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 -癌及其前体发育不良病变的分子基础将带来几个重要的好处。具体来说，新的分子改变将为uc相关肿瘤转化的途径提供线索，从而建立更好的疾病模型。这些事件可能演变成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，特别是在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、更广泛地研究UCN的分子基础。通过利用这些进展，我们现在可以通过发现miRs表达的独特变化，确定其在体外和体内的功能影响，以及确定其失调可能致癌的途径，来评估miRs在uc相关炎症、发育不良和癌性病变中的作用。假设：我们假设mir -失调与uc相关的肿瘤进展有关。为了证明这一假设，我们将追求以下具体目标：1)鉴定参与UCN的肿瘤抑制miRs （ts-miRs）和致癌miRs （oncomiRs）。1a)通过miR微阵列对非UC对照与UC相关的非肿瘤性粘膜、不典型增生和癌的非肿瘤性粘膜进行比较，确定在UC肿瘤各阶段失调的miR。1b)在Aim 1a的每个UC-肿瘤阶段，通过在更大样本队列中使用qRT-PCR和原位杂交分析，确认优先上调和下调的miRs的失调和上皮细胞定位。2)确定优先候选ts-miRs和oncomir在体外和体内uc相关肿瘤进展中的生物学影响。2a)通过将miR-mimics（用于ts-miRs）或antagomiRs（用于oncomir）转染到ucn衍生的细胞系中，然后进行生长、增殖、细胞周期和凋亡分析，在体外测试优先失调的miRs的生物学影响。2b)通过将miR-mimics或antagomiRs转染UCN细胞并植入裸鼠体内，检测体外有效miRs （Aim 2a）在体内的生物学效应。3)采用双管齐下的方法，发现和研究涉及UCN- mir及其假定的同源UCN-基因转录物的途径。3a)从候选mir开始，通过质谱筛选从转染了候选miR-mimics或antagomir的UCN细胞中提取的itraq标记蛋白来发现其靶基因转录物。3b)从先前建立的ucn相关基因转录本开始，使用荧光素酶表达载体和Western blotting技术，记录它们的3'- utr与在ucn中也失调的硅选择的假定同源miRs的结合。公共卫生相关性：将研究一组独特的microRNAs （miRs）在溃疡性结肠炎相关肿瘤病变（ucn）发展中的作用。MiR微阵列和定量逆转录酶PCR （qRT-PCR）检测将确定MiR失调。将进行体外和体内研究，以确定ucn中失调的miRs的致癌生物学效应，并将使用计算机和体外方法来显示哪些信使rna是选定的ucn失调的miRs的靶标。最终，这些致癌机制的发现和研究将为未来miR激动剂和拮抗剂在预防和治疗该疾病中的应用奠定基础。

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.