Hypomethylation of Noncoding DNA Regions and Overexpression of the Long Noncoding RNA, AFAP1-AS1, in Barrett's Esophagus and Esophageal Adenocarcinoma

Abstract

BACKGROUND & AIMS Alterations in methylation of protein-coding genes are associated with Barrett’s esophagus (BE) and esophageal adenocarcinoma (EAC). Dys-regulation of noncoding RNAs occurs during carcinogen-esis but has never been studied in BE or EAC. We applied high-resolution methylome analysis to identify changes at genomic regions that encode noncoding RNAs in BE and EAC. METHODS We analyzed methylation of 1.8 million CpG sites using massively parallel sequencing-based HELP tagging in matched EAC, BE, and normal esophageal tissues. We also analyzed human EAC (OE33, SKGT4, and FLO-1) and normal (HEEpic) esophageal cells. RESULTS BE and EAC exhibited genome-wide hypomethylation, significantly affecting intragenic and repetitive genomic elements as well as noncoding regions. These methylation changes targeted small and long noncoding regions, discriminating normal from matched BE or EAC tissues. One long noncoding RNA, AFAP1-AS1, was extremely hypomethylated and overexpressed in BE and EAC tissues and EAC cells. Its silencing by small interfering RNA inhibited proliferation and colony-forming ability, induced apoptosis, and reduced EAC cell migration and invasion without altering the expression of its protein-coding counterpart, AFAP1. CONCLUSIONS BE and EAC exhibit reduced methylation that includes noncoding regions. Methylation of the long noncoding RNA AFAP1-AS1 is reduced in BE and EAC, and its expression inhibits cancer-related biologic functions of EAC cells.

Novel Aberrations Uncovered in Barrett's Esophagus and Esophageal Adenocarcinoma Using Whole Transcriptome Sequencing

“…Therefore, we sought to systematically characterize lncRNA dysregulation at different stages of Barrett's disease. Previously, only two lncRNAs (HNF1A-AS1 and AFAP-AS1) have been reported as upregulated in EAC (16,17). Although these were upregulated in EAC compared with NE, we observed no difference between EAC and NDBE/LGD (Supplementary Fig.…”

“…Most microarray-based transcriptome analyses will for example miss a large proportion of long noncoding RNAs (lncRNAs), which are involved in almost all known cellular processes (10)(11)(12)(13)(14), and are important in cancer biology, as shown by the number of functional characterized lncRNAs involved in carcinogenesis (15). In EAC, only two lncRNAs have been reported (16,17), and no systematic approach identifying lncRNAs dysregulation at different stages of Barrett's disease and EAC development has been published so far.…”

Human Schlafen 5 (SLFN5) Is a Regulator of Motility and Invasiveness of Renal Cell Carcinoma Cells

“…Assignments were made using known transcripts in the organism's ENSEMBL GTF annotation file using the union strategy, and alignments with a quality score lower than 20 were excluded. Differential expression analysis between tumor and normal samples was performed by edgeR, a bioconductor package specifically for the analysis of replicated count-based expression data (27). Gene counts were normalized by the trimmed mean of M component method.…”

Early Diagnostic Biomarkers for Esophageal Adenocarcinoma—The Current State of Play

“…Very recently published high-resolution methylome analysis has provided first evidence for methylation changes at genomic regions that encode noncoding RNAs. The authors identified long noncoding RNA, AFAP1-AS1, to be severely hypomethylated in Barrett's esophagus and EAC tissue samples, silencing of which significantly reduced aggressiveness of EAC cell lines OE33 and SKGT4 (66).…”