Gene-environment interaction signatures by quantitative mRNA profiling in exfoliated buccal mucosal cells
Gene-environment interaction signatures by quantitative mRNA profiling in exfoliated buccal mucosal cells
复制标题
DOI：
10.1158/0008-5472.can-04-1771
复制
发表时间：
2004-09-15
期刊：
影响因子：
11.2
通讯作者：
Kaminsky, LS
中科院分区：
文献类型：
--
作者：
Spivack, SD;Hurteau, GJ;Kaminsky, LS
从口腔（颊）上皮脱落的细胞学标本可能含有活细胞，从而可以对基因表达进行分析，以直接和无创地测量基因与环境的相互作用，例如吸入（例如烟草烟雾）暴露。我们使用最近开发的 RNA 特异性实时定量逆转录 PCR 策略，测定了细胞刷收集的脱落颊细胞中的特定 mRNA 水平。在一项初步研究中，通过体外 3-[4,5-二甲基噻唑-2-基]-2,5-二苯基四唑测定验证了脱落口腔细胞的代谢活性。在体内定时暴露于主流烟草烟雾后，在连续收集的一名受检受试者的口腔样本中诱导了 CYP1B1 的诱导，观察到转录活性。对于一组 11 名受试者，在口腔细胞中定性检测到的九个编码致癌物和氧化剂代谢酶的基因的 mRNA 表达与同一个体的激光显微解剖肺中的 mRNA 表达相关（chi(2) = 52.91，P < 0.001）。最后，对 42 名受试者的口腔标本进行了七个目标基因（AhR、CYP1A1、CYP1B1、GSTM1、GSTM3、GSTP1 和 GSTT1）和三个参考基因 [3-磷酸甘油醛脱氢酶 (GAPDH)、β-肌动蛋白和 36B4] 转录物的定量实时逆转录 PCR 测定。在多变量分析中，性别、烟草烟雾暴露和其他因素与 CYPIBI、GSTP1 和其他基因转录本的表达水平相关，但 mRNA 表达的显着个体间变异仍然无法解释。在该试点研究的功率限制内，基因表达特征不能清楚地预测肺癌病例或对照状态。这种非侵入性定量方法可以纳入高通量人类应用中，用于探测与癌症相关的基因-环境相互作用。
Exfoliated cytologic specimens from mouth (buccal) epithelium may contain viable cells, permitting assay of gene expression for direct and noninvasive measurement of gene-environment interactions, such as for inhalation (e.g., tobacco smoke) exposures. We determined specific mRNA levels in exfoliated buccal cells collected by cytologic brush, using a recently developed RNA-specific real-time quantitative reverse transcription-PCR strategy. In a pilot study, metabolic activity of exfoliated buccal cells was verified by 3-[4,5-dimethylthiazol-2-yl]-2,5- diphenyltetrazolium assay in vitro. Transcriptional activity was observed, after timed in vivo exposure to mainstream tobacco smoke resulted in induction of CYP1B1 in serially collected buccal samples from the one subject examined. For a set of 11 subjects, mRNA expression of nine genes encoding carcinogenand oxidant-metabolizing enzymes qualitatively detected in buccal cells was then shown to correlate with that in laser-microdissected lung from the same individuals (chi(2) = 52.91, P < 0.001). Finally, quantitative realtime reverse transcription-PCR assays for seven target gene (AhR, CYP1A1, CYP1B1, GSTM1, GSTM3, GSTP1, and GSTT1) and three reference gene [glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta-actin, and 36B4] transcripts were performed on buccal specimens from 42 subjects. In multivariate analyses, gender, tobacco smoke exposure, and other factors were associated with the level of expression of CYPIBI, GSTP1, and other transcripts on a gene-specific basis, but substantial interindividual variability in mRNA expression remained unexplained. Within the power limits of this pilot study, gene expression signature was not clearly predictive of lung cancer case or control status. This noninvasive and quantitative method may be incorporated into high-throughput human applications for probing gene-environment interactions associated with cancer.