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Whole saliva expression data from human populations stratified by psychosocial stress exposure

ABSTRACT: The objective of this study was analysis of the whole saliva transcriptome to search for biomarkers of psychosocial stressor exposure and substance use in young adults drawn from a population-based longitudinal cohort, the Oregon Youth Substance Use Project. We conducted genome-wide gene expression analysis on whole saliva RNA from 48 individuals stratified by psychosocial stressor exposure using an Affymetrix Gene ST 1.0 array. We applied Weighted Gene Co-expression Network Analysis (WCGNA) to characterize the high-level structure in the data and to relate expression patterns among samples to participant clinical characteristics. This approach finds clusters of correlated genes (modules) which can reflect clinical, histological, or intracellular organization and function. Using WCGNA, we constructed a gene expression network from saliva genome-wide gene expression data, which we found to be similar to a network we constructed from publicly available cell-free saliva genome-wide gene expression data. Functional characterization of the WGCNA modules suggested that the samples varied in composition between an expression profile similar to circulating antigen presenting cell types and a glycoprotein-containing expression profile. We identified a significant relation between one module of whole saliva genome-wide gene expression and smoking behavior, and one with psychosocial stressor exposure. The smoking-related module was related to mitochondrial gene expression, and ever-smokers were found to have reduced overall mitochondrial gene expression. To determine whether reduced expression could be explained by reduction in mitochondrial DNA, we measured mtDNA copy number variation within genomic DNA from 400 OYSUP participants. Surprisingly, we found that more frequent smoking is associated with increased mtDNA copy number. The network structure of whole saliva and its association with clinically assessed exposures and behaviors are reported here for the first time.

ORGANISM(S): Homo sapiens

PROVIDER: GSE40013 | GEO | 2014/09/04

SECONDARY ACCESSION(S): PRJNA172385

REPOSITORIES: GEO

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Project description:Background: We seek to understand correlates of chronic stress and their influence on the development of substance use in a population-based cohort of young adults (OYSUP). Methodology: We evaluated clinical features, salivary RNA metrics and differential expression of candidate genes in unfractionated saliva samples from 48 individuals (31% female, 55% ever-smoking) randomly selected from two groups stratified by high versus low severe negative life events and difficulties within the last year as as measured by the Life Events and Difficulties Schedule (LEDS, Brown & Harris, 1978). We used multiple analytical platforms, multiple reference genes and 18 replicates of all gene expression assays (assays). We chose 38 candidate genes previously identified as differentially expressed in a genome-wide scan of monocyte RNA in 11 familial caregivers of glioblastoma multiforme patients and 10 control subjects matched on age (middle-aged), gender (57% female), ethnicity and marital status and free of major stressors during the previous year. Principal Findings: We observed significant differences in smoking prevalence (Pearson’s χ22d.f.=4.5, P=0.035) and RNA integrity score (t=2.12, P=0.039) between stress strata. We analyzed quantitative PCR data using both the comparative standard curve (CSC) and relative standard curve (RSC) methods because 34% of assays had efficiency estimates <90%. Assay data exhibited excellent linearity and low variability. We observed significant under expression of five assays in the high stress stratum using both methods, four of which interrogate glucocorticoid receptor regulated genes. Post-hoc analyses identified significant associations of RNA yield, integrity score, gender, ever-smoking and/or stress with results of normalized gene expression in 22/37 assays. IL8 expression remained significantly associated with chronic stress after adjustment for clinical and salivary RNA variables. Salivary IL8 expression has been previously associated with oropharyngeal squamous cell cancer. Conclusions: A gene expression signature of chronic stress previously observed in adult hematopoietic samples is observed in the unfractionated saliva of young adults. Future investigation will need to analyze associated clinical, metabolic, meta-genomic, and proteomic components of unfractionated saliva to elucidate factors influencing the salivary transcriptome.

