Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Correlates of chronic stress and their influence on the development of substance use in a population-based cohort of young adults (OYSUP)

ABSTRACT: 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).

ORGANISM(S): Homo sapiens

SUBMITTER: Denise Nishita

PROVIDER: E-GEOD-27144 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Chronic psychosocial stressors and salivary biomarkers in emerging adults.

Bergen Andrew W AW Mallick Aditi A Nishita Denise D Wei Xin X Michel Martha M Wacholder Aaron A David Sean P SP Swan Gary E GE Reid Mark W MW Simons Anne A Andrews Judy A JA

Psychoneuroendocrinology 20111214 8

We investigated whole saliva as a source of biomarkers to distinguish individuals who have, and who have not, been chronically exposed to severe and threatening life difficulties. We evaluated RNA and DNA metrics, expression of 37 candidate genes, and cortisol release in response to the Trier Social Stress Test, as well as clinical characteristics, from 48 individuals stratified on chronic exposure to psychosocial stressors within the last year as measured by the Life Events and Difficulties Sch ...[more]

PMID: 22172638

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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:Pancreatic cancer is the fourth leading cause of cancer death. Lack of early detection technology for pancreatic cancer invariably leads to a typical clinical presentation of incurable disease at initial diagnosis. Oral fluid (saliva) meets the demand for non-invasive, accessible, and highly efficient diagnostic medium. The level of salivary analytes, such as mRNA and microflora, vary upon disease onset; thus possess valuable signatures for early detection and screening. In this study, we evaluated the performance and translational utilities of the salivary transcriptomic and microbial biomarkers for non-invasive detection of early pancreatic cancer. Two biomarker discovery technologies were used to profile transcriptome in saliva supernatant and microflora in saliva pellet. The Affymetrix Human Genome U133 Plus 2.0 Array was used to discover altered gene expression in saliva supernatant. The Human Oral Microbe Identification Microarray (HOMIM) was used to investigate microflora shift in saliva pellet. Biomarkers selected from both studies were subjected to an independent clinical validation using a cohort of 30 early pancreatic cancer, 30 chronic pancreatitis and 30 healthy matched-control saliva samples. Two panels of salivary biomarkers, including eleven mRNA biomarkers and two microbial biomarkers were discovered and validated for pancreatic cancer detection. The logistic regression model with the combination of three mRNA biomarkers (ACRV1, DMXL2 and DPM1) yielded a ROC-plot AUC value of 0.974 (95% CI, 0.896 to 0.997; P < 0.0001) with 93.3% sensitivity and 90% specificity in distinguishing pancreatic cancer patients from healthy subjects. The logistic regression model with the combination of two bacterial biomarkers (Neisseria elongata and Streptococcus mitis) yielded a ROC-plot AUC value of 0.895 (95% CI, 0.784 to 0.961; P < 0.0001) with 96.4% sensitivity and 82.1% specificity in distinguishing pancreatic cancer patients from healthy subjects. Importantly, the logistic regression model with the combination of four biomarkers (mRNA biomarkers, ACRV1, DMXL2 and DPM1; bacterial biomarker, S. mitis) could differentiate pancreatic cancer patients from all non-cancer subjects (chronic pancreatitis and healthy control), yielding a ROC-plot AUC value of 0.949 (95% CI, 0.877 to 0.985; P < 0.0001) with 92.9% sensitivity and 85.5% specificity. This study comprehensively compared the salivary transcriptome and microflora between pancreatic cancer and control subjects. We have discovered and validated eleven mRNA biomarkers and two microbial biomarkers for early detection of pancreatic cancer in saliva. The logistic regression model with four salivary biomarkers can detect pancreatic cancer specifically without the complication of chronic pancreatitis. This is the first report demonstrating the value of multiplex salivary biomarkers for the non-invasive detection of a high impact systemic cancer. Keywords: Salivary biomarker, pancreatic cancer, early detection, salivary transcriptome, salivary microflora This study, which was approved by the UCLA Institutional Review Board, started sample collection in February 2006 and ended in April 2008. It had a discovery and verification phase, followed by an independent validation phase. All patients with clinically diagnosed early or locally advanced pancreatic cancer, chronic pancreatitis and matched healthy control were recruited from the UCLA Medical Center. The controls were matched for gender, age, ethnicity and smoking history. We chose early and locally advanced pancreatic cancers because they were considered early stage diseases. All patients were recently diagnosed with primary disease, and had not received any prior treatment in the form of chemotherapy, radiotherapy, surgery, or alternative remedies. No subjects had a history of prior malignancy, immunodeficiency, autoimmune disorders, hepatitis, or HIV infection. Written informed consent was obtained from all patients who agreed to serve as saliva donors. Unstimulated saliva samples were collected and processed as previously described. The saliva bank of pancreatic disease at the UCLA Dental Research Institute has collected 283 saliva samples since 2006. Of these, 114 samples, from 42 pancreatic cancer patients, 30 chronic pancreatitis patients and 42 healthy control subjects, met the following eligibility criteria: the disease subjectsâ?? samples were obtained immediately after primary diagnosis from patients without evidence of metastasis; the saliva sample was free of blood (Table 1 in accompanying manuscript). Of the 114 samples, 12 pancreatic cancer samples and 12 healthy control samples were chosen for the discovery and verification phase. The transcriptomic approach profiled the saliva supernatant samples from 12 pancreatic cancer patients and 12 healthy control subjects using the Affymetrix Human Genome U133 Plus 2.0 Array platform.

