The Role of microRNA Alterations in Barrett's Carcinogenesis

描述（由申请方提供）：Barrett食管（BE）患者发生食管腺癌（EAC）的风险增加，EAC是发达国家增长最快的癌症之一。BE相关肿瘤进展（BEAN）的分子遗传学基础尚不清楚，对它们更彻底的了解将产生几个好处。其中包括：1）BEAN潜在的生物学途径的线索; 2）早期癌症检测、疾病进展或最终预后的有用生物标志物;和3）干预该过程的预防治疗的治疗靶点。被称为microRNA（miR）的小的非编码RNA种类参与许多人类癌症，并且miR调节的翻译调节是与mRNA表达的转录控制一起沿着考虑的重要基因调节机制。因此，miR表达分析将为BEAN提供生物学和临床见解。此外，miR本身可能最终导致靶向分子疗法。我们将通过发现miR表达的独特改变并通过定义其体外和体内生物学影响来评估miR在BE相关化生、异型增生和癌性病变中的参与。假设：我们假设一组独特的miR参与BEAN。为了证明这一假设，我们将比较BE相关化生、异型增生和腺癌以及正常鳞状食管中所有阶段的miR表达水平。此外，我们将探索这些miR在BEAN中调节和发挥作用的功能途径。为了实现这些更广泛的目标，我们将追求以下具体目标：1）鉴定BEAN特异性肿瘤抑制miR（ts-miR）和致癌miR（oncomiR）。1a）对NE vs. BE vs. LGD vs. HGD vs. EAC进行基于miR微阵列的比较，以鉴定在每个肿瘤前转变中差异表达的miR。1b）使用miR RT-PCR确认通过Aim 1a中的微阵列鉴定的miR的失调。1c）评估目标1b中证实的miR失调的潜在机制，包括miR母基因的DNA扩增和启动子甲基化。2)确定关键miR对BE相关肿瘤进展的生物学影响。2a）通过将miR-模拟物和miR-125 b和miR-205转染到BEAN衍生的细胞系中，然后进行增殖、细胞周期和凋亡测定，在体外测试miR-25、miR-93、miR-106 b、miR- 100、miR-125 b和miR-205的生物学作用。2b）通过将miR-模拟物和miR-125 b和miR-205转染到BEAN衍生的细胞中并将细胞植入裸鼠中来测试miR-25、miR-93、miR-106 b、miR-100、miR-125 b和miR-205的体内生物学效应。3)使用互补方法，探索关键miR与其靶基因转录本之间的相互作用。3a）通过组合计算机数据库搜索、mRNA阵列数据和iTRAQ数据来鉴定miR-25、-93、-106 b、-100、-125 b和-205的靶基因转录物。3b）使用含有这些miR靶mRNA的3 '-UTR的荧光素酶表达载体，研究在Aim 3a中鉴定的BEAN-miR靶基因转录物，包括p21和Bim。公共卫生相关性：我们将通过发现miR表达的独特改变并确定其在体外和体内的功能影响来评估miR在BE相关化生、异型增生和癌性相邻转变中的参与。通过这种方式，我们将全面了解BEAN的分子基础，同时为未来潜在的预测和诊断分析以及治疗干预策略奠定基础。

DESCRIPTION (provided by applicant): Patients with Barrett's esophagus (BE) are at increased risk of developing esophageal adenocarcinoma (EAC), one of the most rapidly increasing cancers in developed nations. The molecular genetics underlying BE- associated neoplastic progression (BEAN) remain unclear, and a more thorough understanding of them would yield several benefits. These include: 1) clues to biological pathways underlying BEAN; 2) useful biomarkers of early cancer detection, disease progression, or ultimate prognosis; and 3) therapeutic targets to intervene in the prevention treatment of this process. Small noncoding RNA species known as microRNAs (miRs) are involved in many human cancers, and miR-modulated translational regulation is an important gene-regulatory mechanism to consider along with transcriptional control of mRNA expression. Thus, miR expression analyses will provide biologic and clinical insights into BEAN. In addition, miRs themselves may eventually lead to targeted molecular therapies. We will evaluate the involvement of miRs in BE-associated metaplastic, dysplastic, and cancerous lesions by discovering unique alterations in the expression of miRs and by defining their biologic impact in vitro and in vivo. Hypothesis: We hypothesize that a unique set of miRs is involved in BEAN. To prove this hypothesis, we will compare miR expression levels at all stages of BE-associated metaplasia, dysplasia, and adenocarcinoma as well as in normal squamous esophagus. In addition, we will explore functional pathways by which these miRs are regulated and exert effects in BEAN. To achieve these broader goals, we will pursue the following Specific Aims: 1) To identify BEAN-specific tumor-suppressive miRs (ts-miRs) and oncogenic miRs (oncomiRs). 1a) To perform miR microarray-based comparisons of NE vs. BE vs. LGD vs. HGD vs. EAC to identify miRs that are differentially expressed at each preneoplastic transition. 1b) To confirm dysregulation of miRs identified by microarrays in Aim 1a, using miR RT-PCR. 1c) To evaluate potential mechanisms underlying dysregulation of miRs confirmed in Aim 1b, including DNA amplification and promoter methylation of miR mother genes. 2) To determine the biologic impact of key miRs on BE-associated neoplastic progression. 2a) To test the biologic effects of miRs -25, -93, -106b, - 100, -125b, and -205 in vitro by transfecting miR-mimics and antagomiRs into BEAN-derived cell lines, followed by proliferation, cell cycle, and apoptosis assays. 2b) To test the biologic effects of miRs -25, -93, - 106b, -100, -125b, and -205 in vivo by transfecting miR-mimics and antagomiRs into BEAN-derived cells and implanting the cells into nude mice. 3) Using complementary approaches, to explore interactions between key miRs and their target gene transcripts. 3a) To identify target gene transcripts of miRs -25, -93, -106b, - 100, -125b, and -205 by combining in-silico database searches, mRNA array data, and iTRAQ data. 3b) To study BEAN-miR target gene transcripts identified in Aim 3a, including p21 and Bim, using luciferase expression vectors containing the 3'-UTRs of these miR target mRNAs. PUBLIC HEALTH RELEVANCE: We will evaluate the involvement of miRs in BE-associated metaplastic, dysplastic, and cancerous adjacent transitions by discovering unique alterations in the expression of miRs and defining their functional impact in vitro and in vivo. In this fashion, we will gain comprehensive insights into the molecular basis of BEAN, while simultaneously establishing a foundation for future potential predictive and diagnostic assays and therapeutic intervention strategies.