Differential Expression of miRNAs in Colorectal Cancer: Comparison of Paired Tumor Tissue and Adjacent Normal Mucosa Using High-Throughput Sequencing

We present the results of a global study of dysregulated miRNAs in paired samples of normal mucosa and tumor from eight patients with colorectal cancer. Although there is existing data of miRNA contribution to colorectal tumorigenesis, these studies are typically small to medium scale studies of cell lines or non-paired tumor samples. The present study is to our knowledge unique in two respects. Firstly, the normal and adjacent tumor tissue samples are paired, thus taking into account the baseline differences between individuals when testing for differential expression. Secondly, we use high-throughput sequencing, thus enabling a comprehensive survey of all miRNAs expressed in the tissues. We use Illumina sequencing technology to perform sequencing and two different tools to statistically test for differences in read counts per gene between samples: edgeR when using the pair information and DESeq when ignoring this information, i.e., treating tumor and normal samples as independent groups. We identify 37 miRNAs that are significantly dysregulated in both statistical approaches, 19 down-regulated and 18 up-regulated. Some of these miRNAs are previously published as potential regulators in colorectal adenocarcinomas such as miR-1, miR-96 and miR-145. Our comprehensive survey of differentially expressed miRNAs thus confirms some existing findings. We have also discovered 16 dysregulated miRNAs, which to our knowledge have not previously been associated with colorectal carcinogenesis: the following significantly down-regulated miR-490-3p, -628-3p/-5p, -1297, -3151, -3163, -3622a-5p, -3656 and the up-regulated miR-105, -549, -1269, -1827, -3144-3p, -3177, -3180-3p, -4326. Although the study is preliminary with only eight patients included, we believe the results add to the present knowledge on miRNA dysregulation in colorectal carcinogenesis. As such the results would serve as a robust training set for validation of potential biomarkers in a larger cohort study. Finally, we also present data supporting the hypothesis that there are differences in miRNA expression between adenocarcinomas and neuroendocrine tumors of the colon

Clinical and histopathological characteristics of patients in the study.

<p>Clinical and histopathological characteristics of patients in the study.</p

Intersect of significant miRs from the adenocarcinoma cases when using non-paired (DESeq) and paired (edgeR) analysis approach.

<p>Adjusted for multiple testing using Benjamini and Hochberg, false discovery rate (FDR) < 0.1. Logarithmic fold change (FC) relative to normal mucosa and FDR from paired analysis using edgeR. miRs also significant in the analysis of the neuroendocrine tumor (NET) is indicated.</p

Figure 2

<p><b>Schematic illustration of statistical approach.</b> Panels A and C show approach using non-paired statistics and the DESeq tool. Panel B shows approach using paired statistics and the edgeR tool. See text for further details.</p

Read classification as predicted by miRanalyzer and miRBase.

<p>Panel A with percentage of sequencing reads mapped to mature miRs (black) of the total reads per experiment. Panel B with number of mature miRs identified per sequencing experiment. The total number of mature human miRs in miRBase release 16 (n = 1212) is included as reference.</p

Venn diagram shows the number of significant miRs identified using the non-paired (DESeq) and paired (edgeR) analysis approach.

<p>Venn diagram shows the number of significant miRs identified using the non-paired (DESeq) and paired (edgeR) analysis approach.</p