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Purpose: Recent microarray and RNA-sequencing studies have uncovered aberrantly expressed microRNAs (miRNA) in Barrett's esophagus–associated esophageal adenocarcinoma. The functional significance of these miRNAs in esophageal adenocarcinoma initiation and progression is largely unknown.Experimental Design: Expression levels of miR-199a/b-3p, -199a-5p, -199b-5p, -200b, -200c, -223, and -375 were determined in microdissected tissues from cardiac mucosa, Barrett's esophagus, dysplastic Barrett's esophagus, and esophageal adenocarcinoma using quantitative real-time PCR. miR-223 expression was validated in precursors and esophageal adenocarcinomas from 95 patients with esophageal adenocarcinoma by in situ hybridization (ISH). miR-223 was transfected into two esophageal adenocarcinoma cell lines, and in vitro assays were conducted. Target genes were identified using Illumina microarray, and results were validated in cell lines and human specimens.Results: miR-199 family members and miR-223 were significantly overexpressed in esophageal adenocarcinoma, however, only miR-223 showed a stepwise increase during esophageal adenocarcinoma carcinogenesis. A similar trend was observed by ISH, which additionally showed that miR-223 is exclusively expressed by the epithelial compartment. miR-223–overexpressing cells had statistically significantly more migratory and invasive potential than scramble sequence–transfected cells. PARP1 was identified as a direct target gene of miR-223 in esophageal adenocarcinoma cells. Increased sensitivity to chemotherapy was observed in cells with enforced miR-223 expression and reduced PARP1.Conclusions: miR-223 is significantly upregulated during the Barrett's esophagus–dysplasia–esophageal adenocarcinoma sequence. Although high miR-223 levels might contribute to an aggressive phenotype, our results also suggest that patients with esophageal adenocarcinoma with high miR-223 levels might benefit from treatment with DNA-damaging agents. Clin Cancer Res; 19(15); 4067–78. ©2013 AACR.

Supplementary Tables - PDF file 55K, This file contains supplementary tables S1, S2 and S3. Table S1: Literature‐based miR selection for qRT‐PCR validation in a chemo‐radiation na�ve EAC cohort. Two types of miRs were chosen; miRs that may play a role in the development of BE‐ND (BE‐miRs) and miRs that may be involved in the neoplastic progression of BE (Neo‐miRs). Table S2: Quantitative real‐time PCR primers used for the validation of the Illumina microarray results. Table S3: this table contains the microarray results on two biological replicate experiments in OE33 cells. Only the genes that were more than 1.5 fold lower expressed upon miR-223 transfection are listed in this table.

Supplementary Figure S1 - PDF file 28K, This file contains supplementary figure S1. An overview of the study design for miR validation using FFPE and frozen specimens is provided in this flowchart