Human Lung Tissue Transcriptome:Influence of Sex and Age

Background Sex and age strongly influence the pathophysiology of human lungs, but scarce information is available about their effects on pulmonary gene expression. Methods We followed a discovery-validation strategy to identify sex-and age-related transcriptional differences in lung. Results We identified transcriptional profiles significantly associated with sex (215 genes; FDR <0.05) and age at surgery (217 genes) in non-involved lung tissue resected from 284 lung adenocarcinoma patients. When these profiles were tested in three independent series of non-tumor lung tissue from an additional 1,111 patients, we validated the association with sex and age for 25 and 22 genes, respectively. Among the 17 sex-biased genes mapping on chromosome X, 16 have been reported to escape X-chromosome inactivation in other tissues or cells, suggesting that this mechanism influences lung transcription too. Our 22 age-related genes partially overlap with genes modulated by age in other tissues, suggesting that the aging process has similar consequences on gene expression in different organs. Finally, seven genes whose expression was modulated by sex in non-tumor lung tissue, but no age-related gene, were also validated using publicly available data from 990 lung adenocarcinoma samples, suggesting that the physiological regulatory mechanisms are only partially active in neoplastic tissue. Conclusions Gene expression in non-tumor lung tissue is modulated by both sex and age. These findings represent a validated starting point for research on the molecular mechanisms underlying the observed differences in the course of lung diseases among men and women of different ages

Genes whose expression in lung tissue associated with sex in the discovery series and in all three validation series.

<p>Genes whose expression in lung tissue associated with sex in the discovery series and in all three validation series.</p

Intersection of the lists of genes significantly associated with sex in each of the four datasets.

<p>Four-way Venn diagram analysis of sex-related transcriptional alterations in non-tumor lung tissue. Each of the circles depicts the number of different transcripts based on a sex comparison for each of the labeled data series (yellow, INT, Italian discovery series; green, Laval validation series; blue, Groningen validation series; red, UBC validation series) among the 215 transcripts identified as statistically significant in the Italian discovery series. Shared transcripts are represented in the areas of intersection between two or more circles. Genes whose expression levels were positively and negatively associated with sex in the different series are called “contra-regulated”; these genes were not included among the number of validated genes.</p

Intersection of the lists of genes significantly associated with age in each of the four datasets.

<p>Four-way Venn diagram analysis of age-related transcriptional alterations in non-tumor lung tissue. As in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167460#pone.0167460.g003" target="_blank">Fig 3</a>, each of the circles depicts the number of different transcripts based on age comparison for each of the labeled data series (yellow, INT, Italian discovery series; green, Laval validation series; blue, Groningen validation series; red, UBC validation series) among the 217 transcripts identified as statistically significant in the Italian discovery series. Shared transcripts are represented in the areas of intersection between two or more circles. Genes whose expression levels were positively and negatively associated with age in the different series are called “contra-regulated”; these genes were not included among the number of validated genes.</p

Genes whose expression in tissue associated with patients’ age at surgery in the discovery series and in all three validation series.

<p>Genes whose expression in tissue associated with patients’ age at surgery in the discovery series and in all three validation series.</p

Two major functional themes displayed an altered expression in older patients.

<p>Network-based visualization of gene sets enriched in lung tissue in association with age, in 284 patients who underwent lobectomy for lung adenocarcinoma; the analyzed lung tissue was not involved by cancer. Gene set enrichment analysis (GSEA) was carried out using GSEA v2.0.13 software [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167460#pone.0167460.ref024" target="_blank">24</a>]. Briefly, we first ranked genes of the discovery dataset according to the t-statistic of age and sex. Then, gene sets derived from the Gene Ontology database (C5.all.v4.0) were retrieved from the Molecular Signature Database (MSigDB, <a href="http://www.broadinstitute.org/gsea/msigdb/index.jsp" target="_blank">http://www.broadinstitute.org/gsea/msigdb/index.jsp</a>) and tested for enrichment using a Kolmogorov-Smirnov statistic. Only the 653 gene sets having between 15 and 500 genes were tested for enrichment. An FDR < 0.05 identified significantly enriched gene sets. GSEA results were visualized using Cytoscape v2.8.3 software [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167460#pone.0167460.ref025" target="_blank">25</a>] and the Enrichment Map plugin [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167460#pone.0167460.ref026" target="_blank">26</a>], with an overlap coefficient cut-off of 0.5. Circles (nodes) represent C5 Gene Ontology gene sets connected by lines (edges) whose thickness is proportional to the number of genes shared between the connected nodes. Circle sizes are proportional to the number of genes annotated in each gene set. Two independent clusters of functionally related gene sets were detected, one (on the left) involving extracellular matrix and the other one (on the right) involving pro-inflammatory response and wound healing.</p

Clinical characteristics of patients from whom a sample of resected non-tumor lung tissue was included in the discovery and validation series.

<p>Clinical characteristics of patients from whom a sample of resected non-tumor lung tissue was included in the discovery and validation series.</p

Age distributions of discovery and validation series.

<p>Histogram of age at surgery of male (left panels) and female (right panels) lung cancer patients of the discovery (A) and validation series (B: Laval University; C: University of British Columbia; D: University of Groningen).</p