The Role of microRNA Alterations in Barrett's Carcinogenesis

描述(由申请人提供)：患有巴雷特食道(BE)的患者患食管腺癌(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(BAN)背后的分子遗传学仍不清楚，更彻底地了解它们将产生几个好处。这些包括：1)潜在Bean的生物途径的线索；2)早期癌症检测、疾病进展或最终预后的有用生物标志物；以及3)干预这一过程的预防和治疗的治疗靶点。被称为microRNAs(MiRs)的小的非编码RNA物种参与了许多人类癌症，miR调节的翻译调节是一种重要的基因调控机制，与转录控制的mRNA表达一起考虑。因此，miR的表达分析将为Bean提供生物学和临床方面的见解。此外，MIR本身可能最终导致靶向分子疗法。我们将通过发现miRs表达的独特变化，并通过确定它们在体外和体内的生物学影响，来评估miRs在BE相关化生、异型增生和癌症病变中的作用。假设：我们假设Bean中涉及一组独特的MIR。为了证明这一假设，我们将比较BE相关化生、异型增生和腺癌的所有阶段以及正常鳞状食道中miR的表达水平。此外，我们还将探索调控这些MIR的功能途径，并在菜豆中发挥作用。为了实现这些更广泛的目标，我们将追求以下具体目标：1)鉴定豆类特异的肿瘤抑制MIR(ts-miRs)和致癌MIR(OncomiRs)。1A)进行基于miR微阵列的NE与BE、LGD与HGD与EAC的比较，以确定在每个癌前转变中差异表达的miR。1b)利用miR RT-PCR，确认Aim 1a中微阵列鉴定的miRs的失调。1c)评估在Aim 1b中证实的miR的潜在调控机制，包括DNA扩增和miR母基因的启动子甲基化。2)确定关键的MIR在BE相关肿瘤进展中的生物学影响。2a)通过将miR-模拟物和反义体导入菜豆细胞系，进行细胞增殖、细胞周期和细胞凋亡检测，以检测miRs-25、-93、-106b、-100、-125b和-205的体外生物学效应。2b)将miRs-25、-93、-106b、-100、-125b和-205分别导入豆源性细胞并植入裸鼠体内，检测miRs-25、-93、-106b、-100、-125b和-205的体内生物学效应。3)采用互补的方法，探索关键miRs与其靶基因转录本之间的相互作用。3a)结合电子数据库搜索、mRNA阵列数据和iTRAQ数据，鉴定miRs-25、-93、-106b、-100、-125b和-205的靶基因转录本。3b)利用含有这些miR靶基因3‘-UTRs的荧光素酶表达载体，研究Aim 3a中鉴定的Bean-miR靶基因转录本，包括p21和Bim。公共卫生相关性：我们将通过发现miRs表达的独特变化并确定它们在体外和体内的功能影响，来评估miRs在BE相关的化生、异常增生和癌症相邻转变中的作用。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性和诊断性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.