miR-24-3p regulates CDX2 during intestinalization of cardiac-type epithelium in a human model of Barrett’s esophagus

2021 ◽  
Author(s):  
Gabriel Gil-Gómez ◽  
Matteo Fassan ◽  
Lara Nonell ◽  
Marta Garrido ◽  
Marta Climent ◽  
...  
Keyword(s):  
Barrett’S Esophagus ◽  
Esophageal Adenocarcinoma ◽  
Cell Lines ◽  
Messenger Rna ◽  
Barrett's Esophagus ◽  
Human Model ◽  
Adenocarcinoma Cell ◽  
Intestinal Phenotype ◽  
Cdx2 Expression

Summary Background Cardiac-type epithelium has been proposed as the precursor of intestinal metaplasia in the development of Barrett’s esophagus. Dysregulation of microRNAs (miRNAs) and their effects on CDX2 expression may contribute to intestinalization of cardiac-type epithelium. The aim of this study was to examine the possible effect of specific miRNAs on the regulation of CDX2 in a human model of Barrett’s esophagus. Methods Microdissection of cardiac-type glands was performed in biopsy samples from patients who underwent esophagectomy and developed cardiac-type epithelium in the remnant esophagus. OpenArray™ analysis was used to compare the miRNAs profiling of cardiac-type glands with negative or fully positive CDX2 expression. CDX2 was validated as a miR-24 messenger RNA target by the study of CDX2 expression upon transfection of miRNA mimics and inhibitors in esophageal adenocarcinoma cell lines. The CDX2/miR-24 regulation was finally validated by in situ miRNA/CDX2/MUC2 co-expression analysis in cardiac-type mucosa samples of Barrett’s esophagus. Results CDX2 positive glands were characterized by a unique miRNA profile with a significant downregulation of miR-24-3p, miR-30a-5p, miR-133a-3p, miR-520e-3p, miR-548a-1, miR-597-5p, miR-625-3p, miR-638, miR-1255b-1, and miR-1260a, as well as upregulation of miR-590-5p. miRNA-24-3p was identified as potential regulator of CDX2 gene expression in three databases and confirmed in esophageal adenocarcinoma cell lines. Furthermore, miR-24-3p expression showed a negative correlation with the expression of CDX2 in cardiac-type mucosa samples with different stages of mucosal intestinalization. Conclusion These results showed that miRNA-24-3p regulates CDX2 expression, and the downregulation of miRNA-24-3p was associated with the acquisition of the intestinal phenotype in esophageal cardiac-type epithelium.

385 MIR-24-3P REGULATES CDX2 DURING THE PROCESS OF INTESTINALIZATION OF THE CARDIAC-TYPE EPITHELIUM IN A HUMAN MODEL OF BARRETT’S ESOPHAGUS

2021 ◽  
Vol 34 (Supplement_1) ◽  
Author(s):  
Manuel Pera ◽  
Marta Garrido ◽  
Gabriel Gil ◽  
Matteo Fassan ◽  
Marta Climent ◽  
...  
Keyword(s):  
Real Time ◽  
Barrett’S Esophagus ◽  
Esophageal Adenocarcinoma ◽  
Cell Lines ◽  
Real Time Pcr ◽  
Barrett's Esophagus ◽  
Intermediate Stage ◽  
Human Model ◽  
Adenocarcinoma Cell ◽  
Cdx2 Expression

Abstract   Cardiac-type epithelium has been proposed as an intermediate stage between normal squamous epithelium and intestinal metaplasia in the development of Barrett’s esophagus. Deregulation of certain miRNAs and their effects on CDX2 expression might contribute to the intestinalization process of cardiac-type epithelium. The aim of this study was to identify miRNAs differentially expressed between CDX2 positive and negative glands of Barrett’s esophagus and to examine the function of specific miRNAs on the regulation of CDX2. Methods miRNA expression profiling using OpenArrayTM analysis in microdissected cardiac-type glands with and without fully CDX2 expression was performed in biopsies from patients who developed cardiac-type epithelium in the remnant esophagus after esophagectomy. Data were validated using real-time PCR in esophageal adenocarcinoma cell lines and in situ and real-time PCR miRNA/CDX2/MUC2 co-expression analysis in cardiac-type mucosa samples. The effect of miR-24-3p precursor transfection on CDX2 expression was assessed in the esophageal adenocarcinoma cell lines FLO-1 and KYAE-1. Results CDX2 positive glands were characterized by an unique miRNA profile with a significant downregulation of miR-24-3p, miR-520e-3p, miR-548a-1, miR-597-5p, miR-133a-3p, miR-30a-5p, miR-638, miR-625-3p, miR-1255b-1, miR-1260a and upregulation of miR-590 (Figure 1A). miRNA-24-3p was identified as potential regulator of CDX2 gene expression in three bioinformatics algorithms, and this was confirmed in esophageal adenocarcinoma cell lines (Figure 1C). Furthermore, miR-24-3p expression negatively correlates with CDX2 in cardiac-type mucosa samples with different stages of mucosal intestinalization (Figure 1B). Conclusion These results imply that miRNA-24-3p directly targets CDX2, and downregulation of miRNA-24-3p is associated with the acquisition of an intestinal phenotype in cardiac-type epithelium.

