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.
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.
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