Background

Canine inflammatory bowel disease (IBD) is a group of chronic or recurrent gastrointestinal (GI) disorders with histopathologic evidence of inflammation in intestinal and/or colonic tissue. The exact pathogenesis of these disorders is still unknown; however, genetic factors, intestinal microbiota, environmental factors and deregulated host immune response may contribute to the pathogenesis of the disease [1]. The prevalence of IBD is unknown, but it is the most common histopathologic diagnosis of dogs with chronic vomiting and/or diarrhea. The term IBD should be used if no underlying cause for the inflammation can be documented [2]. Histopathologic confirmation of intestinal inflammation is the gold standard of diagnosis. The establishment of non-invasive biomarkers would help diagnosis, prognosis and disease-monitoring.

According to Bartel (2009), microRNAs (miRs) are small (≈22 nucleotides in length), non-coding RNA molecules with post-transcriptional gene regulatory function via translational repression or messenger (m)RNA degradation [3]. miRs are highly conserved even among distantly related species and are involved in the regulation of many biological processes such as the cell life cycle [4,5,6]. miRs have a complementary binding site on numerous mRNAs and each gene can be regulated by many miRs [3, 4, 7]. In humans, many studies indicate several miRs as regulators of important pathways of the immune response and immune cell development, which are crucial to the pathogenesis of a variety of diseases, including IBD [8, 9]. In addition, miRs are released extracellularly by immune and non-immune cells. Circulating miRs were firstly identified in serum and plasma samples and later in other body fluids (saliva, urine and semen) [10,11,12,13,14]. miRs are remarkably stable in rough conditions (low or high pH, extended storage conditions, boiling, and several freeze-thaw cycles) and are early dysregulated during the course of a broad spectrum of diseases ranging from neoplastic to inflammatory [10, 11, 15,16,17,18,19]. Their expression levels can be easily measured by quantitative polymerase chain reaction (PCR), which has high-precision signal amplification, allowing measurement of very low amounts of miRs in small volumes of body fluids. Furthermore, the expression of some miRs is specific to tissues, and type and/or stage of the disease [20,21,22,23,24,25].

At present, in small animal medicine, little is known about miRs expression in health and disease, including canine IBD. Regarding other canine digestive system diseases some studies have evaluated miRs expression, especially miR-122, and its dysregulation in hepatobiliary diseases [26, 27]. In human IBD, more than 100 miRs have shown altered expression in GI mucosa and/or peripheral blood and serum (circulating miRs) [28,29,30,31], with the latter being a promising non-invasive biomarker for the disease. The aim of the present study was to determine the colonic mucosal and serum relative expression of a selected panel of miRs (miR-16, miR-21, miR-122, miR-146a, miR-147, miR-185, miR-192 and miR-223), as well as to evaluate their correlation in dogs with the degree of histopathological inflammation compared to healthy controls. The miRs selection for this study was based on a combination of their role in the intestinal inflammation in humans and the relevant findings of studies on canine IBD pathogenesis regarding changes in the mucosal immune system [1, 8, 25, 28, 32,33,34,35,36]. Possible correlations of clinical disease activity, endoscopic grading and histopathologic scoring with miRs relative expression in colonic mucosa and serum in dogs with large intestinal IBD were also investigated.

Results

Characteristics of dogs with large intestinal IBD and healthy control dogs

Epidemiological data, canine chronic enteropathy clinical activity index (CCECAI) score and quantitative colonoscopy score of 26 dogs with large intestinal IBD of the study are presented in Table 1. Median age of the dogs was 7 years (range = 1–15 years). Most of the dogs (7/26) with large intestinal IBD were mongrels. Body weight ranged from 1.72 to 36.5 kg (median = 23 kg). Median CCECAI score of dogs with IBD was 7 (range = 4–11). All dogs with large intestinal IBD included in this study had clinical signs of large bowel diarrhea, with mucus and fresh blood in the feces as well as tenesmus. Most dogs with large intestinal IBD had moderate CCECAI score (10/26, range = 6–8), 9/26 had mild (range = 4–5) and 7/26 had severe (range = 9–11). Median quantitative endoscopy score of dogs with IBD was 4 (range = 1–6). All dogs with large intestinal IBD had evidence of primarily lymphoplasmacytic inflammation (Table 2).

Table 1 Epidemiological data, canine chronic enteropathy clinical activity index (CCECAI) score and quantitative endoscopy score of 26 dogs with large intestinal inflammatory bowel disease (IBD)
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Table 2 Histopathologic scores of 26 dogs with large intestinal inflammatory bowel disease (IBD)
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Table 3 shows the epidemiological data of controls. Their median age was 3 years (range = 2–7 years). Most of them (9/16) were mongrels. Body weight ranged from 5 to 40 kg (median = 27.5 kg).