P160 miR-24-3p REGULATES CDX2 DURING THE PROCESS OF INTESTINALIZATION OF THE CARDIAC-TYPE EPITHELIUM IN A HUMAN MODEL OF BARRETT´S ESOPHAGUS

2019 ◽  
Vol 32 (Supplement_2) ◽  
Author(s):  
Pera Manuel ◽  
Garrido Marta ◽  
Gil Gabriel ◽  
Fassan Matteo ◽  
Climent Marta ◽  
...  
Keyword(s):  
Esophageal Adenocarcinoma ◽  
Cell Lines ◽  
Squamous Epithelium ◽  
Intermediate Stage ◽  
Human Model ◽  
Positive Staining ◽  
Adenocarcinoma Cell ◽  
Cdx2 Expression ◽  
Mirna Expression Profiling

Abstract Aim The aim of this study was to identify miRNAs differentially expressed between CDX2 positive and negative glands of Barrett’s esophagus and, based on this, to examine the function of specific miRNAs on the regulation of CDX2. Background & Methods Cardiac-type epithelium has been proposed as an intermediate stage between normal squamous epithelium and intestinal metaplasia (IM) in the development of Barrett´s esophagus. Deregulation of certain miRNAs and their effects on CDX2 expression might contribute to the intestinalization process of cardiac-type epithelium. miRNA expression profiling using OpenArray technology in microdissected cardiac-type glands with and without fully CDX2 expression was performed in biopsies from patients who developed cardiac-type epithelium in the remnant esophagus after esophagectomy. Data were validated using real-time PCR in esophageal adenocarcinoma cell lines and in situ miRNA/CDX2 co-expression analysis in cardiac-type mucosa samples. The effect of miR-24-3p precursor transfection on CDX2 expression was assessed in the esophageal adenocarcinoma cell lines FLO-1 and KYAE-1. Results CDX2 positive glands were characterized by an unique miRNA profile with a significant downregulation of miR-24-3p, miR-520e-3p, miR-548a-1, miR-597-5p, miR-133a-3p, miR-30a-5p, miR-638, miR-625-3p, miR-1255b-1, miR-1260a and upregulation of miR-590 (Figure A). miRNA-24-3p was identified as potential regulator of CDX2 gene expression in three bioinformatics algorithms, and this was confirmed in esophageal adenocarcinoma cell lines (Figure C). Furthermore, miR-24-3p expression negatively correlates with CDX2 in cardiac-type mucosa samples with different stages of mucosal intestinalization (Figure B). Conclusion These results imply that miRNA-24-3p directly targets CDX2, and downregulation of miRNA-24-3p is associated with the acquisition of an intestinal phenotype in cardiac-type epithelium. Figure: (A) OpenArray results showing downregulation of 10 miRNAs in CDX2 positive glands and upregulation of one miRNA (miR-590). (B) miR-24-3p in situ hybridization: intestinal metaplastic glands were characterized by a positive staining for CDX2 (red) and faint to negative miR-24-39. Adjacent non-intestinalized CDX2-negative glands showed a moderate miR-24-3p expression. (C) Transfection of KYAE-1 cells with miR-24-3p and miR-24-39 inhibitor. A significant CDX2 decrease was observed.

