chronic obstructive pulmonary disease ( copd ) is characterized by chronic airflow limitation that is not fully reversible and is highly prevalent worldwide .
although cigarette smoking is a major risk factor for copd , the mechanism by which inhaled smoke contributes to airflow obstruction is not fully understood .
current theories for this include persistent airway inflammation that is modified by oxidative stress , excess proteinases , autoimmunity , and apoptosis of alveolar cells .
gene expression studies of diseased lungs can generate high - throughput results to shed light on the molecular processes underlying copd pathogenesis .
whole - genome expression of copd in humans has been studied using airway epithelium and resected lung tissue in several groups [ 39 ] .
background and cross - hybridization are not issues in rna - seq and the technology can quantify both lowly and highly abundant transcripts .
in addition , this method can provide information about splice variants , allele - specific expression , and fusion transcripts .
rna - seq data of airways was recently published [ 13 , 14 ] ; however , the number of subjects in these studies was relatively small .
we performed gene expression profiling using rna - seq of lung tissues resected from large numbers of subjects with copd or with control subjects to better understand the molecular mechanisms responsible for the pathogenesis of copd .
subjects were patients who required resection for lung cancer and who were registered in the asan biobank from january 2008 to november 2011 . the inclusion criteria were a postbronchodilator fev1/fvc ratio ( ratio of forced expiratory volume in the first second to forced vital capacity ) of less than 0.7 for the copd group and normal spirometry for the control group in accordance with american thoracic society / european respiratory society criteria .
this study was approved by the institutional review board of asan medical center ( 2011 - 0711 ) and written informed consent was obtained from all patients .
total rna was isolated from apparently normal fresh frozen lung tissue that was remote from the lung cancer .
rna integrity was assessed using an agilent bioanalyzer and rna purity was assessed using a nanodrop spectrophotometer .
one g of total rna was used to generate cdna libraries using the truseq rna library kit .
the protocol consisted of poly a - selected rna extraction , rna fragmentation , reverse transcription using random hexamer primers , and 100  bp paired - end sequencing using the illumina hiseq 2000 system .
all data have been deposited in the ncbi gene expression omnibus ( geo ) public repository and can be accessed through the accession number gse57148 . for quality control ,
differential gene expression ( deg ) analysis was performed using tophat and cufflinks software . to estimate expression levels ,
the rna - seq reads were mapped to the human genome using tophat ( version 1.4.1 )   and quantified using cufflinks software 2.0.0 .
the numbers of isoform and gene transcripts were calculated and the relative abundance of transcripts was measured in fragments per kilobase of exon per million fragments mapped ( fpkm ) .
expression levels were extracted as a fpkm value for each gene of each sample using cufflinks software .
statistical significance was determined using student 's t - test . the false discovery rate ( fdr )
the analysis steps used are summarized in figure 1 . to investigate whether degs are related to clinical phenotypes , we performed a linear regression analysis for 5 clinical phenotypes with respect to its gene expression .
we considered each clinical phenotype as the responder for regression and each gene expression as the predictor .
several genes whose expression level was found to be related to copd status by rna - seq were validated using taqman real - time pcr .
functional enrichment analysis was performed using gene set enrichment analysis ( version 2.0.8 ) , which combines information from previously defined gene sets obtained from the molecular signature database ( version 3.1 ) .
biological gene functional annotation analysis was performed using david ( version 6.7 ) with a list of degs .
biolattice ( version 1.1 ) was used to annotate coexpressed gene groups to go biological process terms and visualize their relations . to detect differential alternative splicing between the two groups , subjects from each group
were evaluated using a multivariate bayesian algorithm called  multivariate analysis of transcript splicing  .
differential alternative splicing , including exon skipping , mutually exclusive exons , alternative 5 or 3 splice site usage , and intron retention , was investigated .
a connectivity map   was used to identify potential drugs that might reverse the gene expression pattern associated with the pathogenesis of early copd .
the connectivity map is a collection of genome - wide transcriptional expression data from cultured human cells treated with bioactive small molecules .
the basic assumption of the connectivity map is that transcriptional perturbation can occur or be treated by certain drugs that intrigue similar changes .
the demographic characteristics of 98 subjects with copd and 91 control subjects are shown in table 1 .
all subjects were male and the mean age and the mean pack years of cigarette smoking history were higher in the copd group than in the control group .
as expected , pulmonary function was significantly lower in the copd group than in the control subjects .
in copd group , 28 subjects took inhaled corticosteroid , 40 subjects took tiotropium , and 22 subjects took short - acting beta - agonist .
the total number of reads produced from each sample was 38,742,474  7,332,014 reads ( mean  standard deviation ) .
the difference in the number of reads between copd samples and control samples was not statistically significant .
