Plasma miRNAs as potential biomarkers of chronic degenerative valvular disease in Dachshunds
© Hulanicka et al.; licensee BioMed Central Ltd. 2014
Received: 7 May 2014
Accepted: 28 August 2014
Published: 26 September 2014
Endocardiosis is the most common heart disease in Dachshunds and is therefore an important cause of cardiac morbidity and death. In recent years we have observed an increasing interest in the development of new genetic and genomic markers of heart disease. The discovery of miRNAs circulating in biofluids such as plasma or serum aroused researchers’ interest in using them as potential biomarkers. In the present study we analysed the expression of 9 miRNAs described in literature as being involved in cardiovascular pathology in the plasma of dogs suffering from endocardiosis.
Expression analysis using the Real-time PCR method revealed that two out of nine miRNAs were significantly downregulated: the expression of miR-30b differed between ACVIM stage B and stage A (control) dogs; the expression of mi-133b differed ACVIM stage C and stage A dogs. 5 miRNAs (miR-125, miR-126, miR-21, miR-29b and miR-30b) showed a trend of downregulation in the ACVIM C group. Levels of miR-423 were the same in healthy and diseased dogs. Expression of miR-208a and 208b was not detected.
miR-30b could be a potential biomarker of ACVIM stage B heart failure in Dachshunds with endocardiosis and miR-133b could be a potential biomarker of ACVIM stage C. The lack of expression or lack of significant changes in expression in 7 miRNAs which are potential biomarkers of heart diseases in humans proves that findings from human medicine are not always directly reflected in veterinary medicine.
Endocardiosis is the most common heart disease in Dachshunds and is therefore an important cause of cardiac morbidity and death. It is recognised mainly in middle-aged and older dogs. It is also referred to as myxomatous atrioventricular valvular degeneration, chronic degenerative valvular heart disease (CVHD), or chronic valvular fibrosis. Endocardiosis is characterised by progressive lesions affecting primarily the mitral valve, and less frequently the tricuspid valve. Affected valve leaflets are grossly shortened and thickened and have nodular areas along the free valvular edges. During disease progress, lesions extend and occupy larger areas of the valve surface, sometimes encompassing the chordae tendineae. In most cases atrioventricular valvulopathy occurs in the dog spontaneously and is age-related -.
In recent years significant development of new diagnostic methods allowing precise diagnosis of animal diseases as well as better understanding of their background has been observed. Genetics is a very important aspect of dog breeding and reproduction, and another significant reason why we have been observing a great interest in the development of new genetic and genomic markers of specific diseases. One group of such markers could be miRNA.
miRNAs inhibit gene expression at the post-transcriptional level by directing the RISC complex to target mRNAs resulting in translational repression or cleavage . The transcription profile of many genes can be influenced by miRNAs. This is why they have emerged as crucial regulators of a wide range of physiological and pathological processes.
Several studies have recently discovered circulating miRNAs in blood, not only in platelets, nucleated blood cells, and erythrocytes, but also in plasma. Moreover, plasma miRNAs were found to be unexpectedly stable even under conditions such as boiling, long time storage at room temperature and low or high pH, whereas exogenously added synthetic miRNAs are quickly degraded because of RNAse activity in the plasma -. This means that endogenous miRNAs present in plasma must be shielded in some way from degradation. Recent studies have showed that miRNAs are packaged in lipid vesicles (exosomes, microvesicles, apoptotic bodies) or associated with RNA-binding proteins or lipoprotein complexes -. These properties make miRNAs ideal stable novel diagnostics biomarkers, which can be measured in easy accessible samples and can be used to diagnose different pathological conditions. Plasma and serum miRNAs are currently being intensively investigated and specific miRNA expression patterns are being reported for various pathological conditions in humans. The results of recent studies show that circulating miRNAs are potential biomarkers for detection of cancer - and heart disease -.