Project description:Endogenous damage associated molecular pattern molecules (DAMPs) released from necrotic, damaged or stressed cells are associated with an inflammatory response. Whether the microRNA expression signature of this response is different from that of a PAMP-stimulated inflammatory response is unknown. We report here that miR-34c and miR-214 are significantly expressed in fresh human PBMCs exposed to DAMPcontaining freeze-thaw lysates, or to conditioned media from serum-starved and glucose-deprived cells (p<6x10-4 and p<3.7x10-3), respectively. Interestingly, only miR-34c expression was differentially expressed in PBMCs exposed to freeze-thaw lysates or conditioned media from HMGB1+/+ mouse embryonic fibroblast (MEF) cells, when compared to cultures exposed to lysates or conditioned media from HMGB1-/- MEFs. miR-155 expression in these cultures was negligible, but was significantly higher in PBMCs stimulated with LPS or most other TLR ligands, making it the prototypic PAMPmiR. Exposure to a damaged human colorectal carcinoma cell line lysate (HCT116) similarly resulted in increased miR-34c and miR-214 levels. When PBMCs were pre-transfected with anti-miR-34c and then exposed to lysate, expression levels of IKKgamma mRNA, a putative target of miR-34c, increased, while protein levels of IKKgamma in cultures transfected with a pre-miR-34c were abrogated. Levels of miR-34c expression (as well as pro-inflammatory cytokines, IL-1beta and TNFalpha) decreased when PBMC cultures were briefly pre-incubated with the K+ channel (inflammasome) inhibitor, glybenclamide, suggesting that miR-34c is involved in the inflammasome pathway in response to DAMPs. Our findings suggest that a specific microRNA expression signature is associated with the inflammatory response to damaged/injured cells and carries implications for many acute and chronic inflammatory disorders. Human PBMC (peripheral blood mononuclear cells) were exposed to 4 conditions for 48 hours. In the first condition, PBMCs were exposed to conditioned media from serum-starved and glucose-deprived and heat shocked HMGB1-/- MEF cells (mouse embryonic fibroblast cells). In the second condition, PBMCs were exposed to conditioned media from serum-starved and glucose-deprived and heat shocked HMGB1+/+ MEF cells (mouse embryonic fibroblast cells). In the third condition, PBMCs were exposed to LPS (Lipopolysaccharide). In the fourth condition, the PBMCs where left untreated. Four biological repeats were done for each condition for a total of 16 samples.

Dataset Information

Correlates of chronic stress and their influence on the development of substance use in a population-based cohort of young adults (OYSUP)

Publications

Chronic psychosocial stressors and salivary biomarkers in emerging adults.

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