after read alignment with tophat and read quantification with cufflinks using ucsc hg19 transcriptome reference , a total of 189 samples and 23,146 genes were analyzed . a total of 248 genes had zero fpkm values in all samples .
after filtering for genes with zero counts in whole samples , noncoding genes , and low variance genes , 16,676 genes were analyzed .
out of these genes , 2,312 genes were differentially expressed between the two groups ( fdr corrected q < 0.01 ) ( table 3 ) .
there were many overlaps between t - test and edger ( see supplementary table 1 in the supplementary material available online at  http://dx.doi.org/10.1155/2015/206937 ) .
regression analysis of the degs with clinical phenotypes revealed that genes were more associated with fev1 ( forced expiratory volume in the first second ) and fev1/fvc ratio than with age and smoking history ( figure 2 ) . to validate the rna - seq results , we performed qrt - pcr of the fgg , mcl1 , pde4a , s100a6 , serpine1 , sftpc , and tmsb4x genes using the same rna samples that were used for rna - seq .
the rna - seq and qrt - pcr results were in good agreement ( figure 3 ) .
a heat map shows the genes that were differentially expressed between the two groups ( figure 4 ) .
david revealed that the expression of genes involved in protein catabolism , oxidative phosphorylation , and chromatin modification differed most significantly between the two groups ( table 4 ) .
expression of genes encoding proteasome components including psma2 , psmb1 , psmc5 , and psmd4 was lower in the copd group than in the control group .
gene set enrichment analysis revealed that the most significantly downregulated pathways in the copd group were oxidative phosphorylation and biosynthetic process ( fdr q < 0.25 ) .
we used k - means clustering to construct 30 groups of coexpressed genes for biolattice . after matching with hypergeometric distributions to annotate those gene sets to go terms ,
concept lattice for coexpressed gene clusters with go biological process terms was visualized showing that genes related to transcription and oxidative phosphorylation are enriched in the major clusters ( figure 5 ) . in the copd group ,
the heat map shows hierarchical clustering of two subgroups using euclidean distance ( figure 6 ) .
four hundred and four genes that were differentially expressed between these two subgroups in copd subjects ( q < 0.05 ) are enriched in the mitochondrial and steroid biosynthesis pathway .
group 1 showed tendency for lower fev1 and lower dlco ( carbon monoxide diffusing capacity ) . when comparing each group with the control ,
degs between group 1 and the controls only consisted of 18 genes , and their p values were negligible .
meanwhile , degs between group 2 and the controls consisted of 4072 genes and their p values were even higher than those of degs between the copd group and the controls .
isoforms that were differentially expressed between the copd and control groups were evaluated by cufflinks .
however , among the deis , 310 were not in the deg list , which are enriched in genes encoding proteins that function in cell junctions and focal adhesions .
the multivariate analysis of transcript splicing program ( mats ) revealed that specific alternative splicing events were significantly more common in copd subjects than in control subjects .
five categories of differentially expressed isoforms were identified by mats : skipped exon , alternative 5 splice site , alternative 3 splice site , mutually exclusive exons , and retained introns .
significantly different events between the copd group and the control group are shown in the supplementary table 2 ( fdr q < 0.01 ) . intron retention of hnrnph1 and vim occurred significantly more in the copd group than in the control group .
mutually exclusive exons occurred more frequently in the copd group than in the control group in 78 genes .  
a connectivity map   was used to identify potential drugs that might reverse the gene expression pattern associated with the pathogenesis of early copd .
gene expression changes arising from treatment with several drugs were negatively correlated with the expression patterns that differ in the copd group compared to the control group .
gene expression changes arising from treatment with mg-262 ( p = 0.00004 ) and puromycin ( p <
in this study , we used rna - seq to identify genes whose expression differs between copd and control subjects . in total , 2312 genes were identified with fdr q < 0.01 .
we validated a subset of rna - seq data with qrt - pcr , and the results were in good agreement .
previous studies have investigated the gene expression profiles of copd patients using microarray , serial analysis of gene expression , and rna - seq .
microarray data indicates that mical2 and notch2 are upregulated in the resected lung tissue of copd patients ; the expression of these genes was higher in the copd group than in the control group in the present study .
the expression of genes encoding ribosomal proteins and s100a6 was lower in the copd group than in the control group , and rna - seq previously indicated that the expression of these genes is reduced in the small airway epithelium of smokers .
however , there is little overlap between studies in terms of the genes that are differentially expressed between people with reduced lung function and those with normal lung function .
this may be because different methods were used or because different phenotypes were examined . of interest ,
previous studies of the copd transcriptome reported a high degree of overlap in the biological processes affected . in the current study ,
the expression of mitochondrial genes was previously shown to be reduced in the lung tissues of copd subjects using serial analysis of gene expression .
