The Correlation With Single Nucleotide Polymorphism In Promoter,Transcriptional Regulatory Mechanisms And Biomarkers In Patients With Severe Eosinophilic Asthma

Objective:The precise diagnosis,treatment and molecular mechanism of severe eosinophilic asthma?EA?are prevalently in recent years.The comprehensive analysis of genomics,transcriptomics,and proteomics is a new approach to study the molecular mechanisms of severe and refractory diseases.Our study explores the molecular mechanism of severe eosinophilic asthma?EA?in patients and its correlation with clinical features and biomarkers through Multi-omics analysis.It can provide scientific basis with the pathogenesis,precise diagnosis and treatment of severe EA.Methods:1.Study on the Single Nucleotide Polymorphism?SNP?in promoter of severe EA patients and its correlation with clinical characteristics and biomarkers?1?Subjects were selected according to the inclusion and exclusion criteria.From June 1,2016 to June 1,2017,30 cases of severe EA patients?chronic persistent period?and healthy persons in Chinese PLA Genenal Hospital and Chinese Armed Police General Hospital were selected as experimental subjects.?2?The subjects'basic situation,ACT score,ACQ score,were determined by blood routine,IgE,pulmonary function and FeNO,induced sputum,blood and induced sputum inflammatory factors?IL-5,IL-8,IL-13,IL-17 and Periostin?;the control group and patients were collected venous blood from the target area DNA target capture sequencing.?3?The analysis of bioinformatics and DNA mutation sites and the correlation analysis with biomarker levels.2.Differential expression of transcriptome?mRNA and lncRNA?in severe EA patients?1?The patients and control group were selected:12 cases of severe EA were selected randomly,and 6 cases were healthy.?2?High throughput sequencing of mRNA and IncRNA two generations in the transcriptional group.?3?The differential analysis of mRNA and IncRNA expression profiles and the analysis with the pathway of promoter mutation.3.The Pathogenesis and Mechanism of Novel LncRNA?LNC₀00062?in Severe EA and its regulation and research mechanism?1?The application of small interfering RNA?siRNA?silencing human airway epithelial cells?16HBE?model in the LNC 000062 gene,designed and synthesized3 knockdown sequences?Y6036,Y6037,Y6038?and 1?Y007?antisense sequence,were constructed lentiviral transfer and stability through RT-qPCR identification and verification.?2?The cell proliferation activity and cell apoptosis were detected.?3?RT-qPCR and Western-Blot were used to determine the effect of new LNC₀00062 on gene regulation.Result:1.Study on the Single Nucleotide Polymorphism?SNP?in promoter of severe EA patients and its correlation with clinical characteristics and biomarkers?1?DNA biological mutation analysis:selected mutation frequency is greater than or equal to 5%and P<0.05 in promoter and exon 32 of SNP,which is located in the promoter region of the SNP 10 of the 8 genes located in the;?2?Related pathways in patients with severe EA mutation screening:10 promoter SNP?8 genes?pathway associated mainly KEGG pathway and asthma cross matching,PI3K,MMP9,TSLP,MS4A2 and CTLA4 5 genes of 6 SNP associated with asthma,selected for subsequent analysis;?3?Compared with the normal control group,the levels of IL-5,IL-13 and periosteal protein in asthmatic serum and sputum specimens were statistically higher?P<0.05?,and the levels of IL-8 and IL-17 had no statistical difference.?4?Correlation analysis between promoter region mutation site and biomarker:correlation analysis of 6 SNP loci of 5 genes and blood and induced sputum biomarkers level in severe EA patients.Among them,the level of rs751930632 in the promoter region of PI3K gene and the level of phlegm periosteum and the level of blood IL-8 were statistically significant?P<0.05?.2.Differential expression of transcriptome?mRNA and IncRNA?in severe EA patients?1?LncRNA screening:according to the IncRNA screening process,we screened and screened 332150 transcripts of severe EA patients,and finally got 3972 transcripts of IncRNA numbers.?2?Differential expression of IncRNA or mRNA:in severe EA group,the expression of IncRNA was up to 107,and the expression was down regulated by 130,and the difference of mRNA was 3217,of which 1527 were up-regulated and 1690 were down regulated.?3?The sequencing results verify:numerical/log2FC/top 10 differential expression of IncRNA and GAPDH as the reference gene for RT-qPCR verification,inspection samples and expand the sample test results were consistent with the sequencing results;?4?DNA,mRNA and lncRNA sequencing multi omics analysis:filtering and channel correlation for DNA sequencing results,5 genes?PI3K,MMP9,retention of TSLP,MS4A2 and CTLA4?and screened 3217 differentially expressed mRNA cross filtration,selected PI3K and MMP9 two gene expression,weight the up regulation of PI3K and MMP9 gene in EA patients;further for PI3K and MMP9 two gene co expression or co location to compare IncRNA cross screening,1 new lncRNA LNC₀00062 correlated with PI3K and MMP9 both,the expression decreased in patients with severe EA,log2FC value is-2.2348.3.The Pathogenesis and Mechanism of Novel LncRNA?LNC 000062?in Severe EA and its regulation and research mechanism?1?The application of siRNA on human airway epithelial cells?16HBE?model of lncRNA LNC₀00062 gene,identified by RT-qPCR knockdown results,three knockdown sequences were the decreased expression of LNC₀00062,which Y6038 knockdown efficiency is the highest;the antisense sequence has no effect on the expression of LNC 000062 Y007,the two selected for subsequent cell experiment;?2?The cell proliferation activity of 16HBE-KD knockdown group was higher than that of 16HBE-NC negative control group and 16-HBE blank control group?P<0.05?,while the rate of apoptosis decreased?P<0.05?,indicating that LNC 000062 could inhibit the proliferation of 16-HBE cells and promote 16-HBE cell apoptosis.?3?Compared with 16HBE-KD negative group and 16HBE-NC negative control group and 16-HBE blank control group,it was found that the new lncRNA LNC 000062 could up regulate the expression of PI3K,MMP9,TSLP,IL4 and GATA3,and down regulate the expression of IL4 and GATA3?P<0.05?.?4?Western-Blot detected the protein level of PI3K and MMP9 gene:the level of PI3K and MMP9 protein in 16HBE-KD group was higher than that in 16HBE-NC and 16-HBE?P<0.05?.Conclusion:?1?Six SNP,rs751930632,rs190171340,rs3806933,rs1441585,rs574700 and rs16840252,were found in patients with severe EA,and PI3K,MMP9,TSLP,MS4A2 and CTLA4 5 genes;the promoter rs751930632 and PI3K gene in patients with induced sputum and blood IL-8 level Periostin levels of inflammatory factors,suggesting that PI3K promoter rs751930632 mutations may be associated with severe airway inflammation in EA.?2?Severe EA were first found in the 1 new lncRNA LNC 000062 associated with PI3K and MMP9 two genes,the gene expression was down regulated in peripheral blood of patients with severe EA,involved in the regulation of the expression and regulation of protein PI3K and MMP9 gene of mRNA,suggesting that lncRNA LNC₀00062 may not only control model by PI3K and TSLP GATA3,IL4,Th2 in patients with severe EA cell activation and cytokine secretion,and may be involved in airway remodeling mediated by MMP9.?3?The expression of new IncRNA LNC₀00062 gene in the peripheral blood of severe EA patients is significantly down regulated.It may be used as a new biomarker for molecular targeted diagnosis of severe EA patients,providing a basis for accurate diagnosis of asthma.?4?The expression of new IncRNA LNC₀00062 is obviously reduced in severe EA patients.It is possible that the enhancer may be used as a target therapy for severe EA patients in the future.