Discovery and validation of Barrett's esophagus microRNA transcriptome by next generation sequencing

Abstract

Objective: Barrett's esophagus (BE) is transition from squamous to columnar mucosa as a result of gastroesophageal reflux disease (GERD). The role of microRNA during this transition has not been systematically studied.

Design: For initial screening, total RNA from 5 GERD and 6 BE patients was size fractionated. RNA <70 nucleotides was subjected to SOLiD 3 library preparation and next generation sequencing (NGS). Bioinformatics analysis was performed using R package "DEseq". A p value<0.05 adjusted for a false discovery rate of 5% was considered significant. NGS-identified miRNA were validated using qRT-PCR in an independent group of 40 GERD and 27 BE patients. MicroRNA expression of human BE tissues was also compared with three BE cell lines.

Results: NGS detected 19.6 million raw reads per sample. 53.1% of filtered reads mapped to miRBase version 18. NGS analysis followed by qRT-PCR validation found 10 differentially expressed miRNA; several are novel (-708-5p, -944, -224-5p and -3065-5p). Up- or down- regulation predicted by NGS was matched by qRT-PCR in every case. Human BE tissues and BE cell lines showed a high degree of concordance (70-80%) in miRNA expression. Prediction analysis identified targets that mapped to developmental signaling pathways such as TGFβ and Notch and inflammatory pathways such as toll-like receptor signaling and TGFβ. Cluster analysis found similarly regulated (up or down) miRNA to share common targets suggesting coordination between miRNA.

Conclusion: Using highly sensitive next-generation sequencing, we have performed a comprehensive genome wide analysis of microRNA in BE and GERD patients. Differentially expressed miRNA between BE and GERD have been further validated. Expression of miRNA between BE human tissues and BE cell lines are highly correlated. These miRNA should be studied in biological models to further understand BE development.

Figure 1. Flowchart depicting sequential mapping of the reads.

As demonstrated, the unmapped reads were remapped to miRbase after relaxing the criteria to allow 2–3 mismatches leaving only ∼1% of the reads unmapped. fRNAdb, functional RNA database version 3.4; ‘ambiguous’ represents those reads that mapped to multiple different non-coding RNA in the fRNAdb; ‘others’ includes unclassified ncRNAs in fRNAdb; * these miRNA were not included in the final analysis of differential expression.

Figure 2. Normalized read counts and their distribution according to the nucleotide length.

Fig. 1A shows that majority of trimmed reads were 21–23 nucleotides in length, the same size as miRNA. Fig. 1B shows the distribution of trimmed reads based on their mapping to miRNA, human genome, non-coding RNA (besides miRNA etc) and E coli genome. mm1, mm2 and mm3 represent alignment to miRBase with 0 or 1, 2 and 3 mismatches respectively. Note that the majority of aligned miRNA with 0 or 1 mismatch are distributed around 22 base pairs, the expected size of miRNA. hg19, human genome version 19; fRNAdb, functional RNA database.

Figure 3. Prediction of target genes for similarly regulated miRNA.

Note that miR -3065, -944 and -149-5p all were down-regulated and share multiple common targets (Panel A) and miR-192 and -215 all were up-regulated and share common targets (Panel B). These results of common targets for similarly up- or down- regulated miRNA suggest coordination between miRNA.