MicroRNA and protein profiling in melanocytes exposed to solar UVR in situ

描述（由申请人提供）：多年来，流行病学和科学研究都支持太阳紫外线辐射（UVR）在黑色素瘤发展中的作用。我们的新数据表明，紫外线可以不同地影响健康人与有黑色素瘤病史的人黑素细胞中microRNAs （miRNAs）的表达。因此，我们假设，当暴露在太阳紫外线下时，黑色素瘤易发黑色素细胞与健康黑色素细胞在mirna的失调方面存在显著差异，这种异常导致分子通路的扰动，这在一定程度上促进了黑色素瘤的发展。利用新技术和蛋白质组学技术，我们将通过以下研究来验证这一假设：目的1 -评估健康人和黑色素瘤患者原位暴露于太阳紫外线照射后黑色素细胞之间mirna的表达。具体来说，我们将：a)使用生物学相关剂量的模拟太阳紫外线（ssUVR）照射统计数量的人类志愿者（包括健康和黑色素瘤患者），然后通过激光捕获显微解剖（LCM）分离照射或未照射的黑色素细胞。b)量化来自每个志愿者的辐照和未辐照黑素细胞之间mirna表达的相对变化，然后使用生物信息学方法鉴定两组中通常不受管制的mirna。c)确认microRNA阵列卡上观察到的表达模式，并利用现有的软件数据库初步预测miRNA靶基因。d)进行平行实验，分析同一组辐照和未辐照黑素细胞之间的mrna，作为鉴定候选紫外响应miRNA-mRNA关系的第一步。目的2 -鉴定黑色素瘤患者黑色素细胞中受异常表达的紫外线反应性mirna潜在影响的蛋白质组学途径。具体而言，我们将：a)使用逆相蛋白微阵列（RPMA）测量黑色素瘤患者或健康个体辐照和未辐照黑素细胞之间单个蛋白（miRNA靶标）的数量。b)使用这种新的蛋白质组学分析技术来分析与黑色素瘤早期发展相关的分子通路的潜在改变状态。

DESCRIPTION (provided by applicant): For years, epidemiological and scientific studies have supported a role for solar ultraviolet radiation (UVR) in the development of melanoma. Our new data suggests that UV rays can differentially affect the expression of microRNAs (miRNAs) in the melanocytes of healthy persons versus those who have a history of melanoma. We therefore hypothesize that melanoma prone melanocytes, when exposed to solar UVR, differ markedly from healthy melanocytes in the deregulation of miRNAs and that this aberrancy leads to the perturbation of molecular pathways that contribute in part to the development of melanoma. With the use of novel techniques and proteomic technology, we will test this hypothesis by performing the following studies: Aim 1 - Evaluate the expression of miRNAs between the melanocytes of healthy persons and melanoma patients after exposure to solar UVR in situ. Specifically we will: a) Use biologically relevant doses of simulated solar UVR (ssUVR) to irradiate a statistical number of human volunteers (both healthy and melanoma patients), and thereafter isolate irradiated or un-irradiated melanocytes by laser capture microdissection (LCM). b) Quantify relative changes in the expression of miRNAs between the irradiated and un-irradiated melanocytes from each volunteer and then use bioinformatic approaches to identify miRNAs that are commonly deregulated amongst each of the two groups. c) Confirm the expression patterns observed on the microRNA array cards and use currently available software databases to tentatively predict miRNA target genes. d) Perform parallel experiments to profile mRNAs between the same set of irradiated and un-irradiated melanocytes as a first step in identifying candidate UV-responsive miRNA-mRNA relationships. Aim 2 - Identify proteomic pathways potentially affected by aberrantly expressed UV-responsive miRNAs in melanocytes of melanoma patients. Specifically, we will: a) Use reverse phase protein microarrays (RPMA) to measure the quantity of individual proteins (miRNA targets) between the irradiated and un-irradiated melanocytes of melanoma patients or healthy individuals. b) Use this new class of proteomic profiling technology to analyze the potentially altered state of molecular pathways relevant to the early development of melanoma.