Gene-promoters And LncRNAs Analysis In Patients With Eosinophilic Asthma And Correlation With Clinical Features And Biomarkers

Objective:This study aimed to analysis the differential SNPs in the promoter and exon regions in patients with eosinophilic asthma,to explore the correlation between SNPs and clinical features,biomarkers,and to explore the function of SNPs.Furthermore,this study also aimed to detect lncRNA in patients with eosinophilic asthma,to explore the correlation between novel lncRNA and clinical features,biomarkers,and to explore the regulation mechanism of novel lncRNA.The results of this study will provide a theoretical basis for mechanism,personalized diagnosis and treatment of patients with eosinophilic asthma.Method:Part Ⅰ:The study of SNP in the promoter and exon regions in patients with eosinophilic asthma and its correlation with clinical features,biomarkers.1.A total of 57 patients with eosinophilic asthma were enrolled and their clinical data and biomarkers（age of onset,smoking history,family and individual allergy history,total IgE,peripheral blood Eos%and induced sputum Eos%,lung function,FeNO,ACT,ACQ score,anxiety and depression score,IL-5,IL-6,IL-8,IL-13,TNF-α,etc）were collected.High-throughput sequencing technology was used to detect SNPs in the promoter and exon regions（109 genes）of patients with eosinophilic asthma.Differential SNPs in patients with eosinophilic asthma were identified using a case-control study;2.Sanger sequencing technology was utilized to verify the rs 1269486 A>G mutation in the promoter region of GATA3 gene which is the key transcription factor in Th2 inflammatory pathway;3.The luciferase reporter gene was utilized to investigate the function of rs 1269486 A>G mutation on GATA3 gene expression;4.The association of rs 1269486 A>G mutation with clinical features and biomarkers was detected in the study.Part Ⅱ:The study of IncRNA in patients with eosinophilic asthma and its correlation with clinical features,biomarkers.1.Twelve patients with eosinophilic asthma,6 patients with neutrophilic asthma and 6 healthy controls were enrolled.The clinical characteristics and biomarkers of these subjects were collected.The lncRNA in patients with eosinophilic asthma was screened using high-throughput sequencing and the biological function of this IncRNA was also analysed;2.PCR technology was used to verify lncRNA in patients with eosinophilic asthma,and the correlation of this IncRNA with clinical features and biomarkers was analyzed;3.JURKAT cells and human CD4+T lymphocytes were used to investigate the lncRNA involved with Th2 imflammatory pathway;Part Ⅲ:The regulation of novel lncRNA（LNC₀00127）in patients with eosinophilic asthma in Asthma-Th2 imflammatory pathway1 The FISH experiment was used to identify the location of new;ncRNA（LNC₀00127）;2.The interfering RNA was used to knock down the novel lncRNA（LNC 000127）in JURKAT cells and to construct a lentiviral stable cell line.3.PCR array and Western-Blot were used to explore the role of novel lncRNA（LNC₀00127）in Th2 imflammatory pathway.Result:Part Ⅰ:The study of SNP in the promoter and exon regions in patients with eosinophilic asthma and its correlation with clinical features,biomarkers.1.A total of 47 SNPs were screened out from 57 patients with eosinophilic asthma（P<0.05）.There were 16 SNPs in the promoter region and 31 SNPs in the exon region.Two new SNPs which were located in IRF2,NFKB1 gene were newly discovered in the exon region.2.GATA3 rsl269486 A>G mutation（P<0.05）was detected in 12 of 57 patients with eosinophiclic asthma for the first time;3.The luciferase activity of allele G increased significantly（P<0.05）,so that GATA3 rs1269486 A>G mutation may increase its transcriptional activity.4.Compared with the patients with allele A,allele G had a high incidence of family asthma and severe eosinophiclic asthma,a low incidence of FEV₁/FVC%、PEF%and a high proportion of sustained airflow limitation and severe anxiety;IL-5 in patients with allele G increased significantly（P<0.05）.Blood IL-5 was negatively correlated with FEV₁/FVC%and PEF%in patients with allele GPart Ⅱ:The study of lncRNA in patients with eosinophilic asthma and its correlation with clinical features,biomarkers.1.Compared with neutrophilic asthma and control group,there were 190 differentially expressed IncRNAs in eosinophilic asthma,of which 81 were up-regulated and 109 were down-regulated;3 of the 190 lncRNAs（RP11-408H1.3,LNC 000127,OIP5-AS1）were found to be co-expressed with eosinophilic asthma-related genes.LNC 000127 is a novel lncRNA;2.Verified by PCR,novel LNC₀00127 was elevated in patients with eosinophilic asthma.The group with high expression level of LNC 000127 had a high incidence of family asthma,a high proportion of total IgE,FeNO,Eos%,IL-5 and a low incidence of FEV₁%,FEV₁/FVC%（P<0.05）.Blood IL-5 was negatively correlated with FEV₁/FVC%and FEV₁%in paient with high expression level of LNC 000127.3.Novel LNC 000127 expression was elevated in PMA/CD28-stimulated JURKAT cells and human CD4+T cells（P<0.05）.Part Ⅲ:The regulation of novel IncRNA（LNC₀00127）in patients with eosinophilic asthma in Asthma-Th2 imflammatory pathway1.Novel LNC 000127 is localized in the nucleus of JURKAT cells2.Interfering RNA lentiviral vector was successfully constructed,and Y6472 knocked down novel LNC 000127 in JURKAT with the highest efficiency（P<0.05）;3.Knockdown of LNC 000127 decreased the expression of key transcription factors GATA3,STAT5A and cell surface receptors CD40L,CCR8 and CRLF2 during the differentiation of JURKAT cells into Th2 cells（P<0.05）.Conclusion:1.High-throughput sequencing technology was used to detect SNPs in the promoter and exon regions（109 genes）of patients with eosinophilic asthma.GATA3 rs1269486 A>G mutation was detected in 12 patients with eosinophiclic asthma for the first time.GATA3 rs 1269486 A>G mutation can increase the transcriptional activity of GATA3.Compared with the patients with allele A,allele G had a high incidence of family asthma and severe eosinophiclic asthma,a low incidence of lung function and a high proportion of severe anxiety;IL-5 in patients with allele G increased significantly;Blood IL-5 was negatively correlated with FEV₁/FVC%and PEF%in patients with allele G,suggesting that GATA3 rs 1269486 A>G mutation may be related to the pathogenesis of eosinophilic asthma and may be a biomarker of eosinophilic asthma;2.We firstly found that the novel lncRNA（LNC₀00127）significantly increased in patients with eosinophilic asthma.The group with high expression level of LNC₀00127 had a high incidence of family asthma a high proportion of total IgE,FeNO,Eos%,IL-5 and a low incidence of FEV₁%,FEV₁/FVC%.Blood IL₅ was negatively correlated with FEV₁/FVC%and FEV₁%in paient with high expression level of LNC 000127.LNC 000127 may become a biomarker in the diagnosis of eosinophilic asthma in the future;3.The novel LNC 000127 in patients with eosinophilic asthma is involved in the regulation of the transcription factors（GATA3,STAT5A）and surface receptors（CCR8,CD40L and CRLF2）.The novel LNC₀00127 may influence the differentiation of Th0 lymphocytes into Th2 cells through the TCR-STAT-GATA3 pathway,leading to the onset of asthma.The novel LNC₀00127 may become a therapeutic target for eosinophilic asthma that GATA3 mediated in the future.