Patients with Barrett's esophagus (BE) are at increased risk of developing esophageal adenocarcinoma (EAC), one of the most rapidly increasing cancers in developed nations. The molecular genetics underlying BE- associated neoplastic progression (BEAN) remain unclear, and a more thorough understanding of them would yield several benefits. These include: 1) clues to biological pathways underlying BEAN;2) useful biomarkers of early cancer detection, disease progression, or ultimate prognosis;and 3) therapeutic targets to intervene in the prevention treatment of this process. Small noncoding RNA species known as microRNAs (miRs) are involved in many human cancers, and miR-modulated translational regulation is an important gene-regulatory mechanism to consider along with transcriptional control of mRNA expression. Thus, miR expression analyses will provide biologic and clinical insights into BEAN. In addition, miRs themselves may eventually lead to targeted molecular therapies. We will evaluate the involvement of miRs in BE-associated metaplastic, dysplastic, and cancerous lesions by discovering unique alterations in the expression of miRs and by defining their biologic impact in vitro and in vivo. Hypothesis: We hypothesize that a unique set of miRs is involved in BEAN. To prove this hypothesis, we will compare miR expression levels at all stages of BE-associated metaplasia, dysplasia, and adenocarcinoma as well as in normal squamous esophagus. In addition, we will explore functional pathways by which these miRs are regulated and exert effects in BEAN. To achieve these broader goals, we will pursue the following Specific Aims: 1) To identify BEAN-specific tumor-suppressive miRs (ts-miRs) and oncogenic miRs (oncomiRs). 1a) To perform miR microarray-based comparisons of NE vs. BE vs. LGD vs. HGD vs. EAC to identify miRs that are differentially expressed at each preneoplastic transition. 1b) To confirm dysregulation of miRs identified by microarrays in Aim 1a, using miR RT-PCR. 1c) To evaluate potential mechanisms underlying dysregulation of miRs confirmed in Aim 1b, including DNA amplification and promoter methylation of miR mother genes. 2) To determine the biologic impact of key miRs on BE-associated neoplastic progression. 2a) To test the biologic effects of miRs -25, -93, -106b, - 100, -125b, and -205 in vitro by transfecting miR-mimics and antagomiRs into BEAN-derived cell lines, followed by proliferation, cell cycle, and apoptosis assays. 2b) To test the biologic effects of miRs -25, -93, - 106b, -100, -125b, and -205 in vivo by transfecting miR-mimics and antagomiRs into BEAN-derived cells and implanting the cells into nude mice. 3) Using complementary approaches, to explore interactions between key miRs and their target gene transcripts. 3a) To identify target gene transcripts of miRs -25, -93, -106b, - 100, -125b, and -205 by combining in-silico database searches, mRNA array data, and iTRAQ data. 3b) To study BEAN-miR target gene transcripts identified in Aim 3a, including p21 and Bim, using luciferase expression vectors containing the 3'-UTRs of these miR target mRNAs.
We will evaluate the involvement of miRs in BE-associated metaplastic, dysplastic, and cancerous adjacent transitions by discovering unique alterations in the expression of miRs and defining their functional impact in vitro and in vivo. In this fashion, we will gain comprehensive insights into the molecular basis of BEAN, while simultaneously establishing a foundation for future potential predictive and diagnostic assays and therapeutic intervention strategies.
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