P143 RNA-BASED LIQUID BIOPSIES FOR BETTER CLINICAL MANAGEMENT OF BARRETT’S ESOPHAGUS AND ESOPHAGEAL ADENOCARCINOMA

2019 ◽  
Vol 32 (Supplement_2) ◽  
Author(s):  
Philippron Annouck ◽  
Schoofs Kathleen ◽  
Nafteux Philippe ◽  
Depypere Lieven ◽  
Vandesompele Jo ◽  
...  
Keyword(s):  
Barrett’S Esophagus ◽  
Esophageal Adenocarcinoma ◽  
Messenger Rna ◽  
Barrett's Esophagus ◽  
Differentially Expressed ◽  
Circular Rnas ◽  
Proof Of Concept ◽  
Liquid Biopsies ◽  
Tissue Samples ◽  
Concept Study

Abstract Aim In this study we will explore several RNA types in blood, including microRNA, messenger RNA, long non-coding RNA and circular RNA as a potential liquid biomarker to facilitate early diagnosis, prognosis and monitoring of esophageal adenocarcinoma Background & Methods In the past decades the incidence of esophageal adenocarcinoma (EAC) has increased dramatically in most Western populations. Due to the lack of symptoms EAC is often detected in a late stage, contributing to a poor 5-year survival rate. The potential of RNA (coding and miRNA) as circulating biomarker in blood has already been shown for many cancer entities but requires further investigation for EAC. Blood and tissue samples from patients with non-dysplastic Barrett’s esophagus (NDBE), high-grade dysplasia (HGD) and EAC have been collected. Currently, our biobank includes >5000 samples from 120 patients. A proof-of-concept study was conducted including 17 patients from three groups (EAC, HGD and NDBE). For each patient, biopsies from diseased tissue and healthy tissue as well as blood were collected and analyzed using small RNA and total RNA sequencing. Results Gene expression analysis was performed to identify differentially expressed genes across the three groups. The highest number of significantly differentially expressed m(i)RNAs were present in the tissues of EAC versus NDBE patients, while these differences were much lower or even absent in the plasma samples. Moreover, we have identified between 1500 and 7500 unique circular RNAs in individual EAC cancer patients’ plasma, indicating promising opportunities for a blood-based liquid biomarker for BE and EAC. Conclusion An exploration of several RNA types in blood and tissue biopsies has been done in patients with Barrett’s Esophagus and esophageal adenocarcinoma. Additional samples are needed to significantly increase the power of the differential expression study as well as to verify the results of our proof-of-concept study.

Prioritization and functional analysis of GWAS risk loci for Barrett’s esophagus and esophageal adenocarcinoma

2021 ◽  
Author(s):  
Jianhong Chen ◽  
Mourad Wagdy Ali ◽  
Li Yan ◽  
Shruti G Dighe ◽  
James Y Dai ◽  
...  
Keyword(s):  
Barrett’S Esophagus ◽  
Esophageal Adenocarcinoma ◽  
Cell Lines ◽  
Barrett's Esophagus ◽  
Luciferase Reporter ◽  
Genome Wide Association Studies ◽  
Potential Candidate ◽  
Functional Potential ◽  
Functional Variants ◽  
Causal Variants

Abstract Genome-wide association studies (GWAS) have identified ~ 20 genetic susceptibility loci for esophageal adenocarcinoma (EAC), and its precursor, Barrett’s esophagus (BE). Despite such advances, functional/causal variants and gene targets at these loci remain undefined, hindering clinical translation. A key challenge is that most causal variants map to non-coding regulatory regions such as enhancers, and typically, numerous potential candidate variants at GWAS loci require testing. We developed a systematic informatics pipeline for prioritizing candidate functional variants via integrative functional potential scores consolidated from multi-omics annotations, and used this pipeline to identify two high-scoring variants for experimental interrogation: chr9q22.32/rs11789015 and chr19p13.11/rs10423674. Minimal candidate enhancer regions spanning these variants were evaluated using luciferase reporter assays in two EAC cell lines. One of the two variants tested (rs10423674) exhibited allele-specific enhancer activity. CRISPR-mediated deletion of the putative enhancer region in EAC cell lines correlated with reduced expression of two genes—CREB-regulated transcription coactivator 1 (CRTC1) and Cartilage oligomeric matrix protein (COMP); expression of five other genes remained unchanged (CRLF1, KLHL26, TMEM59L, UBA52, RFXANK). Expression quantitative trait locus (eQTL) mapping indicated that rs10423674 genotype correlated with CRTC1 and COMP expression in normal esophagus. This study represents the first experimental effort to bridge GWAS associations to biology in BE/EAC, and supports the utility of functional potential scores to guide variant prioritization. Our findings reveal a functional variant and candidate risk enhancer at chr19p13.11, and implicate CRTC1 and COMP as putative gene targets, suggesting that altered expression of these genes may underlie the BE/EAC risk association.

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