Integrated miRNA and mRNA Expression Profiling in Inflamed Colon of Patients with Ulcerative Colitis

<div><p>Background</p><p>Ulcerative colitis (UC) is associated with differential colonic expression of genes involved in immune response (e.g. <i>IL8</i>) and barrier integrity (e.g. cadherins). MicroRNAs (miRNAs) are regulators of gene expression and are involved in various immune-related diseases. In this study, we investigated (1) if miRNA expression in UC mucosa is altered and (2) if any of these changes correlate with mucosal mRNA expression. Integration of mRNA and miRNA expression profiling may allow the identification of functional links between dysregulated miRNAs and their target mRNA.</p><p>Methodology</p><p>Colonic mucosal biopsies were obtained from 17 UC (10 active and 7 inactive) patients and 10 normal controls. Total RNA was used to analyze miRNA and mRNA expression via Affymetrix miRNA 2.0 and Affymetrix Human Gene 1.0ST arrays, respectively. Both miRNA and gene expression profiles were integrated by correlation analysis to identify dysregulated miRNAs with their corresponding predicted target mRNA. Microarray data were validated with qRT-PCR. Regulation of <i>IL8</i> and <i>CDH11</i> expression by hsa-miR-200c-3p was determined by luciferase reporter assays.</p><p>Results</p><p>When comparing active UC patients <i>vs.</i> controls, 51 miRNAs and 1543 gene probe sets gave significantly different signals. In contrast, in inactive UC <i>vs.</i> controls, no significant miRNA expression differences were found while 155 gene probe sets had significantly different signals. We then identified potential target genes of the significantly dysregulated miRNAs and genes in active UC <i>vs.</i> controls and found a highly significant inverse correlation between hsa-miR-200c-3p and <i>IL8</i>, an inflammatory marker, and between hsa-miR-200c-3p and <i>CDH11</i>, a gene related to intestinal epithelial barrier function. We could demonstrate that hsa-miR-200c-3p directly regulates <i>IL8</i> and <i>CDH11</i> expression.</p><p>Conclusion</p><p>Differential expression of immune- and barrier-related genes in inflamed UC mucosa may be influenced by altered expression of miRNAs. Integrated analysis of miRNA and mRNA expression profiles revealed hsa-miR-200c-3p for use of miRNA mimics as therapeutics.</p></div

Top 10 most significantly correlated miRNA-mRNA pairs.

<p>Top 10 most significantly correlated miRNA-mRNA pairs.</p

Heatmap of mRNA expression in mucosal colonic biopsies of UC patient and control cohorts.

<p>Unsupervised hierarchical clustering of all samples based on the log2 expression values of the top 20 most variable mRNAs. Samples are shown in the columns and mRNAs in the rows. The boxes in color indicate the log2 intensities of the mRNAs, with blue indicating low expression and yellow indicating high expression.</p

Scatter plot of expression levels of hsa-miR-200c-3p and its predicted target genes.

<p>Correlation between hsa-miR-200c-3p expression values and <i>IL8</i> (A) and <i>CDH11</i> (B) expression values, with their respective Spearman correlation coefficient.</p

Baseline characteristics.

<p>IQR: interquartile range; BMI: body mass index; SD: standard deviation.</p><p>Baseline characteristics.</p

Validation of expression levels of 7 selected mRNAs in human colon.

<p>Boxplots of the expression level of <i>IL8</i>, <i>CDH11</i>, <i>SLC6A14</i>, <i>CD55</i>, <i>CDH3</i>, <i>AQP8</i> and <i>PDZD3</i> in controls (n = 8), active UC (n = 7), inactive UC (n = 6) and IC (n = 5) patients, as assessed by qRT-PCR (box, 25%–75%; whisker, 5%–95%; *p<0.05; ** p<0.01).</p

Venn diagram of the overlap of miRNA profiles in comparative analyses between (in)active UC and controls.

<p>The differentially expressed miRNAs in the comparative analyses active UC <i>vs.</i> controls and active UC <i>vs.</i> inactive UC are depicted in two overlapping circles. An overlap of 24 miRNAs (14 up- and 10 downregulated) was observed between both analyses.</p

Unsupervised hierarchical cluster analysis based on log2 expression values of top 50 most variable gene probe sets.

<p>Individual samples are shown in columns and gene probe sets in rows. The log2 expression values for individual genes are indicated by color, as shown in the scale (color key), with yellow indicating a high level of expression and blue a low level of expression. The abbreviations of the individual genes and gene probe IDs are explained in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068876#pone.0068876.s002" target="_blank">Table S1</a>.</p

Biological function of selected genes in chronic and acute colitis and after additional recovery.

<p>Biological function of selected genes in chronic and acute colitis and after additional recovery.</p

Colonic gene expression in acute DSS colitis.

<p>(A) Venn diagram of the differentially expressed probe sets of the top 50 significantly upregulated genes in acute DSS colitis (fold change compared to controls) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068876#pone.0068876.s004" target="_blank">Table S3</a>) (FDR<0.05, FC>2). These genes were compared with the 72 genes identified in chronic colitis. In total, 30 genes were uniquely upregulated in acute colitis. The biological functions of these 30 genes uniquely upregulated in acute colitis are summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068876#pone-0068876-t001" target="_blank">Table 1</a>. Out of these 50 genes, 21 genes were common in acute and chronic colitis (underlined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068876#pone-0068876-g007" target="_blank">Figure 7</a>). Only 11 out of these 21 genes were common with the 27 common upregulated genes in chronic colitis. For simplicity of the figure, only these 27 genes are shown in the venn diagram for the chronic condition. (B) Individual fold change expression of the top 50 most upregulated genes in acute DSS colitis vs. controls. The abbreviations of the individual genes and gene probe IDs are explained in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068876#pone.0068876.s004" target="_blank">Table S3</a>.</p