Inflammatory Bowel Disease-Associated Malignant Transformation

描述(由申请人提供)：溃疡性结肠炎(UC)患者患结直肠癌的风险增加。更全面地了解UC癌症及其前驱发育不良病变的分子基础将带来几个重要的好处。具体地说，新的分子改变将为UC相关肿瘤转化的潜在途径提供线索，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microRNAs(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，定义它们在体外和体内的功能影响，并确定它们的失调可能致癌的途径，来评估miRs在UC相关的炎症、发育不良和癌症病变中的参与。假设：我们假设miR-失调与UC相关的肿瘤进展有关。为了证明这一假说，我们将追求以下具体目标：1)确定与UCN相关的肿瘤抑制MIR(ts-miRs)和致癌MIR(OncomiRs)。1A)通过基于miR微阵列的非UC对照非肿瘤性粘膜与UC相关的非肿瘤性黏膜、异型增生和癌的比较，确定在UC-肿瘤性疾病的每个阶段中失调的miR。1b)在更大的样本队列中使用qRT-PCR和原位杂交分析，确认在Aim 1a的每个UC肿瘤阶段，优先显著上调和下调的miRs的调节失调和上皮细胞定位。2)确定优先候选基因ts-miRs和oncomiRs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模拟物(针对ts-miRs)或抗配子(针对oncomiRs)导入UCN来源的细胞系，然后进行生长、增殖、细胞周期和凋亡检测，来测试优先调控失调miRs的体外生物学影响。2b)通过将miR模拟物或抗配子导入UCN细胞，并将其植入裸鼠体内，检测体外有效miR(Aim 2a)的生物学效应。3)采用双管齐下的方法，发现和研究涉及UCN-miRs及其推测的同源UCN-基因转录本的途径。3a)从候选miR开始，通过对从已转染候选miR模拟物或反交配子的UCN细胞中提取的iTRAQ标记蛋白进行质谱筛选，发现其靶基因转录本。3b)从先前建立的UCN相关基因转录本开始，利用荧光素酶表达载体和Western blotting，证明它们的3‘-UTRs与电子选择的MIR中推测的同源物的结合，这些MIR也在UCN中表达异常。公共卫生相关性：将调查一组独特的microRNAs(MiRs)在溃疡性结肠炎相关肿瘤性病变(UCNs)发展中的作用。MIR芯片和定量逆转录聚合酶链式反应(qRT-PCR)检测将建立miR失调。体外和体内研究将确定UCN中失调的miRs的致癌生物学效应，并将在电子和体外方法中显示哪些信使RNA是选定的UCN调控失调的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本病中的使用奠定基础。

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.