Development of transcriptome-based biomarkers from melanoma in hybrid fish

描述（由申请人提供）：黑色素瘤是一种严重的皮肤癌，在美国正在上升。我们的研究和其他研究表明，种间剑尾鱼杂交表现出自发和环境诱导的几种类型的肿瘤，包括黑色素瘤和其他癌症。剑尾鱼和人类黑色素瘤之间的生物化学和组织学相似性已经揭示，使这些杂交瘤的黑色素瘤研究的一个有吸引力的模型。我们将测试的假设，这些不同的黑色素瘤模型中的每一个共享一组共同的调制遗传因素（由于种间杂交），易患种间杂交个体诱导黑色素瘤的发展。这些遗传因素和途径，他们代表可能会被确定在这些剑尾鱼杂交模型中的黑色素瘤范例的研究，并可能被翻译到黑色素瘤研究在其他生物体，包括人类。我们采用传统的克隆以及下一代454钛（454-Ti）分析，以检查剑尾鱼转录组。我们已经确定了10，679个独特的EST序列，编码来自成年雄性和雌性X的七个不同器官的蛋白质。maculatus Jp 163 A.我们建议调查两个种间杂交剑尾鱼产生的动物代表一个自发的黑色素瘤模型和诱导模型，黑色素瘤的形式，只有在治疗后与MNU或UVB光。在目标1中，将分离来自黑素瘤和正常组织的RNA，然后用于合成标准化的全长cDNA。454-Ti将用于开发这些样品的深度转录物序列覆盖。从黑色素瘤和对照组织的454-Ti获得的EST将被添加到我们的EST数据库中，并用于数据挖掘那些在黑色素瘤中显示特异性表达的基因。在目标2中，目标1中收集的每种RNA将用于构建SAGE文库。454-Ti将用于这些SAGE文库的深度转录组序列覆盖，以开发每个样品的转录谱。SAGE标签将被映射到目标1中获得的EST，以鉴定在黑素瘤中特异性表达的基因和似乎在每个不同的黑素瘤模型（自发的、UVB诱导的和MNU诱导的）中表达的基因子集。对于目标3，将使用真实的时间PCR和原位杂交来验证和确认我们的结果。这些研究的结果将进一步阐明可能导致诱发黑色素瘤遗传易感性的遗传因素的范围。 公共卫生相关性：黑色素瘤是一种严重的皮肤癌。这项建议的重点是了解各种遗传途径的黑色素瘤，在不同的剑尾鱼种间杂交模型将进一步阐明的遗传因素，可能是人类共享的，可能会导致遗传易感性诱发黑色素瘤的范围。在本研究中可以鉴定出稳健的黑素瘤生物标志物候选物。

DESCRIPTION (provided by applicant): Melanoma is a serious form of skin cancer which is rising in the United States. Our studies plus others demonstrated that interspecies Xiphophorus hybrids exhibit spontaneous and environmentally induced tumors of several types, including melanoma and other cancers. Biochemical and histological similarities between Xiphophorus and human melanoma have been revealed making these hybrids an attractive model for melanoma research. We will test the hypothesis that each of these different melanoma models share a common set of modulated genetic factors (due to the interspecies cross) that predispose interspecies hybrid individuals to induced melanoma development. These genetic factors and pathways they represent may be identified by study of melanoma paradigms in these Xiphophorus hybrid models and may be translated to melanoma study in other organisms, including humans. We have employed traditional cloning as well as next generation 454 titanium (454-Ti) analyses to examine the Xiphophorus transcriptome. We have identified 10,679 unique EST sequences encoding proteins from seven different organs of adult male and female X. maculatus Jp 163 A. We propose to investigate two inter-species crosses Xiphophorus that produce animals representing a spontaneous melanoma model and an induced model whereby melanoma forms only after treatment with MNU or UVB light. In aim 1, RNA from melanoma and normal tissues will be isolated then used to synthesis normalized full-length cDNAs. 454-Ti will be employed to develop deep transcript sequence coverage of these samples. ESTs obtained from the 454-Ti from melanoma and control tissues will be added to our EST database and used to data mine those genes showing specific expression in the melanoma. In aim 2, each of the RNAs collected in aim 1 will be used to construct SAGE libraries. 454-Ti will be used for deep transcriptome sequence coverage of these SAGE libraries to develop transcript profiles of each sample. SAGE tags will be mapped to the ESTs obtained in aim 1 to identify genes that are specifically expressed in melanoma and those subsets of genes that appear to be expressed in each distinct melanoma model (spontaneous, UVB-induced, and MNU-induced). For aim 3, real time PCR and in situ hybridization will be used to validate and confirm our results. Results from these studies will further illuminate the range of genetic factors that may lead to genetic predisposition to induced melanoma. PUBLIC HEALTH RELEVANCE: Melanoma is a serious form of skin cancer. This proposal focuses on understanding the various genetic routes to melanoma, among varied Xiphophorus interspecies hybrid models will further illuminate the range of genetic factors that may be shared in humans that may lead to genetic predisposition to induced melanoma. Robust melanoma biomarker candidates can be identified in this investigation.