Identification, amplification and characterization of miR-17-92 from canine tissue.
ABSTRACT Recently, a novel group of genes encoding small RNA molecules, termed microRNAs (miRNAs), has been discovered to play a vital role in eukaryotic gene expression. Known to act in a post-transcriptional fashion, miRNAs can inhibit translation by binding to messenger RNA (mRNA) or by targeting mRNA for degradation. A search of genetic databases revealed significant conservation of miRNA genes between the domestic dog and the human. This finding suggests that expression patterns may also be conserved. Proof of principle experiments, including serial dilutions and sequencing, were performed to verify that primers made to amplify human mature miRNAs can be used to amplify canine miRNAs, providing that the mature sequences are conserved. TaqMan Real-time PCR techniques were used to isolate the first miRNA mature products from canine tissues. The expression levels of miR-17-3p, miR-17-5p, miR-18, miR-19a, miR-19b, miR-20, and miR-92 were evaluated in five canine tissues (heart, lung, brain, kidney, and liver) using the delta-delta Ct (critical threshold) method.
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ABSTRACT: Osteosarcoma (OS) is the most common primary malignant bone tumour in dogs and humans. MicroRNAs are short non-coding RNA molecules involved in post-transcriptional gene expression. Here, we compared the effects of miR-196a deregulation in human and canine OS cells after having observed a more uniform distribution and stronger down-expression in the human specimens. Cell response to miR-196a transfection was different in human and canine OS. A decreased proliferation rate was seen in human MG63 and 143B OS cells, while no appreciable changes occurred in canine DAN cells. Transient decrease of motility was highly remarkable and longer in MG63, concomitant with decreased levels of annexin1, a target of miR-196a promoting cell migration and invasion.Research in Veterinary Science 12/2014; 99. DOI:10.1016/j.rvsc.2014.12.017 · 1.51 Impact Factor
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ABSTRACT: MicroRNA-19b (miR‑19b) is part of the miR‑17‑92 cluster which is associated with cardiac development. It has previously been reported that the overexpression of miR‑19b increases proliferation, inhibits apoptosis and promotes differentiation of embryonic carcinoma cells (P19 cells). The aim of the current study was to investigate the effects of miR‑19b knockdown on the proliferation, apoptosis, differentiation and regulation of the Wnt/β‑catenin signaling pathway in P19 cells. P19 cells were transfected with an miR‑19b knockdown plasmid or an empty vector. MiR‑19b knockdown or vector control stable cell lines were selected using puromycin. Cell Counting kit‑8 and flow cytometry were used to analyze the levels of cellular proliferation, cell cycle progression and the levels of apoptosis, respectively. Caspase‑3 activity and mitochondrial function assays were also used to analyze apoptosis. An inverted microscope was used to observe the morphological changes of P19 cells during differentiation. Reverse transcription‑quantitative polymerase chain reaction and western blot analysis were used to detect P19 cell differentiation markers and Wnt/β‑catenin signaling pathway‑related genes and their corresponding proteins. The results demonstrated that miR‑19b knockdown inhibited the proliferation and apoptosis of P19 cells. However, the levels of expression of Wnt and β‑catenin increased. MiR‑19b knockdown activated the Wnt/β‑catenin signaling pathway, which may regulate cardiomyocyte differentiation. The results of this study indicate that miR‑19b is a novel therapeutic target for cardiovascular diseases and provide insight into the mechanisms underlying congenital heart diseases.Molecular Medicine Reports 12/2014; 11(4). DOI:10.3892/mmr.2014.3037 · 1.48 Impact Factor
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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.BMC Veterinary Research 01/2013; 9(1):12. DOI:10.1186/1746-6148-9-12 · 1.74 Impact Factor