However, in veterinary medicine the situation is quite different–there are only few reports about research on circulating miRNAs research in animal diseases. To our best knowledge there is only one report concerning circulating miRNA levels in dogs with heart disease. This study compares expression of miRNAs in the serum of Doberman Pinschers with dilated cardiomyopathy (DCM) in comparison to healthy control dogs.
Therefore the aim of the present paper was to compare levels of miRNAs in the plasma of Dachshunds with endocardiosis and healthy control dogs. Moreover, dogs with endocardiosis were divided in two groups according to the ACVIM (American College of Veterinary Internal Medicine) classification scheme . The results of our previous study revealed that the biggest differences in expression of genes in peripheral blood nuclear cells were observed when healthy dogs and ACVIM stage C (mild to moderate heart failure) dogs where compared, so we decided to include the ACVIM stage C group in the present study. Furthermore, we also included the ACVIM stage B group in our study, which is a group of great importance since this is the asymptomatic stage of the disease and deemed to be important for early diagnostics. The potential identification of any prognostic marker of canine endocardiosis would be very important for the staging of disease progress, therapy assessment as well as for breeding purposes. It could give new insight into the pathogenesis and staging of the disease which could help in the effective planning of treatment.
Clinical characteristics of the study population in the control, ACVIM B and ACVIM C groups
ACVIM A–unaffected (mean ± SD)
ACVIM B (mean ± SD)
ACVIM C (mean ± SD)
Numer of dogs
Age range (years)
5.458 ± 2.51
10.17 ± 3.36
10.40 ± 4.73
Body weight (kg)
10.38 ± 3.26
9.46 ± 2.13
9.20 ± 1.83
71.25 ± 12.26
69.5 ± 8.21
77.14 ± 3.39
1.223 ± 0.10
1.479 ± 0.41
1.774 ± 0.21*
1.938 ± 0.47
2.348 ± 0.54
2.811 ± 0.32*
2.863 ± 0.24
2.993 ± 0.43
3.529 ± 0.42**
5.466 ± 1.05*** (n = 5)
5.963 ± 0.78***
0.9375 ± 0.17
0.9275 ± 0.14
0.6057 ± 0.21**
0.3988 ± 1.13
0.925 ± 1.39
2.379 ± 1.19*
Overview of miRNAs in various cardiovascular diseases in humans, rats and mouse
Chronic Systolic Heart Failure
Acute Myocardial Infarction
Human and mice
Congestive Heart Failure
Coronary Artery Disease
Ischemic Dilated Cardiomyopathy
Peripheral blood mononuclear cells
Acute Myocardial Infarction
This study reports the results of an evaluation of the expression of miRNAs as potential biomarkers in the plasma of dogs suffering from endocardiosis. Endocardiosis (also referred to as chronic degenerative valvular disease or myxomatous valve disease) can lead to congestive heart failure and the associated clinical signs. Since the molecular background of this disease is still far from fully elucidated, the search for any specific markers (prognostic, therapeutic) of this disease would be of great value and importance.
Although miRNA are currently intensively investigated in human medicine because of their diagnostic potential in many different conditions, there are only few reports related to circulating miRNA studies in animal diseases. One of these is the study about expression of miRNAs in serum of Doberman Pinschers with dilated cardiomyopathy (DCM). The authors compared the expression of 1368 miRNAs using miRNA microarrays. They identified 404 different miRNAs but the results did not show statistical significance after multiple testing correction (FDR) and Real-time PCR validation of 5 miRNAs which were differentially regulated without FDR correction showed no statistical differences . Another study conducted by Mizuno et al. revealed that the level of muscle-specific miRNAs are inceresed in serum of canine X-linked muscular dystrophy in Japan dog model (CXMDJ) in comparison to wild type control dogs . Moreover, there were miRNAs quantitated in peripheral blood from dogs with chronic lymphocytic leukemia. The results revealed distinct miRNA patterns in chronic lymphocytic leukemia depending on the immunophenotype . The number of studies is not sufficient and much more data are needed to determine whether miRNAs can be used as biomarkers in veterinary medicine.