a recent report showed that expression of the mitochondrial membrane protein phb1 is downregulated in lung tissue of copd patients , suggesting that mitochondrial stability is reduced .
another report revealed that mitochondrial mass is reduced in airway epithelial cells exposed to particulate matter , and this defect is also observed in the daughter cells .
one possible explanation may be related to the fact that an increase in the co2 level can reduce oxidative phosphorylation . however , considering that our copd subjects were in relatively early stages of the disease , they are not expected to have a clinically meaningful increase in co2 levels . in the current study ,
many genes related to the 20s proteasome including psma2 , psmb1 , psmc5 , psmd4 , and psmd13 , as well as ubiquitin ligase complex genes including stub1 , sels , and derl2 , were downregulated in copd subjects .
the ubiquitination - proteasome pathway is dysregulated in copd , but the mechanism by which this occurs is not fully understood .
the expression of 26s proteasome - associated genes is lower in lung tissues of moderate copd subjects than in those of the smoker control subjects with normal lung function .
the expression and activity of the proteasome are reduced in lung tissues of copd subjects due to dysregulation of nrf2 .
interestingly , the proteasome inhibitor mg-262 was on top of a list of drugs that reverse the gene expression pattern of copd . in recent experiments in mice ,
a proteasome inhibitor was suggested to be a therapeutic agent for pulmonary arterial hypertension via inhibition of pulmonary vascular smooth muscle cell proliferation and correction of endothelial dysfunction .
inhibition of proteasome inhibitors can reverse diaphragmatic function in a copd mouse model . in the current study
chromatin modification is an important mechanism in epigenetics . in the current study , the expression of mll , which plays an important role in h3k4 methylation , and chd , which is important for chromatin modification and opening of chromatin to allow transcription ,
the expression of hdac10 was decreased in the lung tissue of copd subjects , while the expression of hdac2 was previously reported to be decreased in copd .
several studies report the mechanism by which the expression of hdac2 is reduced in copd , but further studies are required to explain how the expression of several genes related to chromatin modification is increased in copd . in a recent study investigating genes associated with the severity of emphysema , the major pathways affected were inflammation and tissue repair .
however , the inflammation pathway was not majorly affected in copd subjects in the current study , probably because the subjects were at relatively early stages of the disease .
pathway analysis results of the deis were similar to those of the degs ; however , genes encoding proteins that function in cell junctions and cell migration were found specifically in the deis and not in the degs .
alternative splicing of genes is tissue - specific and has important roles in development , physiological responses , and the pathogenesis of diseases .
interestingly , the gene encoding vimentin , which is a structural protein , had more retained introns in the copd group than in the control group , which may alter the sequence of the protein .
copd subjects were older and had more pack years of smoking than the control group .
however , regression analysis results showed that most of degs had stronger correlation with lung function than age or smoking amount .
the results of this study may not be applicable to nonsmoking or female copd subjects .
microdissection of lung tissue or single cell sequencing would be required to determine whether the differential expression is altered in all lung cells or only in a specific subset of cells . finally , it is difficult to determine whether the dysregulated pathways identified in this study are a cause or a consequence of the pathogenesis of copd .
experiments in which the increase / decrease of the degs is reversed and shown to slow disease progression are needed to confirm that these pathways are causally involved in the pathogenesis of copd . in conclusion , reduced oxidative
phosphorylation , modulation of protein catabolism , and dysregulation of transcription are important molecular features of early stages of copd .
genes and splicing variants were identified that were differentially expressed between copd subjects and control subjects .
 
 background and objectives . 
 chronic obstructive pulmonary disease ( copd ) is a complex disease characterized by airflow limitation . 
 although airway inflammation and oxidative stress are known to be important in the pathogenesis of copd , the mechanism underlying airflow obstruction is not fully understood . 
 gene expression profiling of lung tissue was performed to define the molecular pathways that are dysregulated in copd . 
 methods . 
 rna was isolated from lung tissues obtained from 98 subjects with copd and 91 control subjects with normal spirometry . 
 the rna samples were processed with rna - seq using the hiseq 2000 system . 
 genes expressed differentially between the two groups were identified using student 's t - test . results . after filtering for genes with zero counts and noncoding genes , 
 16,676 genes were evaluated . 
 a total of 2312 genes were differentially expressed between the lung tissues of copd and control subjects ( false discovery rate corrected q < 0.01 ) . 
 the expression of genes related to oxidative phosphorylation and protein catabolism was reduced and genes related to chromatin modification were dysregulated in lung tissues of copd subjects . conclusions . 
 oxidative phosphorylation , protein degradation , and chromatin modification were the most dysregulated pathways in the lung tissues of copd subjects . 
 these findings may have clinical and mechanistic implications in copd . 
1. Introduction
2. Methods
3. Results
4. Discussion