Specific Gene- and MicroRNA-Expression Pattern Contributes to the Epithelial to Mesenchymal Transition in a Rat Model of Experimental Colitis

Abstract

The aim of this study was to determine the gene- and microRNA-expression profile contributing to epithelial to mesenchymal transition in a rat model of experimental colitis. For this, inflammation was induced by injecting 2,4,6-trinitrobenzene sulphonic acid to the colon of male Wistar rats. Samples were taken from both inflamed and uninflamed regions of the same colon, total RNA was isolated, and the mRNA and microRNA expressions were monitored. We have determined that the expression of genes responsible for inducing mesenchymal phenotype, such as Egr1, Fgf2, Fgf7, Jak2, Notch2, Hif1α, Zeb2, Mmp9, Lox, and Vim, was all significantly induced in the inflamed regions of the affected colons while the epithelial marker E-cadherin (Cdh1) was downregulated. In contrast, the expression of microRNAs miR-192, miR-143, miR-375, miR-30a, miR-107, and miR-200b responsible for the regulation of the above mentioned genes was significantly downregulated in inflamed colon. Importantly, we detected moderate induction in the expression of five out of six tested microRNAs in the uninflamed regions. In summary, we identified numerous interacting genes and microRNAs with mutually exclusive expression pattern in inflamed regions of colitis-induced rats. These findings suggest that-among others-an important step in the epithelial to mesenchymal transition in experimental colitis is the dysregulated microRNA expression.

Figure 1

Representative images of noninduced (a) and colitis-induced (b) colons. Green box in (a) indicates control sampling; red and blue boxes in (b) indicate sampling from colitis-induced inflamed and uninflamed regions, respectively.

Figure 2

Opposing expression of growth factors and their regulating miRNAs at the site of colon inflammation. The relative gene expression of growth factors Egr1 (a), Fgf2 (b), and Fgf7 (c) shows significant upregulation in inflamed colons as compared to both control and uninflamed colons of colitis-induced animals. In contrast, the expression pattern of both miR-192 (d) and miR-143 (e) shows slight upregulation in uninflamed colons and a significant downregulation in inflamed colons. Data are presented as the mean ± SEM; the significance of differences between sets of data was determined by one-way analysis of variance (ANOVA) using SPSS Statistics; ^∗p < 0.05.

Figure 3

Induced expression of genes regulating proliferation occurs due to decreased expression of miRNAs. The relative gene expression of Jak2 (a), Notch2 (b), and Hif1α (c) shows significant upregulation in inflamed colons as compared to both control and uninflamed colons of colitis-induced animals. In contrast, the expression of miR-375 (d), miR-30a (e), and miR-107 (f) is significantly downregulated in inflamed colons. Data are presented as the mean ± SEM; the significance of differences between sets of data was determined by one-way analysis of variance (ANOVA) using SPSS Statistics; ^∗p < 0.05.

Figure 4

Induced expression of Zeb2 in inflamed colon downregulates the expression of its targets Cdh1 and miR-200b. The relative gene expression of Zeb2 (a) is significantly induced in inflamed colons as compared to both control and uninflamed colons of colitis-induced animals. In contrast, epithelial markers Cdh1 (b) and miR-200b (c) are both significantly downregulated in inflamed colons of colitis-induced animals. Data are presented as the mean ± SEM; the significance of differences between sets of data was determined by one-way analysis of variance (ANOVA) using SPSS Statistics; ^∗p < 0.05.

Figure 5

The expression of mesenchymal markers Mmp9, Lox, and Vim is upregulated at the site of colon inflammation. The relative gene expression of Mmp9 (a), Lox (b), and Vim (c) is significantly induced in inflamed colons as compared to both control and uninflamed colons of colitis-induced animals. Conversely, mir-125a (d) shows slight but not significant downregulation in both uninflamed and inflamed colons (p = 0.285 and p = 0.135, resp.). Data are presented as the mean ± SEM; the significance of differences between sets of data was determined by one-way analysis of variance (ANOVA) using SPSS Statistics; ^∗p < 0.05.

Figure 6

Characteristic features of epithelial to mesenchymal transition at the site of colon inflammation in TNBS-induced rat model of experimental colitis. Because of the downregulated expression, microRNAs are no longer able to regulate the expression of their target genes at the site of colonic inflammation; hence, the expression of genes specific for mesenchymal phenotype increases. Upregulated Zeb2 expression, in turn, downregulates the expression of Cdh1 which further promotes the loss of epithelial phenotype.