Differential gene expression patterns in lung carcinogenesis mediated by loss of mouse tumor supressor Gprc5a. Mus musculus

Increasing the understanding of the impact of changes in oncogenes and tumor suppressor genes is essential for improving the management of lung cancer. Recently, we identified a new mouse lung-specific tumor suppressor - the G-protein coupled receptor 5A (Gprc5a). We sought to understand the molecular consequences of Gprc5a loss and towards this we performed microarray analysis of the transcriptomes of lung epithelial cells cultured from normal tracheas of Gprc5a knockout and wild-type mice to define a loss-of-Gprc5a gene signature. Moreover, we analyzed differential gene expression patterns between Gprc5a knockout normal lung epithelial cells as well as lung adenocarcinoma cells isolated and cultured from tumors of NNK-exposed Gprc5a knockout mice. Overall design: Gprc5a wild type cells (WT-NLE) and Gprc5a knockout cells (NULL-NLE) were isolated and cultured from trachea of three week old Gprc5a wild type and knockout mice, respectively. MDA-F471 mouse lung adenocarcinoma cells were derived de novo from an adenocarcinoma that was found in the lung of a female Gprc5a-knockout mouse sacrificed 16 months after NNK tobacco carcinogen i.p. injection based on a protocol approved by the M.D. Anderson Cancer Center Institutional Animal Care and Use Committee. Following RNA extraction and purification, the transcriptome of the Gprc5a wild type and knockout cells were analyzed by microarray analysis using the Affymetrix MG-430 2.0 murine array platform.

加深对致癌基因与肿瘤抑制基因变异影响的认知，对优化肺癌诊疗管理至关重要。近期，本团队鉴定出一种新型小鼠肺特异性肿瘤抑制基因——G蛋白偶联受体5A（G-protein coupled receptor 5A, Gprc5a）。为阐明Gprc5a缺失引发的分子层面效应，我们对从Gprc5a敲除型与野生型小鼠正常气管中分离培养的肺上皮细胞转录组开展微阵列分析，以明确Gprc5a缺失相关的基因表达特征。此外，我们还分析了两类样本的差异基因表达模式：一类为Gprc5a敲除型正常肺上皮细胞，另一类为从经NNK烟草致癌物暴露的Gprc5a敲除小鼠肿瘤中分离培养的肺腺癌细胞。

实验整体设计如下：分别从3周龄的Gprc5a野生型与敲除型小鼠气管中分离培养细胞，得到Gprc5a野生型肺上皮细胞（WT-NLE）与Gprc5a敲除型肺上皮细胞（NULL-NLE）。MDA-F471小鼠肺腺癌细胞系为从头构建所得：该细胞系源自一只经腹腔注射NNK烟草致癌物16个月后处死的雌性Gprc5a敲除小鼠肺部发现的腺癌组织，构建流程符合M.D.安德森癌症中心（M.D. Anderson Cancer Center）机构动物护理与使用委员会批准的实验方案。完成RNA提取与纯化后，我们采用Affymetrix MG-430 2.0小鼠基因芯片平台，对Gprc5a野生型与敲除型细胞的转录组进行微阵列分析。