Abstract Although alterations in microRNA (miRNA; miR) expression have been implicated in the pathogenesis of a variety of human malignancies, limited information is available regarding mechanisms by which these noncoding RNAs contribute to initiation and progression of tobacco-induced esophageal cancers. In order to examine this issue, array and RT-PCR techniques were used to examine miRNA expression profiles in immortalized esophageal epithelial cells (Het-1A), as well as NCI-SB-EsC1 (EsC1), NCI-SB-EsC2 (EsC-2), OE19, and OE33 esophageal cancer cells cultured in normal media (NM) with or without cigarette smoke condensate (CSC). Under relevant exposure conditions, CSC significantly decreased miR-217 expression (1.5-9.2 fold) in esophageal cancer cells and Het-1A cells. Endogenous miR-217 expression levels in cultured esophageal cancer cells were significantly lower than those observed in Het-1A cells. Consistent with these findings, miR-217 was significantly down-regulated (3.5-23.5 fold) in resected esophageal cancers relative to adjacent normal esophageal tissues. Software-guided analysis revealed that miR-217 potentially targeted KLK7, encoding a kallikrein family member implicated in invasion and metastasis of several cancers. Constitutive over-expression of miR-217 inhibited expression, whereas depletion of endogenous miR-217 enhanced expression of KLK7 in Het-1A, EsC1 and EsC2 cells. RNA cross-link immunoprecipitation (CLIP) experiments confirmed direct interaction of miR-217 with KLK7 transcripts. Methylated DNA Immunoprecipitation (MeDIP) and chromatin immunoprecipitation (ChIP) experiments demonstrated that CSC increased DNA methylation and decreased H3K4me3 levels within the miR-217 genomic locus in Het-1A, EsC1 and EsC2 cells. Deoxyazacytidine (DAC) induced miR-217 expression in EsC1 and EsC2 cells but not Het-1A cells, and markedly attenuated CSC-mediated miR-217 repression in these cells. Over-expression of miR-217 significantly decreased proliferation of esophageal cancer and Het-1A cells, as well as invasion of esophageal cancer cells. Depletion of miR-217 increased growth of Het-1A cells, but not EsC1 and EsC2 cells presumably due to lower endogenous miR-217 expression in these cancer cells. Experiments are currently in progress to examine the effects of miR-217 and KLK7 expression on tumorigenicity of esophageal cancer cells. Collectively, these data demonstrate that epigenetic repression of miR-217 contributes to the pathogenesis of esophageal carcinomas, and suggest that restoration of miR-217 expression may be a novel strategy for therapy of these malignancies. Citation Format: Sichuan Xi, Suzanne Inchauste, Zuoxiang Xiao, Jigui Shan, Mary Zhang, Julie A. Hong, Manish T Raiji, David G. Beer, David S. Schrump. Epigenetic repression of miR-217 contributes to tobacco-induced esophageal carcinogenesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3071. doi:10.1158/1538-7445.AM2013-3071 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.