Cigarette smoke mediates epigenetic repression of MIR-217 during esophageal adenocarcinogenesis

Abstract

Although microRNAs (miRs) have been implicated in the pathogenesis of various human malignancies, limited information is available regarding mechanisms by which these noncoding RNAs contribute to initiation and progression of tobacco-induced esophageal cancers. In this study, array and quantitative reverse transcriptase-PCR techniques were used to examine miR expression in immortalized esophageal epithelia (IEE) and esophageal adenocarcinoma (EAC) cells cultured in normal media with or without cigarette smoke condensate (CSC). Under relevant exposure conditions, CSC significantly decreased miR-217 expression in these cells. Endogenous levels of miR-217 expression in cultured EAC cells (EACC)/primary EACs were significantly lower than those observed in IEE/ paired normal esophageal tissues. RNA crosslink immunoprecipitation, quantitative reverse transcriptase-PCR (qRT-PCR) and immunoblot experiments demonstrated direct interaction of miR-217 with kallikrein 7 (KLK7), encoding a putative oncogene not previously implicated in EAC. Repression of miR-217 correlated with increased levels of KLK7 in primary EACs, particularly those from smokers. Chromatin and methylated DNA immunoprecipitation experiments demonstrated that CSC-mediated repression of miR-217 coincided with DNMT3b-dependent hypermethylation and decreased occupancy of nuclear factor 1 within the miR-217 genomic locus. Deoxyazacytidine induced miR-217 expression and downregulated KLK7 in EACC; deoxyazacytidine also attenuated CSC-mediated miR-217 repression and upregulation of KLK7 in IEE and EACC. Overexpression of miR-217 significantly decreased, whereas overexpression of KLK7 increased proliferation, invasion and tumorigenicity of EACC. Collectively, these data demonstrate that epigenetic repression of miR-217 contributes to the pathogenesis of EAC via upregulation of KLK7 and suggest that restoration of miR-217 expression may be a novel treatment strategy for these malignancies.