Table 3 Epidemiological data of 16 healthy dogs (C)
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Relative expression of microRNAs in the colonic mucosa and serum of dogs with large intestinal IBD and healthy dogs

In all dogs with large intestinal IBD, colonic mucosal miRs expression analysis was assessed, while serum miRs expression analysis was available for a majority of the dogs with large intestinal IBD (21/26). Nine of 16 control dogs underwent colonoscopy in order to access colonic mucosal miRs expression, with serum miR expression analysis being performed in 14/16 dogs.

Compared to healthy controls, there was a significantly increased relative expression of the following miRs in the colonic mucosa of dogs with large intestinal IBD: miR-16 (p < 0.0005), miR-21 (p = 0.042), miR-122 (p = 0.009) and miR-147 (p < 0.0005), as well as a significantly decreased relative expression of miR-185 (p < 0.0005), miR-192 (p < 0.0005) and miR-223 (p < 0.0005). miR-146a relative expression in the colonic mucosa didn’t show a statistically significant difference between dogs with large intestinal IBD and healthy controls in the colonic mucosa (p = 0.725) (Fig. 1).

Fig. 1
figure 1

Relative expression of miR-16 (a), miR-21 (b), miR-122 (c), miR-146a (d), miR-147 (e), miR-185 (f), miR-192 (g) and miR-223 (h) in the colonic mucosa of healthy dogs (Controls) (n = 9) and dogs with large intestinal inflammatory bowel disease (IBD) (n = 26). Data is presented as median with 25th and 75th quartiles in each box plot. The whiskers indicate the highest and lowest data within 1.5 times the lengths of the quartiles. Statistical significance was defined as p < 0.05. AU = arbitrary units (which represent the expression of each miR normalized toU6sn RNA internal control), miR = microRNA

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Compared to healthy controls, there was a statistically significant increased relative expression of the following miRs in the serum of dogs with large intestinal IBD: miR-16 (p < 0.0005), miR-21 (p = 0.009), miR-122 (p = 0.012), miR146a (p = 0.016) and miR-147 (p < 0.0005), as well as a significantly decreased relative expression of miR-185 (p < 0.0005), miR-192 (p < 0.0005) and miR-223 (p < 0.0005) (Fig. 2).

Fig. 2
figure 2

Relative expression of miR-16 (a), miR-21 (b), miR-122 (c), miR-146a (d), miR-147 (e), miR-185 (f), miR-192 (g) and miR-223 (h) in the serum of healthy dogs (Controls) (n = 14) and dogs with large intestinal inflammatory bowel disease (IBD) (n = 21). Data is presented as median with 25th and 75th quartiles in each box plot. The whiskers indicate the highest and lowest data within 1.5 times the lengths of the quartiles. Statistical significance was defined as p < 0.05. AU = arbitrary units (which represent the expression of each miR normalized toU6sn RNA internal control), miR = microRNA

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Relationship between miRs relative expression, canine chronic enteropathy clinical activity index, colonoscopy score and histopathologic score of dogs with large intestinal IBD

There was a moderate, statistically significant negative correlation of serum miR-192 (r (21) = − 0.466, p = 0.033) (Fig. 3 and Additional file 1: Table S1) and of colonic mucosal miR-185 (r(26) = − 0.559, p = 0.008) (Fig. 3) relative expression with the CCECAI score in dogs with large intestinal IBD. In addition, there was a moderate, statistically significant negative correlation of serum miR-223 relative expression with colonoscopy score (r(21) = − 0.491, p = 0.024) (Fig. 4 and Additional file 1: Table S1). Analysis of the relationship between colonic mucosal (n = 26) and serum (n = 21) miRs relative expression and histopathologic score (Table 2) of dogs with large intestinal IBD revealed that there was a strong, statistically significant positive correlation of colonic mucosal miR-122 relative expression with surface epithelial injury (a subscore of the WSAVA index) (r(26) = 0.789, p = 0.0005) and a moderate, statistically significant negative correlation of colonic mucosal miR-185 expression with mucosal fibrosis and atrophy (other subscores of the WSAVA index) (r(26) = − 0.436, p = 0.048) (Fig. 5 and Additional file 1: Table S1).

Fig. 3
figure 3

Scatter plots of the relative expression of miR-192 in the serum (n = 21) (a) and miR-185 in the colonic mucosa (n = 26) (b), and CCECAI score of dogs with large intestinal inflammatory bowel disease (IBD) as determined by Spearman’s rank correlation. Statistical significance was defined as p < 0.05. AU = arbitrary units (which represent the expression of each miR normalized to U6sn RNA internal control), CCECAI = canine chronic enteropathy clinical activity index, miR = microRNA, r = Spearman’s rank correlation coefficient

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Fig. 4
figure 4

Scatter plot of the relative expression of miR-223 in the serum and colonoscopy score of dogs with large intestinal inflammatory bowel disease (IBD) (n = 26) as determined by Spearman’s rank correlation. Statistical significance was defined as p < 0.05. AU = arbitrary units (which represent the expression of each miR normalized to U6sn RNA internal control), CCECAI = canine chronic enteropathy clinical activity index, miR = microRNA, r = Spearman’s rank correlation coefficient

