in the atherosclerotic cerebral infarction group compared with the healthy control group,and the genes were up-regulated.There are 260,194 down-regulated.LncRNA data: 684up-regulated,of which RP3-368 is the most up-regulated,502 is down-regulated,IL1 B is the most down-regulated,mRNA: Up-regulated There are 536,of which RPL27 A is the largest one,and 523 is down.The biggest reduction is IGLV2-14;2)LncRNA and mRNA differentially expressed in the atherosclerotic cerebral infarction group compared with the normal control group were predicted and screened by lnc TAR,and the top two predicted target genes in the atherosclerotic cerebral infarction group were GCNT1.And KDSR.The first two target genes of the small arterial occlusion type cerebral infarction group were predicted to be C9orf16 and SMARCA5;3)LncRNA differentially expressed in the atherosclerotic cerebral infarction group and the control group was analyzed in the KEGG signaling pathway database.The significantly enriched signaling pathways included sphingolipid metabolism,vasopressin-regulated water absorption pathway,and O-glycan.Biosynthesis,PPAR signaling pathways,metabolic pathways,neuroactive ligand-receptor interactions,biosynthesis of glycosphingolipids-globo series,peroxidase,body glycerophospholipid metabolism,steroid biosynthesis,circadian rhythm,nicotinate And nicotinamide metabolism,glycosphingolipid biosynthesis-lactic acid and neolactate series,leukocyte transendothelial migration,histidine metabolism,propionate metabolism,porphyrin and chlorophyll metabolism,valine,leucine and Leucine degradation pathway solution,Notch signaling pathway,drug metabolism-other enzymes,m TOR signaling pathway,cytoplasmic DNA sensing pathway,glycolysis/gluconeogenesis,epithelial cell signaling in Helicobacter pylori infection,RNA degradation,RIG-I-like receptor signaling pathway,regulation of actin cytoskeleton,pyrimidine metabolism,melanin production,Toll-like receptor signaling pathway,calcium signaling pathway,chemokine signaling pathway,cells The sub-path interaction of the cytokine receptors;4)Using Cytoscape software to map lncRNA and mRNA co-expression network map,screening results LNCRNA and mRNA with the largest clustering coefficient are ENST00000534336(MALAT1)and GCNT1 for the differential diagnosis of large atherosclerotic cerebral infarction LncRNA and its co-expression mRNA.Using a combination of multipleRNA databases,the target miRNA 1205,which ranked first in the total score of the predicted results,was used to screen for the predicted target miRNA of lncRNA for differential atherosclerotic cerebral infarction.Therefore,through this study,LncRNA MALAT1,mRNA GCNT1 and miRNA-1205 are proposed as differentially expressed genes of LAAS;5)The expression of MALAT1 in the atherosclerotic cerebral infarction group and the small artery occlusion cerebral infarction and the control group showed that compared with the control group MALAT1 gene expression,the atherosclerotic cerebral infarction group and The expression of MALAT1 gene in the small arterial occlusion group was significantly increased,and the difference was statistically significant(P<0.001),which was consistent with the sequencing screening results;6)The expression of MALAT1 gene was positively correlated with systolic blood pressure in small artery occlusion cerebral infarction group,the correlation coefficient was 0.578,the expression level of SENCR gene was negatively correlated with diastolic blood pressure(rs=-0.455),and positive with low density lipoprotein and MEG3.Relevant(rs are 0.537/0.615 respectively);7)The expression level of MALAT1 gene in the atherosclerotic cerebral infarction group was related to the history of hypertension,with a correlation coefficient of 0.406.SENCR was positively correlated with homocysteine(rs=0.496);MALAT1 expression in large atherosclerotic cerebral infarction group and small artery occlusion cerebral infarction and control group showed that compared with the control group MALAT1 gene expression level The expression of MALAT1 gene in the atherosclerotic cerebral infarction group and the small artery occlusion group was significantly increased,and the difference was statistically significant(P<0.001),which was consistent with the sequencing screening results.8)Using the receiver operating characteristic curve(ROC)curve to evaluate the relative expression of MALAT1,MEG3,and SENCR in peripheral blood for the diagnosis of aortic atherosclerotic cerebral infarction,MEG3 combined with MALAT1 to diagnose aortic atherosclerosis Type cerebral infarction has the highest efficacy,while MALAT1 combined with SENCR has a high specificity but poor sensitivity.9)In this study,the atherosclerosis cell model was induced by ox-LDL.In the atherosclerotic cell model,lncRNA MALAT1 was highly expressed compared with normal vascular endothelial cells,and mRNA GCNT1 was also highly expressed.The expression of miRNA1205 was decreased,which was consistent with the results of the sequencing and the results in the case control sample.10)MALAT1 was highly expressed in atherosclerotic cerebral infarction population,and the expression of ox-LDL-induced atherosclerotic endothelial cell dysfunction model was also highly expressed,which verified the first part of high-throughput sequencing screening of lncRNA,mRNA and miRNA were differentially expressed in the arteriosclerosis cell model.Conclusions: 1)We obtained a genome-wide LncRNA/mRNA expression profile by high-throughput sequencing using a small number of clinical samples of large atherosclerotic cerebral infarction.Through bioinformatics analysis,differentially expressed lncRNAs involve multiple signaling pathways,multiple diseases and multiple biological functions,providing a theoretical basis for subsequent research;2)The expression of MALAT1 in the atherosclerotic cerebral infarction group and the small artery occlusion cerebral infarction and the control group showed that compared with the control group,the expression of MALAT1 gene was compared with the atherosclerotic cerebral infarction group and small artery.The expression of MALAT1 gene in the occlusion group was significantly increased,and the difference was statistically significant(P<0.001),which was consistent with the sequencing screening results.The expression level of MALAT1 gene in the atherosclerotic cerebral infarction group was related to the history of hypertension,with a correlation coefficient of 0.406;3)In this study,in the atherosclerotic endothelial dysfunction model,MALAT1 expression decreased,while mRNA;GCNT1 and predicted target gene miRNA1205 expression increased.MALAT1 was shown to be highly expressed in atherosclerotic cerebral infarction,and decreased expression in atherosclerotic endothelial dysfunction model,suggesting that MALAT1 may be regulated by multiple signaling pathways.