Association between Hypomethylation of Noncoding Regions and Overexpression of the lnc-RNA, AFAP1-AS1, in Barrett’s Esophagus. Homo sapiens

Background & Aims: Alterations in methylation of protein-coding genes have been associated with development of Barrett’s esophagus (BE) and esophageal adenocarcinoma (EAC). Deregulation of non-coding RNAs has been associated with carcinogenesis, but never studied in BE or EAC cells. We used high-resolution methylome analysis to identify changes at genomic regions that encode non-coding RNAs in BE and EAC cells. Methods: We analyzed the methylation status of 1.8 million CpG sites using the massively parallel sequencing-based HELP-tagging assay in matched sets of BE and healthy esophageal tissues from the same patients. We also analyzed human EAC cell lines (OE-33, SK-GT-4, and FLO-1) and human primary non-immortalized esophageal epithelial cells (HEEpiC). Results: BE was characterized by genome-wide loss of methylation that significantly affected intragenic and repetitive elements of the genome. A high proportion of non-coding regions were hypomethylated in BE tissues. The changes in methylation affected small and long non-coding (lnc) regions, and discriminated healthy esophageal from BE tissues, even in matched samples. One lncRNA, AFAP1-AS1, was highly hypomethylated and overexpressed in EAC cells, compared with primary non-malignant esophageal epithelial cells. Knockdown of AFAP1-AS1 with small interfering RNA inhibited proliferation and colony-forming ability of EAC cells, inducing apoptosis; knockdown also significantly reduced EAC cell migration and invasion in Matrigel assays. Conclusions: BE tissues have reduced genome-wide methylation, compared with healthy esophageal tissues, that includes many non-coding regions. Methylation of the long noncoding RNA AFAP1-AS1 is reduced in EAC cells, increasing its expression and the aggressive behavior of cells in culture, similar to activation of protein-coding oncogenes. Overall design: We did methylation tests for three normal esophagus and three Barrett’s esophagus