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

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE43150 | GEO | 2015/12/26

SECONDARY ACCESSION(S): PRJNA184727

REPOSITORIES: GEO

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Project description:Esophageal cancers (ECs) are highly aggressive tumors with poor prognosis and few treatment options. This study investigated the possibility of treating esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EAC) cells by inhibitors of broad and specific histone deacetylases (HDACi; SAHA, MS-275, FK228) and/or of DNMT (Azacytidine, AZA). Drug targets (HDAC1,2,3 and DNMT1) were present in non-neoplastic (HET-1A), ESCC (OE21) and EAC (OE33) cell lines. All cell lines responded to HDACi by reduced HDAC activity and increased histone acetylation as well as to AZA by up-regulation of p21. Expression of drug targets remained largely unaffected by HDACi and AZA treatment. Importantly, cell viability, apoptosis, cell cycle dynamics and DNA damage were only affected by HDACi and/or AZA in ESCC and EAC, but not the non-neoplastic cells. This was specifically seen for the combination of MS-275 and AZA, leading to enhanced cancer cell selectivity and drug efficiency. By transcriptome analyses of MS-275, AZA and MS-275/AZA treated cells, known (e.g. p21) as well as novel regulated genes significantly associated with the cellular effects post HDACi and/or AZA treatment in ESCC and EAC cells were identified. Finally, human EC tissue specimens frequently expressed the actionable drug targets HDAC1/2/3 and DNMT1. In summary, a combined HDACi (MS-275)/AZA treatment is cancer cell selective and efficient in vitro. Since the majority of ECs express the drug targets in situ, this paves the way for further investigations of HDACi/AZA treatment in esophageal cancer cells and their translation into a clinico-pathological setting. To elucidate the transcriptome response to HDAC inhibitors of normal esophageal cells and esophageal tumor cells, total RNA was isolated from non-neoplastic esophageal epithelial cells (Het1A cells) a well as from two esophageal tumor cell lines (OE21 and OE33), respectively. Cells were treated with either MS-275, Azacytidine (AZA) or in combination of both. DMSO treatment was used as control in each case. Total RNA was isolated from cells 24 h after treatment and experiments were performed in biological triplicates.

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

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