Inflammatory Bowel Disease-Associated Malignant Transformation

炎症性肠病相关的恶性转化

• 批准号: 7929479
• 负责人: Stephen J Meltzer
• 金额: $ 30.01万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-11 至 2014-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7929479
• 关键词: Adenocarcinoma; Agonist; Apoptosis; Binding; Biological; Biological Assay; Cancerous; Carcinoma; Categories; Cell Cycle; Cell Line; Cells; Colorectal Cancer; Computer Simulation; Development; Disease; Disease model; Dysplasia; Epithelial Cells; Event; Foundations; Future; Gene Targeting; Genes; Goals; Growth; Implant; In Situ Hybridization; In Vitro; Inflammation; Inflammatory Bowel Diseases; Label; Lesion; Lesion by Stage; Luciferases; Malignant - descriptor; Malignant Neoplasms; Messenger RNA; Methods; MicroRNAs; Molecular; Mucous Membrane; Neoplastic Cell Transformation; Nude Mice; Oncogenes; Oncogenic; Pathway interactions; Patients; Prevention; Proteins; Quantitative Reverse Transcriptase PCR; Risk; Sampling; Scientific Advances and Accomplishments; Screening procedure; Staging; Testing; Transcript; Translations; Tumor Suppressor Proteins; Ulcerative Colitis; Untranslated Regions; Western Blotting; base; cohort; expression vector; in vivo; insight; neoplastic; novel; novel therapeutics; public health relevance; therapeutic target; tumor; tumor progression

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.

描述（由申请人提供）：溃疡性结肠炎患者（UC）患有结直肠癌的风险增加。对UC-Canters及其前体发育不良病变的分子基础有更全面的了解将带来一些重要的好处。具体而言，新的分子改变将为与UC相关的肿瘤转化的潜在途径提供线索，从而导致更好的疾病模型。这些事件可能会演变为预防和治疗此续集的治疗靶标。最近的技术和科学进步，尤其是microRNA（miR）领域的爆炸性增长，现在使我们能够比以往更深入地研究UCN的分子基础。通过利用这些进步，我们现在可以通过发现MIR表达的独特变化来评估MIR参与UC相关，发炎，发育不良和癌变，从而定义其在体外和体内的功能影响，并定义途径。它们的失调可能是致癌的。假设：我们假设miR-局部调节与UC相关的肿瘤进展有关。为了证明这一假设，我们将追求以下特定目的：1）鉴定参与UCN的肿瘤抑制miR（TS-MIRS）和致癌miR（oncomirs）。 1A）使用基于miR微阵列的非肿瘤粘膜对非UC对照的非肿瘤粘膜的比较与UC相关的非肿瘤粘膜，增生和癌瘤的非肿瘤粘膜相比。 1b）在AIM 1A中的每个UC-肿瘤阶段明显上调和下调的MIR，以确认在AIM 1A中明显上调和下调的上皮细胞定位，并使用QRT-PCR在较大的样品组和原位杂交测定中使用QRT-PCR。 2）确定优先级候选TS-MIR和oncomirs在体外和体内与肿瘤相关的肿瘤进展的生物学影响。 2a）通过转染miR模拟（对于TS-MIRS）或Antagomirs（对于oncomirs），在体外测试优先级失调的MIR的生物学影响，然后进入UCN衍生的细胞系，然后再进行生长，增殖，细胞周期和凋亡分析。 。 2b）通过将miR模拟或Antagomirs转染到UCN细胞中并将细胞植入裸鼠，以测试体外有效miR（AIM 2A）在体内的生物学效应。 3）使用两管沟的方法，发现和研究涉及UCN-MIR及其假定的同源UCN-Gene转录本的途径。 3A）从候选miR开始，通过对已从已转染的UCN细胞中提取的ITRAQ标记的蛋白进行质谱筛选来发现其靶基因转录本，这些蛋白质已被候选miR-Mimimic或antagomirs转染。 3b）从先前建立的UCN相关基因转录物开始，以记录其3'-UTRS与硅酸盐选择的MIR中假定的同源物的结合，这些miR在UCN中也使用荧光素酶表达矢量和蛋白质斑点的结合。公共卫生相关性：将研究一组独特的microRNA（miR）参与与溃疡性结肠炎相关的肿瘤病变（UCN）的发展。 miR微阵列和定量反向转录酶PCR（QRT-PCR）测定法将产生miR失调。将进行体外和体内研究，以确定UCN中MIR的致癌生物​​学作用，并将使用硅和体外方法来证明哪些Messenger RNA是所选UCN-Dyscromented miR的靶标。最终，对这些致癌机制的发现和研究将为未来使用mir激动剂和拮抗剂在预防和治疗这种疾病的情况下建立基础。