Inflammatory Bowel Disease-Associated Malignant Transformation

描述（由申请人提供）：溃疡性结肠炎（UC）患者发生结直肠癌的风险增加。更全面地了解UC癌及其前体发育异常病变的分子基础将带来几个重要的好处。具体来说，新的分子改变将提供线索的途径UC相关的肿瘤转化，导致更好的疾病模型。这些事件可能演变为预防和治疗这种后遗症的治疗靶点。最近的技术和科学进步，特别是microRNA（miRs）领域的爆炸性增长，现在使我们能够比以往任何时候都更深入和广泛地研究UCN的分子基础。通过利用这些进展，我们现在可以通过发现miR表达的独特改变，确定其在体外和体内的功能影响，并确定其失调可能致癌的途径，来评估miR在UC相关炎症，发育异常和癌性病变中的参与。假设：我们假设miR-失调参与UC相关的肿瘤进展。为了证明这一假设，我们将追求以下具体目标：1）鉴定UCN中涉及的肿瘤抑制miR（ts-miR）和致癌miR（oncomiRs）。1a）使用来自非UC对照的非肿瘤性粘膜与UC相关的非肿瘤性粘膜、异型增生和癌的基于miR微阵列的比较，鉴定在每个UC肿瘤性阶段失调的miR。1b）在更大的样本队列中使用qRT-PCR和原位杂交测定，确认目标1a中每个UC肿瘤阶段优先显着上调和下调的miR的失调和上皮细胞定位。2)确定体外和体内优先候选ts-miR和oncomiR对UC相关肿瘤进展的生物学影响。2a）通过将miR模拟物（用于ts-miR）或antagomiR（用于oncomiR）转染到UCN衍生的细胞系中，然后进行生长、增殖、细胞周期和细胞凋亡测定，在体外测试优先失调的miR的生物学影响。2b）通过将miR-模拟物或miRomiR转染到UCN细胞中并将细胞植入裸鼠中来测试体外有效miR（Aim 2a）的体内生物学效应。3)使用双管齐下的方法，发现和研究涉及UCN-miR及其推定的同源UCN基因转录物的途径。3a）从候选miR开始，通过对从已经用候选miR-模拟物或miRomiR转染的UCN细胞中提取的iTRAQ-标记的蛋白进行质谱筛选来发现它们的靶基因转录物。3b）从先前建立的UCN相关基因转录物开始，使用荧光素酶表达载体和蛋白质印迹记录它们的3 '-UTR与推定的同源计算机选择的miR的结合，所述miR也在UCN中失调。公共卫生相关性：将研究一组独特的microRNA（miR）参与溃疡性结肠炎相关肿瘤性病变（UCN）的发展。miR微阵列和定量逆转录酶PCR（qRT-PCR）测定将建立miR失调。将进行体外和体内研究以确定UCN中失调的miR的致癌生物学效应，并将使用计算机模拟和体外方法来显示哪些信使RNA是选定的UCN失调miR的靶标。最终，这些致癌机制的发现和研究将为未来使用miR激动剂和拮抗剂预防和治疗这种疾病奠定基础。

DESCRIPTION (provided by applicant): Patients with ulcerative colitis (UC) are at increased risk of developing colorectal cancer. A more complete understanding of the molecular basis of UC-cancers and their precursor dysplastic lesions will result in several important benefits. Specifically, novel molecular alterations will provide clues to pathways underlying UC-associated neoplastic transformation, leading to better disease models. These events may evolve into therapeutic targets for both the prevention and treatment of this sequela. Recent technical and scientific advances, particularly explosive growth in the field of microRNAs (miRs), now enable us to delve more deeply and broadly than ever previously possible into the molecular underpinnings of UCN. By leveraging these advances, we can now evaluate the involvement of miRs in UC-associated inflamed, dysplastic, and cancerous lesions by discovering unique alterations in the expression of miRs, defining their functional impact both in vitro and in vivo, and defining pathways by which their dysregulation may be carcinogenic. Hypothesis: We hypothesize that miR-dysregulation is involved in UC-associated neoplastic progression. To prove this hypothesis, we will pursue the following Specific Aims: 1) To identify tumor-suppressive miRs (ts-miRs) and oncogenic miRs (oncomiRs) that are involved in UCN. 1a) To identify miRs that are dysregulated at each UC- neoplastic stage using miR microarray-based comparisons of non-neoplastic mucosae from non-UC controls vs. UC-associated non-neoplastic mucosa, dysplasia, and carcinoma. 1b) To confirm dysregulation and epithelial cell localization of prioritized significantly upregulated and downregulated miRs at each UC- neoplastic stage in Aim 1a, using qRT-PCR in a larger sample cohort and in situ hybridization assays. 2) To determine the biologic impacts of prioritized candidate ts-miRs and oncomiRs in UC-associated neoplastic progression in vitro and in vivo. 2a) To test the biologic impacts of prioritized dysregulated miRs in vitro by transfecting either miR-mimics (for ts-miRs) or antagomiRs (for oncomiRs) into UCN-derived cell lines, followed by growth, proliferation, cell cycle, and apoptosis assays. 2b) To test the biologic effects of in vitro effective miRs (Aim 2a) in vivo by transfecting miR-mimics or antagomiRs into UCN cells and implanting the cells in nude mice. 3) Using a two-pronged approach, to discover and investigate pathways involving UCN- miRs and their putative cognate UCN-gene transcripts. 3a) Starting from candidate miRs, to discover their target gene transcripts by performing mass spectrometric screening of iTRAQ-labeled proteins extracted from UCN cells that have been transfected with candidate miR-mimics or antagomiRs. 3b) Starting from previously established UCN-related gene transcripts, to document binding of their 3'-UTRs to putative cognate in silico- selected miRs that are also dysregulated in UCNs, using luciferase expression vectors and Western blotting. PUBLIC HEALTH RELEVANCE: The involvement of a unique set of microRNAs (miRs) in the development of ulcerative colitis-associated neoplastic lesions (UCNs) will be investigated. MiR microarray and quantitative reverse-transcriptase PCR (qRT-PCR) assays will establish miR dysregulation. In vitro and in vivo studies will be performed to determine the carcinogenic biologic effects of miRs dysregulated in UCNs, and in silico and in vitro methods will be used to show which messenger RNAs are targets of selected UCN-dysregulated miRs. Ultimately, the discovery and study of these carcinogenic mechanisms will establish a foundation for the future use of miR agonists and antagonists in the prevention and treatment of this disease.