One of the difficulties researchers face related to circulating miRNA studies is the impossibility of reliable large–scale expression screening using the microarray method. Microarray analysis is not a suitable method for studying miRNAs expression in biofluids because of insufficient sensitivity which cannot ensure reproducible detection of samples with low miRNA levels . The results of a study conducted by Jensen et al. revealed that the best specificity, sensitivity reproducibility, recovery and linearity in circulating miRNA studies is attained with the Real-time PCR method, which is based on Locked Nucleic Acid (LNA™) technology . This is why we decided to apply this method in our analysis.
Our study included 24 client-owned dogs with endocardiosis classified into groups according to the ACVIM (American College of Veterinary Internal Medicine) classification scheme. The results revealed that among nine miRNAs that have been previously connected to heart disease in literature (Table 2), there were two miRNAs significantly downregulated in plasma of Dachshunds with progression of endocardiosis. miR-30b was downregulated in ACVIM stage B dogs when compared to ACVIM stage A dogs (this miRNA also showed a downregulatory trend in ACVIM stage C dogs) and miR-133b was downregulated in ACVIM stage C dogs. These miRNAs could be potential diagnostic biomarkers of endocardiosis in Dachshunds. miR-30 and miR-133 are cardiomyocyte-enriched miRNAs which regulate connective tissue growth factor (CTGF)–a key molecule in the process of fibrosis and therefore an attractive therapeutic target of heart diseases. A decreased expression of these two miRNAs in human pathological left ventricular hypertrophy and rodent models of heart disease allow for the increase of CTGF levels, which contributes to collagen synthesis . Downregulation of miR-30 is related to endoplasmic reticulum stress in cardiac muscle and vascular smooth muscle cells . On the other hand the results of the study conducted by Goren et al. revealed elevated levels of miR-30 in the serum of stable chronic systolic heart failure patients . The second miRNA with decreased levels in our study–miR-133 was also downregulated in human infarcted heart tissue . Another study showed that the level of miR-133 in the plasma of patients with acute coronary syndrome was associated with high-sensitivity troponin T levels .
The next four analyzed miRNAs–miR-21, miR-29b, miR-125, miR-126 showed a trend of downregulation in the mild to moderate heart failure group–ACVIM B but the results did not reach statistical significance. The literature data for these miRNAs are present in Table 2. It is worthwhile to mention that during the study conducted on dogs by Steudeman et al. miR-21 was one of the 22 miRNAs that showed changed expression in the serum of Doberman Pinchers with Dilated Cardiomyopathy–a 1.67 fold change in microarray (without false discovery rate control), but the results of qPCR validation showed no difference between healthy and DCM dogs .
Although miR-208a and 208b are proposed as potential biomarkers of heart disease in humans ,, their expression has not been detected in any of the samples analyzed in our study. The reason might be that the etiopathogenesis of the most frequent heart disease in dogs-myxomatous valve disease may be different compared to humans . Furthermore, an analysis of the heart transcriptomic profile showed a higher similarity between dog and mouse than between dog and human .
Moreover, levels of miR-423, which were elevated during left ventricular heart failure in humans ,, were equal in the healthy and diseased groups of dogs in our study. The results of the study conducted by Turatel et al. revealed that circulating miR-423_5p was not a useful biomarker for heart failure patients with a systemic right ventricle . This means that we cannot consider miRNAs identified in one kind of heart disease as potential biomarkers of other heart diseases. miRNA patterns differed not only between species but showed different expressions of the same circulating miRNA within the same species and disease but different patient characteristics.
An important aspect of the study is a significant age difference between dogs belonging to different experimental groups. This is a natural phenomenon because the more advanced heart disease is always related to older age. The question is whether the age differences between experimental groups influence the findings of this study. This could be quite an important question since it is known that miRNAs may play a role in the age-related changes in the skeletal muscle phenotype . In order to verify the presence of the age effect we decided to correlate the expression of miRNAs not only with the ACVIM stage of disease, but also with the age of the dogs. We created three groups consisting of the youngest, the oldest and middle age dogs. The results of this analysis (Additional file 1) did not show any significant changes in expression of miRNAs between these three different age groups. Thus we concluded that the cause of the changed expression of miR-30b and miR-133b in our study was the stage of the disease, not the age of the animals.
