Identification of Metastasis-related Gene in Oral Cancer

描述（由申请人提供）：在本文提出的研究中，cDNA微阵列技术结合全基因组功能分析的统计和计算方法将用于在新型淋巴结转移小鼠模型中鉴定口腔鳞状细胞癌（SCC）中的转移相关基因。由于小鼠模型的独特功能，丰富的测试样本以及将用于数据分析的强大统计方法，这项研究有望产生重要的数据，并导致进一步的研究，这将增加对转移基因表达的理解。这是一个探索性/发展性研究补助金的适当申请，如计划公告的目的部分所定义。为了研究口腔鳞状细胞癌的转移，我们最近建立了高转移性口腔鳞状细胞癌细胞系的人口转移性差的口腔鳞状细胞癌细胞通过在体内选择使用一个修改的口底（FOM）的人类肿瘤动物模型。我们使用cDNA微阵列分析、基因表达快速分析（Rapid Analysis of Gene Expression，RT-PCR）、北方和西方印迹分析比较了12个高转移细胞系与其低转移亲本细胞的基因表达。初步实验揭示了几个基因的存在和表达的序列标签，其表达在选定的转移性细胞系中显著改变。基于我们的初步研究结果，我们认为淋巴结转移小鼠模型结合cDNA微阵列分析可用于鉴定负责增强口腔SCC转移潜力和促进SCC细胞迁移至颈部淋巴结并在颈部淋巴结中生长的基因表达的改变。本研究有两个具体目的。第一个是确定在高转移性口腔鳞状细胞癌中上调和下调的基因。将使用DNA微阵列和数据挖掘方法，如聚类分析和特征选择，比较高转移性原发肿瘤细胞与低转移性原发肿瘤细胞和淋巴结转移中的基因表达。将在口腔SCC细胞系和临床标本中进行PCR和北方印迹分析，以进一步证实DNA微阵列分析的结果。二是证实差异表达基因对口腔鳞癌细胞转移表型的影响。将目的基因的cDNA以有义或反义形式插入真核表达载体中，并转染到转移性差的亲本细胞或其高转移性衍生物中。将使用体外和体内测定来检查转染子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 R 01资助申请的基础。

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.