Detection and comparison of microRNA expression in the serum of Doberman Pinschers with dilated cardiomyopathy and healthy controls

Abstract

Background: Dilated cardiomyopathy (DCM) is the most common heart disease in Doberman Pinschers. MicroRNAs (miRNAs) are short non-coding RNAs playing important roles in gene regulation. Different miRNA expression patterns have been described for DCM in humans and might represent potential diagnostic markers. There are no studies investigating miRNA expression profiles in canine DCM. The aims of this study were to screen the miRNA expression profile of canine serum using miRNA microarray and to compare expression patterns of a group of Doberman Pinschers with DCM and healthy controls.

Results: Eight Doberman Pinschers were examined by echocardiography and 24-hour-ECG and classified as healthy (n=4) or suffering from DCM (n=4). Total RNA was extracted from serum and hybridized on a custom-designed 8x60k miRNA microarray (Agilent) containing probes for 1368 individual miRNAs. Although total RNA concentrations were very low in serum samples, 404 different miRNAs were detectable with sufficient signal intensity on miRNA microarray. 22 miRNAs were differentially expressed in the two groups (p<0.05 and fold change (FC)>1.5), but did not reach statistical significance after multiple testing correction (false discovery rate adjusted p>0.05). Five miRNAs were selected for further analysis using quantitative Real-Time RT-PCR (qPCR) assays. No significant differences were found using specific miRNA qPCR assays (p>0.05).

Conclusions: Numerous miRNAs can be detected in canine serum. Between healthy and DCM dogs, miRNA expression changes could be detected, but the results did not reach statistical significance most probably due to the small group size. miRNAs are potential new circulating biomarkers in veterinary medicine and should be investigated in larger patient groups and additional canine diseases.

Figure 1

Matrix of pairwise correlations. Red coefficient of correlation = 1, blue = lowest values; 1A: Correlation matrix showing all samples. Color intensity of DoCM8 is markedly different to other columns indicating strong differences of expression levels compared to other samples. Strong blue colored boxes are illustrating much lower correlation compared to light colored boxes of the other samples. 1B: Correlation matrix after exclusion of DoCM8. All boxes are colored similarly indicating uniform variation between all samples. DoCM: DCM in Doberman Pinschers; Con: Control.

Figure 2

Whisker-Box Plot showing normalized data after exclusion of DoCM8. Y-axis: vsn normalized signal intensities (log2 values). The bar near the middle of the box represents the median, the bottom and the top of the box are representing the 25^th and the 75^th percentile. Circles are displaying outliers. DoCM: DCM in Doberman Pinschers. Con: Control.

Figure 3

Hierarchical cluster analysis (HCL support tree) summarizing miRNA expression differences. Each column represents 1 of 7 samples, each row represents 1 of 22 miRNAs with nominal p-values <0.05. Samples were reproducibly grouped in DoCM and controls after unsupervised hierarchical clustering (MeV 4.7.1, Pearson correlation, HCL support tree, 200 iterations). The different colors of the tree visualize the reliability of branching (black: 100%, grey: 90–100%, blue: 80–90%, green: 70–80%, yellow: 60–70%, ocher: 50–60%, magenta: 0–50%, red: 0% support). Red and blue (rectangles) indicating higher and lower expression levels, respectively, relative to the mean of all samples (log2 mean-centered expression values); DoCM: Dilated Cardiomyopathy in Doberman Pinschers; Con: Control; cfa: canis familiaris, mmu: mus musculus, miR: microRNA.

Figure 4

Whisker-Box Plots showing expression ratios of miRNAs in the qPCR. Expression ratios in the diseased group were compared to healthy control and normalized to the control assays hs_RNU6B and hs_RNU1A. miR-142-3p and miR-144* were up regulated about two-fold in the DCM group compared to the control group, while let-7c appears to be slightly down regulated. The long whiskers represent the high interindividual variance of expression. Expression ratio of miR-92a and miR-21 is nearly 1. No statistically significant differences between the groups are evident. The dotted line in the box are representing the sample median, the bottom and the top of the box are representing the 25^th and 75^th percentile and the whiskers are displaying the outer 50% of observations. miR: microRNA.