The utility of circulating miRNAs as biomarkers of many diseases has attracted considerable attention over recent years. However, it is also worthwhile to point out that the clinical application of miRNAs as biomarkers is still limited. One of the most significant obstacles is the difficulty concerning circulating miRNA normalization. Spiked in synthetic miRNA are widely used to normalize serum and plasma miRNA expression, but this approach does not include effects of pre-analytic variables on circulating miRNA measurement ,. That is why we decided to use a previously described housekeeping gene in our study–miR-16-5p ,. This miRNA is also a reference gene found on the Exiqon miRNome panels for plasma and is used as a control for hemolysis. Since the difficulties associated with hemolysis and platelet contamination of plasma samples are also significant, strategies were proposed to minimize the degree of red blood cell derived miRNA contamination, as well as minimize platelet contamination of plasma samples . It must also be pointed out that the sole presence of specific miRNAs is considered to be a biomarker of disease, infection or inflammation. There are no measuring units, there are no possibilities of disease grading based on miRNA concentration and there are no reference levels of specific miRNAs in specific tissues, both healthy and diseased. Despite all these deficiencies, miRNAs are still very promising tools in the process of disease diagnostics.
miR-30b could be a potential biomarker of ACVIM stage B heart failure in Dachshunds and miR-133b could be a potential biomarker of ACVIM stage C. The lack of expression of miR-208a and 208b in healthy dogs and dogs with heart disease and lack of significant changes in expression in the remaining 5 miRNAs which are potential biomarkers of heart diseases in humans proves that the findings in human medicine are not always directly reflected in veterinary medicine.
Blood test results of the study population in the control, ACVIM B and ACVIM C groups
ACVIM A–unaffected (mean ± SD)
ACVIM B (mean ± SD)
ACVIM C (mean ± SD)
272.5 ± 58.46
371.8 ± 95.81
472 ± 126.9**
1.039 ± 0.14
0.9625 ± 0.13
0.92 ± 0.09
7.568 ± 2.21
6.498 ± 1.28
10.98 ± 3.92
7.4 ± 0.45
7.168 ± 0.47
7.559 ± 0.81
16.71 ± 1.13
16.11 ± 1.20
17.19 ± 1.95
48.85 ± 3.12
48.02 ± 3.30
51.05 ± 5.91
66 ± 2.78
66.75 ± 3.20
67.43 ± 2.76
22.6 ± 0.89
22.46 ± 0.84
22.74 ± 0.63
34.2 ± 0.72
33.54 ± 0.68
33.71 ± 0.91
15.2 ± 0.58
15.36 ± 0.59
15.93 ± 0.61
Segmented neutrophils %
60.63 ± 5.63
70.63 ± 7.41
69.14 ± 7.08
Band neutrophil %
1 ± 1.60
2.88 ± 2.9
3.429 ± 3.64
7.375 ± 2.13
3.875 ± 3.27
6.857 ± 3.13
28.5 ± 6.30
21.75 ± 7.40
17.86 ± 6.91*
2.13 ± 1.25
0.63 ± 0.74
2.71 ± 1.89
0.375 ± 0.74
0.125 ± 0.35
0 ± 0
30.93 ± 3.82
30.53 ± 5.72
35 ± 24.32
28.13 ± 6.67
31.95 ± 6.46
27.97 ± 8.62
Total protein (g/l)
55 ± 3.51
55.13 ± 4.05
58.71 ± 2.69
207.1 ± 55.43
261.7 ± 39.9
267.7 ± 76.69
9.638 ± 0.32
9.763 ± 0.56
9.829 ± 0.58
4.639 ± 0.40
4.676 ± 0.39
4.854 ± 0.48
147.2 ± 2.43
150.2 ± 2.55*
148.9 ± 1.23
111.3 ± 2.258
112.3 ± 2.872
106.5 ± 2.867**
158.7 ± 30.05
183 ± 21.71
179.3 ± 28.75
Clinical and laboratory examinations
24 client-owned dogs were examined cardiologically during routine veterinary examinations. The clinical picture of the animals included: animal history, clinical examination and echocardiographic examination. Dogs with recognized heart disease were classified according the ACVIM (American College of Veterinary Internal Medicine) classification scheme as stage B (asymptomatic)–8 dogs, stage C (mild to moderate heart failure)–8 dogs and stage A–8 dogs (the control group of unaffected dogs).
