Analysis Of The Regulatory Mechanism Of COPD Susceptibility Gene GSTCD And The Exploration Of Now COPD Susceptibility Genes

Chronic Obstructive Pulmonary Disease（COPD）is a comnon respiratory system disease characterized by decreased lung function,with its high morbidity and mortality,this disease is the third leading cause of death in the world today,and has caused a huge economic burden to the society.Genetic factors may increase the risk of COPD.A large number of genome-wide association studies（GWAS）have been conducted in recent years,and it reveal that rs 10516526 which located in the intron region of GSTCD（GIutathine s-transferase,c-terminal domain）is closely related to FEV₁ and COPD.However,due to the linkage imbalance and the complexity of gene regulation,it has not been confirmed whether rs 10516526（or LD SNPs）is related to GSTCD gene or other genes in this region.In this study,the expression of candidate COPD susceptibility gene G.STCD was measured by real-time PCR（RT-PCR）.In order to explore the relationship between rs10516526 and COPD,the information from the 1000 genome database were used to analyze the linkage imbalance of all SNP in the 500kb region near rs 105 16526（r²≥0.8）,all rs10516526 linkage sites were analyzed as candidate functional sites.And the functional verification analysis of candidate functional SNPs was carried out by combining the ENCODE databases with chromosomal conformation capture（3C）,dual luciferase reporter assay,chromatin immunoprecipitation assay（ChIP）and electrophoretic mobility shift assay（EMSA）,so as to explore the possible mechanism of gene expression regulated by rs10516526.In RT-PCR detection,we found that the GSTCD gene may be a COPD susceptibility gene（P=0.037）.A total of 256 highly linked SNP sites（r²≥0.8）were found within the 500kb range of rs10516526,which may be candidate functional regulatory sites of GSTCD gene.We used the information of ENCODE database and 3C technology to locate the candidate functional regulatory sites of GSTCD gene to rs80245547 which located in the promoter region of GSCTD gene and rs72673891 which located in the downstream of GSTCD promoter～67kb.Meanwhile,dual luciferase reporter assay,ChIP and EMSA experiments confirmed that rs80245547 T and rs72673891 G may remotely up-regulate the expression of GSTCD gene by enhancing the binding of transcription factors C-Jun and CREB1,which may enhance the susceptibility of COPD.In addition,COPD is a complex disease regulated by multiple genes.In recent years,the research on gene expression of transcriptome makes it possible for us to deeply understand the pathogenesis of COPD.However,the research progress on the expression of COPD transcriptome gene is slow,and the differentially expressed genes in each study are not overlapped at a large extent.it is not known which genes in these studies have co-existed differences in COPD transcriptome analysis.By integrating published COPD transcriptome data,we expect to find information on the differential expression of COPD susceptibility genes in the COPD whole transcriptome gene expression studies.In this study,we downloaded published COPD transcriptome data,by screening the sample size and sampling source,we found three transcriptome data（GSE8545,GSE57148 and GSE76925）for subsequent integrated analysis.During the analysis,we found four upregulated genes with differential expression in GSE8545,GSE57148 and GSE76925,which were CYP1B1（Cytochrome P450,family 1,subfamily B,polypeptide 1）,NAV1（Neuron navigator 1）,SPP1（Secreted phosphoprotein 1）,NEK6（NIMA related kinase 6）.At the same time,we also used RT-PCR to detect the expression of these four genes in the COPD disease group and the healthy control group,and the results indicated that the expression levels of these four genes in the disease group were significantly higher than those in the control group（P values were less than 0.05）,which may be the susceptible genes to COPD.Based on the above results,we found a possible regulatory mechanism of COPD susceptibility gene GSTCD,namely,rs80245547 T and rs72673891 G may up-regulate the expression of GSTCD gene by enhancing the binding of C-Jun and CREB1 transcription factors,thus affecting COPD.By integrating published COPD transcriptome data,we identified four new COPD susceptibility genes（CYP1B1,NAV1,SPP1,NEK6）.