Project description:Background: We seek to understand correlates of chronic stress and their influence on the development of substance use in a population-based cohort of young adults (OYSUP). Methodology: We evaluated clinical features, salivary RNA metrics and differential expression of candidate genes in unfractionated saliva samples from 48 individuals (31% female, 55% ever-smoking) randomly selected from two groups stratified by high versus low severe negative life events and difficulties within the last year as as measured by the Life Events and Difficulties Schedule (LEDS, Brown & Harris, 1978). We used multiple analytical platforms, multiple reference genes and 18 replicates of all gene expression assays (assays). We chose 38 candidate genes previously identified as differentially expressed in a genome-wide scan of monocyte RNA in 11 familial caregivers of glioblastoma multiforme patients and 10 control subjects matched on age (middle-aged), gender (57% female), ethnicity and marital status and free of major stressors during the previous year. Principal Findings: We observed significant differences in smoking prevalence (Pearson’s ?22d.f.=4.5, P=0.035) and RNA integrity score (t=2.12, P=0.039) between stress strata. We analyzed quantitative PCR data using both the comparative standard curve (CSC) and relative standard curve (RSC) methods because 34% of assays had efficiency estimates <90%. Assay data exhibited excellent linearity and low variability. We observed significant under expression of five assays in the high stress stratum using both methods, four of which interrogate glucocorticoid receptor regulated genes. Post-hoc analyses identified significant associations of RNA yield, integrity score, gender, ever-smoking and/or stress with results of normalized gene expression in 22/37 assays. IL8 expression remained significantly associated with chronic stress after adjustment for clinical and salivary RNA variables. Salivary IL8 expression has been previously associated with oropharyngeal squamous cell cancer. Conclusions: A gene expression signature of chronic stress previously observed in adult hematopoietic samples is observed in the unfractionated saliva of young adults. Future investigation will need to analyze associated clinical, metabolic, meta-genomic, and proteomic components of unfractionated saliva to elucidate factors influencing the salivary transcriptome. Gene expression was performed on a Fluidigm 48 x 48 array on 48 individuals for 38 target genes and 6 possible reference genes (represented by 9 reference assays) in triplicate. Quantitative Q-PCR datas were normalized according to relative levels of four reference genes (CDKN1B, YWHAZ, RPL13A, and UBC).

Project description:Background: Epigenetics is involved in various human diseases. Smoking is one of the most common environmental factors causing epigenetic changes. The DNA methylation changes and mechanisms after quitting smoking have not yet been defined. The present study examined the changes in DNA methylation level before and after short-term smoking cessation and explored the potential mechanism. Methods: Whole blood and clinical data were collected in 8 patients before and after short-term smoking cessation, DNA methylation was assessed, and differentially methylated sites were analyzed, followed by a comprehensive analysis of the differentially methylated sites with clinical data. GO/KEGG enrichment and protein-protein interaction (PPI) network identified the hub genes. The differentially methylated sites were detected by GEO2R between former smoking and current smoking in GSE50660 from the GEO database. Then, a Venn analysis was carried out using the differentially methylated sites. GO/KEGG enrichment analysis was performed on the genes corresponding to the common DNA methylation sites, the PPI network was constructed, and hub genes were predicted. The enriched genes associated with the cell cycle were selected, and the gene expression was analyzed in pan-cancer based on the TCGA database. Results: Most of the DNA methylation levels were decreased after short-term smoking cessation; a total of 694 hypermethylated CPG sites and 3184 hypomethylated CPG sites were identified. The DNA methylation levels altered according to the clinical data (body weight, expiratory, and tobacco dependence score). Enrichment analysis, construction of PPI network, and pan-cancer analysis suggested that smoking cessation may be involved in various biological processes. Conclusions: Smoking cessation leads to epigenetic changes, mainly observed in the decline of most DNA methylation levels. Bioinformatics further identified the biologically relevant changes after short-term smoking cessation.

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Whole saliva expression data from human populations stratified by psychosocial stress exposure

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