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Fig. 5
figure 5

Scatter plots of the relative expression of miR-122 in the colonic mucosa and colonic mucosal epithelial injury score (a), and miR-185 in the colonic mucosa and colonic mucosal fibrosis and atrophy score (b) of dogs with large intestinal inflammatory bowel disease (IBD) (n = 26), as determined by Spearman’s rank correlation. Statistical significance was defined as p < 0.05. AU = arbitrary units (which represent the expression of each miR normalized toU6sn RNA internal control), miR = microRNA, r = Spearman’s rank correlation coefficient

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Relationship between miRs relative expression in the colonic mucosa and serum of dogs with large intestinal IBD

There was a weak, statistically significant negative correlation of colonic mucosal with serum miR-146a relative expression (r(21) = − 0.104, p = 0.047) in dogs with large intestinal IBD (Fig. 6 and Additional file 2: Table S2).

Fig. 6
figure 6

Scatter plots of the relative expression of miR-146a in the colonic mucosa and serum of dogs with large intestinal inflammatory bowel disease (IBD) (n = 21) as determined by Spearman’s rank correlation. Statistical significance was defined as p < 0.05. AU = arbitrary units (which represent the expression of each miR normalized to U6sn RNA internal control), miR = microRNA, r = Spearman’s rank correlation coefficient

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Discussion

This study evaluated the expression of miR-16, miR-21, miR-122, miR-146a, miR-147, miR-185, miR-192 and miR-223 in the colonic mucosa and serum of dogs with large intestinal IBD compared to healthy controls. It is believed that IBD is the result of the interaction between a dysregulated immune response to luminal (intestinal flora) and/or environmental factors in genetically susceptible individuals. In spite of extensive investigation in animals and humans, the pathogenesis of IBD remains unknown. Evaluation of miRs could offer a potential link between genetic susceptibility, environmental and immunological factors involved in the pathogenesis of IBD and may serve as clinically useful biomarkers for diagnosis and treatment. To our knowledge, this is the first study that has explored changes of miRs expression in canine IBD.

We found a significantly increased relative expression of miR-16, miR-21, miR-122 and miR-147 and a significantly decreased relative expression of miR-185, miR-192 and miR-223 in the colonic mucosa of dogs with IBD compared to healthy controls.

The primary function of miR-16 is to regulate the production of inflammatory mediators and immunity through co-operation with other miRs, targeting to tumor necrosis factor- alpha (TNF-α) [37]. It is involved in the induction of apoptosis by targeting B-cell leukemia-lymphoma 2 (Bcl-2). In humans, miR-16 expression is inversely correlated to Bcl-2 expression in chronic lymphocytic leukemia and negatively regulate Bcl-2 at a posttranscriptional level [38]. This finding agrees with two studies that evaluated apoptosis in canine IBD, where the expression of Bcl-2 was greater in the duodenum and colon of healthy control dogs versus dogs with IBD and greater numbers of Bcl-2 cells were found in the colon of healthy control dogs compared to dogs with IBD. It is therefore possible that this could be the reason for the miR-16 increase in the colonic mucosa that was detected in our study [39, 40].

In mice, miR-21 could be involved in the pathogenesis of IBD through at least 3 different mechanisms targeting epithelial barrier function, apoptosis and fibrosis [41]. Intestinal permeability was greater in wild type compared to miR-21 knockout strain mice with experimental dextran sulfate sodium (DSS) colitis, while knockout mice had also less intestinal epithelial cell apoptosis [Data normalization

miR-16, miR-21, miR-122, miR-146a, miR-147, miR-185, miR-192 and miR-223 and U6sn were reliably amplified in tested samples. Amplified miRs showed specific melting temperature, confirming the accuracy and specificity of the method used. Real-time quantitative RT-PCR was conducted on an ABI Prism 7700 apparatus (Applied Biosystems™, Foster City, CA, USA). Data were analyzed with the ABI Prism 7700 SDS software (Applied Biosystems™, Foster City, CA, USA). The expression of each miR was normalized to U6sn RNA internal control. The levels of miRs expression were normalized after subtracting the Ct value of the U6sn RNA internal control from that of each miR Ct value for samples (ΔCt = |CtmiR (samples) − CtU6sn|). The relative mRNA expression level of each miR (in arbitrary units- AU) was calculated by dividing the expression level by the mean value in control samples; the latter was considered equal to 1.

Statistical analysis

Statistical analysis was performed using IBM SPSS 19 software program (USA, Chicago, Illinois). Mann–Whitney U test or T- test was performed to compare differences in miRs levels between dogs with large intestinal IBD and healthy control dogs. Correlations of miRs expression with CCECAI score, canine IBD quantitative endoscopic activity and histopathologic score, as well as correlation of colonic mucosal miRs expression with serum miRs expression of dogs with large intestinal IBD were also statistically analyzed using Spearman’s correlation coefficient test. P values of < 0.05 were considered significant.