A complete physical examination was performed, with a specific emphasis on thoracic auscultation. A standard transthoracic echocardiographic (TTE) examination was performed in all dogs with the Aloka 4000 ultrasound machine, equipped with a cardiology program and 2.5–7-megahertz (mHz) sector transducers according to previously published norms. All examinations were performed at rest without pharmacological restraint. Blood samples for basic morphologic and biochemical analysis as well as the transcription profile analysis were collected from the jugular or cephalic vein. The following laboratory tests were performed in all of the dogs: complete blood count, serum sodium, potassium, calcium, urea, creatinine, albumin, liver enzymes, and cholesterol. The dogs were classified into groups based on clinical signs of heart disease and the results of clinical examination. Echocardiography exam was used to diagnose endocardiosis.
Sample preparation and RNA isolation
Blood samples were collected in EDTA-K3 probes (Eqimed, Poland). Within 2 hours the samples were centrifuged at 1300 RCF for 10 minutes at room temperature. Afterwards the 250 μl plasma aquilots were transferred to 1.5 ml tubes (Eppendorf, Germany) and stored at–80°C until RNA isolation. Before extraction plasma samples were thawed on ice and centrifuged at 3000 × g for 5 min at a room temperature to pellet any debris and insoluble components. An aliquot of 200 μl of plasma per sample was transferred to a new microcentrifuge tube and the total RNA including small RNAs, were purified according to the protocol supplied with miRCURY™ RNA Isolation Kit–Biofluids (Exiqon, Denmark). In brief this procedures involves lyses of membranized particles using the provided Lysis Solution, precipitation of proteins and on column removing of any remaining impurities with provided Wash Solution. During isolation RNA Spike-in template mixture from miRCURY LNA™ Universal RT microRNA PCR, RNA Spike-in kit (Exiqon, Denmark) and carrier-RNA MS2 bacteriophage RNA (Roche Applied Science) were added as recommended by the manufacturer. RNA Spike-in kit was used for quality control of the RNA isolation and cDNA synthesis while MS2 RNA was used to minimize the loss of small RNA during isolation and the technical variation between replicates in further analyses. Total RNA was eluted by adding 50 μl of RNase-free water. The eluted RNA was stored at–80°C until further analysis.
cDNA synthesis and real-time RT-PCR
The sequences of miRNAs and accession numbers of the primers used in the study (Exiqon, Denmark)
hsa-miR-126-5p LNA™ PCR primer set, UniRT
hsa-miR-208a LNA™ PCR primer set, UniRT
hsa-miR-208b LNA™ PCR primer set, UniRT
hsa-miR-423-5p LNA™ PCR primer set, UniRT
hsa-miR-133b LNA™ PCR primer set, UniRT
hsa-miR-125b-5p LNA™ PCR primer set, UniRT
hsa-miR-30b-5p LNA™ PCR primer set, UniRT
hsa-miR-21-5p LNA™ PCR primer set, UniRT
hsa-miR-16-5p LNA™ PCR primer set, UniRT
hsa-miR-29b-3p LNA™ PCR primer set, UniRT
Initial qPCR data analysis was performed using MXPro software (Stratagene). Threshold cycle (Ct) for each miRNA was extracted by setting a threshold and automatically defined baseline. Furthermore, the data were analysed statistically using Exiqon GenEx qPCR analysis software (Exiqon, Denmark). miR-16-5p was used as a housekeeping gene ,. This miRNA is also a reference gene found on Exiqon miRNome panels for plasma and is used as a control for hemolysis. The fold change for each miRNA was calculated using ΔΔCt method . Data are shown as means ± SEM.
