Identification of Metastasis-related Gene in Oral Cancer

描述（由申请人提供）：在这里提出的研究中，将利用cDNA微阵列技术结合全基因组功能分析的统计和计算方法，在一种新型淋巴结转移小鼠模型中鉴定口腔鳞状细胞癌（SCC）的转移相关基因。由于小鼠模型的独特特征，测试样本的丰富程度以及将用于数据分析的强大统计方法，本研究有望产生重要数据，并导致进一步的研究，从而增加对转移基因表达的理解。这是一个适当的申请探索性/发展性研究资助，定义在项目公告的目的部分。为了研究口腔鳞状细胞癌的转移，我们最近使用改良的口底（FOM）人类肿瘤动物模型，通过体内选择，从低转移性口腔鳞状细胞癌群体中建立了高转移性口腔鳞状细胞癌细胞系。我们使用cDNA微阵列分析、快速基因表达分析（RAGE）以及northern和western blot分析，比较了12株高转移细胞系与其低转移亲代细胞的基因表达。初步实验发现，在选择的转移细胞系中存在一些基因和表达序列标签，其表达显著改变。基于我们的初步发现，我们假设淋巴结转移小鼠模型与cDNA微阵列分析相结合，可以用来识别负责增强口腔鳞状细胞癌转移潜力的基因表达的改变，并促进鳞状细胞癌细胞向颈部淋巴结的迁移和生长。本研究有两个具体目的。首先是确定高转移性口腔鳞状细胞癌中上调和下调的基因。DNA微阵列和数据挖掘方法，如聚类分析和特征选择，将用于比较高转移原发肿瘤细胞与低转移原发肿瘤细胞和淋巴结转移的基因表达。我们将对口腔SCC细胞系和临床标本进行RAGE和northern blot分析，以进一步证实DNA微阵列分析的结果。二是确认差异表达基因对口腔SCC细胞转移表型的影响。将目标基因的cdna以正义或反义格式插入真核表达载体中，并转染到低转移性亲本细胞或其高转移性衍生物中。将使用体外和体内试验来检查转染物，以确定这些基因在转移中的作用。这项研究将有助于确定其表达水平对转移行为至关重要的基因。该项目的研究结果将作为NIH R01基金申请的基础。

DESCRIPTION (provided by applicant): In the study proposed here, the cDNA microarray technique in combination with statistical and computational methods for whole-genome functional analysis will be used to identify metastasis-related genes in oral squamous cell carcinoma (SCC) in a novel lymph nodal metastatic mouse model. As a result of a unique feature of the mouse model, the abundance of testing samples, and the powerful statistical methods that will be used for data analysis, this study promises to generate significant data and lead to further studies that will increase understanding of the expression of metastatic genes. It is an appropriate application for an Exploratory/Developmental Research Grant as defined in the Purpose section of the program announcement. To study the metastasis of oral SCC, we recently established highly metastatic oral SCC cell lines from a population of poorly metastatic oral SCC cells through in vivo selection using a modified floor-of-mouth (FOM) human tumor animal model. We compared the gene expression in 12 highly metastatic cell lines with their poorly metastatic parental cells using cDNA microarray analysis, rapid analysis of gene expression (RAGE), and northern and western blot analyses. The preliminary experiments revealed the existence of several genes and expressed sequence tags whose expressions were significantly altered in the selected metastatic cell lines. Based on our preliminary findings, we posit that the nodal metastatic mouse model in combination with cDNA microarray analysis can be used to identify alterations of gene expressions that are responsible for enhancing the metastatic potential of oral SCC and for facilitating the migration of SCC cells to and the growth in cervical lymph nodes. There are two Specific Aims in the study. The first is to identify the genes that are up- and down-regulated in highly metastatic oral SCC. DNA microarray and data mining methods such as cluster analysis and feature selection will be used to compare gene expression in highly metastatic primary tumor cells with that in poorly metastatic primary tumor cells and in lymph node metastases. RAGE and northern blot analysis will be done in both oral SCC cell lines and clinical specimens to further confirm the result from DNA microarray analysis. The second is to confirm the effect of differentially expressed genes on the metastatic phenotype of oral SCC cells. The cDNAs of the genes of interest will be inserted into a eukaryotic expression vector in either a sense or antisense format and transfected into either poorly metastatic parental cells or their highly metastatic derivatives. The transfectant will be examined using in vitro and in vivo assays to determine the roles of these genes in metastasis. This study will help to identify genes whose expression levels are crucial for metastatic behaviors. The findings from this project will serve as the basis for an NIH R01 grant application.