Examining smoking-induced differential gene expression changes in buccal mucosa

Research article Open Access Open Peer Review Examining smoking-induced differential gene expression changes in buccal mucosa Doris M Kupfer1Email author, Vicky L White1, Marita C Jenkins2 and Dennis Burian1 BMC Medical Genomics20103:24 https://doi.org/10.1186/1755-8794-3-24 ©  Kupfer et al; licensee BioMed Central Ltd. 2010 Received: 10 December 2009Accepted: 24 June 2010Published: 24 June 2010 Open Peer Review reports Abstract Background Gene expression changes resulting from conditions such as disease, environmental stimuli, and drug use, can be monitored in the blood. However, a less invasive method of sample collection is of interest because of the discomfort and specialized personnel necessary for blood sampling especially if multiple samples are being collected. Buccal mucosa cells are easily collected and may be an alternative sample material for biomarker testing. A limited number of studies, primarily in the smoker/oral cancer literature, address this tissue's efficacy as an RNA source for expression analysis. The current study was undertaken to determine if total RNA isolated from buccal mucosa could be used as an alternative tissue source to assay relative gene expression. Methods Total RNA was isolated from swabs, reverse transcribed and amplified. The amplified cDNA was used in RT-qPCR and microarray analyses to evaluate gene expression in buccal cells. Initially, RT-qPCR was used to assess relative transcript levels of four genes from whole blood and buccal cells collected from the same seven individuals, concurrently. Second, buccal cell RNA was used for microarray-based differential gene expression studies by comparing gene expression between a group of female smokers and nonsmokers. Results An amplification protocol allowed use of less buccal cell total RNA (50 ng) than had been reported previously with human microarrays. Total RNA isolated from buccal cells was degraded but was of sufficient quality to be used with RT-qPCR to detect expression of specific genes. We report here the finding of a small number of statistically significant differentially expressed genes between smokers and nonsmokers, using buccal cells as starting material. Gene Set Enrichment Analysis confirmed that these genes had a similar expression pattern to results from another study. Conclusions Our results suggest that despite a high degree of degradation, RNA from buccal cells from cheek mucosa could be used to detect differential gene expression between smokers and nonsmokers. However the RNA degradation, increase in sample variability and microarray failure rate show that buccal samples should be used with caution as source material in expression studies. Keywords Transcription Factor Binding SiteGene ListCotinineIngenuity Pathway AnalysisBuccal Mucosa Background Blood has been shown to be a responsive tissue that is useful for monitoring gene expression changes due to disease, environmental, biological or drug effects. However, for studies performed in human subjects, a less invasive tissue source for biomarker monitoring is of interest due to the discomfort, required skill level, and cost of blood collection, especially for repeated-measures studies. Buccal mucosa (from cheek swabs) is an easily accessed tissue and has been used successfully to obtain DNA for genotyping studies [1]. However, the literature is limited as to the usefulness of RNA from buccal cells as a substrate for gene expression testing, presumably due to concern regarding a high concentration of RNases in saliva which are known to rapidly degrade RNA in these cells [2]. qPCR has been used to detect expression changes in genes from the P450 family using snap frozen surgical buccal plug samples [3] and from brushed exfoliated buccal cells [4, 5]. These studies suggested that buccal cells might serve as an alternative to blood in qPCR assays examining gene expression profiles after exposure to environmental toxins, tobacco smoke, drugs, nutrients, or the presence of certain cancers. With RNA purified from brushed exfoliated buccal cells, Sridhar et al. [6] used microarrays from smoker samples they collected and nonsmoker arrays from the Gene Expression Omnibus (GEO) collection to compare expression levels between smokers and nonsmokers, and to compare expression patterns between buccal cells and bronchial epithelium in smokers and nonsmokers from an earlier microarray-based study [7] by Gene Set Enrichment Analysis (GSEA) [8]. To our knowledge, buccal cells have not been used in a whole transcriptome approach to investigate differential gene expression between smokers and nonsmokers using concurrently harvested samples in a manner which directly compares expression differences. A successful study of this type would more clearly suggest that buccal cells have efficacy as source material for biomarker discovery or in a gene expression monitoring system than earlier studies. We describe here, both qPCR and microarray approaches. The RT-qPCR study used matched blood and brushed buccal samples from the same subjects. Relative expression levels of four genes allowed comparison of tissue sources and subject differences. RNA from buccal cells was highly degraded; nonetheless, expression could be detected by qPCR for all four transcripts tested. This was sufficient evidence of the potential of buccal cells to follow up on the work of Sridhar et al. [6] and use microarrays for differential gene expression analysis on the transcriptome level in smokers and nonsmokers. An important consideration was the availability of the Smoking Induced Epithelial Gene Expression Database, (SEIGE) [7] and smoker buccal mucosa-specific gene lists [6] against which results from this study could be compared for confirmation of our method. Our data was first analyzed for differences between smokers and nonsmokers using Significance Analysis of Microarray (SAM) [9] and Rank Product (RP) [10] for detection of significant gene expression differences between smokers and nonsmokers in our study. These analyses resulted in a list of candidate marker genes from each method. Ingenuity Pathway Analysis [11] was used to find functional networks containing the differentially expressed genes. The gene lists were also examined for transcriptional coregulation by searching the promoters of differentially expressed genes for transcription factor binding sites (TFBS) using PAINT [12] to access the TRANSFAC [13] database of known TFBS. Specifically, we identified 103 genes with RP analysis that had increased expression in smokers. Pathway analysis showed five function networks involving 91 of the 103 target genes. Network functions included cell cycle; cell growth, proliferation and movement; gene expression; and immunological disease. Upstream sequence analysis showed 38 target genes containing binding sites for at least one of three widely expressed transcription factors. Twenty-five genes were ident (...truncated)