MH was responsible for all molecular and statistical analyses and preparation of the manuscript. MG was responsible for the clinical part of the study–patient examinations, disease staging and final draft of the text. MP-J assisted with patient clinical examinations and blood sampling. MJ designed the study protocol, coordinated the study and was involved in the final draft of the text. All authors read and approved the final manuscript.
The preparation of this paper was supported by grant no. UMO-2012/05/N/NZ5/02627 received from the Polish Ministry of Science.
- Donnelly KB: Cardiac valvular pathology: comparative pathology and animal models of acquired cardiac valvular diseases. Toxicol Pathol. 2008, 36: 204-217. 10.1177/0192623307312707.View ArticlePubMedGoogle Scholar
- Häggström J, Duelund Pedersen H, Kvart C: New insights into degenerative mitral valve disease in dogs. Vet Clin North Am Small Anim Pract. 2004, 34: 1209-1226. 10.1016/j.cvsm.2004.05.002.View ArticlePubMedGoogle Scholar
- Oyama MA, Levy RJ: Insights into serotonin signaling mechanisms associated with canine degenerative mitral valve disease. J Vet Intern Med. 2010, 24: 27-36. 10.1111/j.1939-1676.2009.0411.x.View ArticlePubMedGoogle Scholar
- Bartel DP: MicroRNAs: target recognition and regulatory functions. Cell. 2009, 136: 215-233. 10.1016/j.cell.2009.01.002.PubMed CentralView ArticlePubMedGoogle Scholar
- Chen X, Ba Y, Ma L, Cai X, Yin Y, Wang K, Guo J, Zhang Y, Chen J, Guo X, Li Q, Li X, Wang W, Zhang Y, Wang J, Jiang X, Xiang Y, Xu C, Zheng P, Zhang J, Li R, Zhang H, Shang X, Gong T, Ning G, Wang J, Zen K, Zhang J, Zhang CY: Characterization of microRNAs in serum: a novel class of biomarkers for diagnosis of cancer and other diseases. Cell Res. 2008, 18: 997-1006. 10.1038/cr.2008.282.View ArticlePubMedGoogle Scholar
- Lawrie CH, Gal S, Dunlop HM, Pushkaran B, Liggins AP, Pulford K, Banham AH, Pezzella F, Boultwood J, Wainscoat JS, Hatton CS, Harris AL: Detection of elevated levels of tumour-associated microRNAs in serum of patients with diffuse large B-cell lymphoma. Br J Haematol. 2008, 141: 672-675. 10.1111/j.1365-2141.2008.07077.x.View ArticlePubMedGoogle Scholar
- Mitchell PS, Parkin RK, Kroh EM, Fritz BR, Wyman SK, Pogosova-Agadjanyan EL, Peterson A, Noteboom J, O’Briant KC, Allen A, Lin DW, Urban N, Drescher CW, Knudsen BS, Stirewalt DL, Gentleman R, Vessella RL, Nelson PS, Martin DB, Tewari M: Circulating microRNAs as stable blood-based markers for cancer detection. Proc Natl Acad Sci U S A. 2008, 105: 10513-10518. 10.1073/pnas.0804549105.PubMed CentralView ArticlePubMedGoogle Scholar
- Arroyo JD, Chevillet JR, Kroh EM, Ruf IK, Pritchard CC, Gibson DF, Mitchell PS, Bennett CF, Pogosova-Agadjanyan EL, Stirewalt DL, Tait JF, Tewari M: Argonaute2 complexes carry a population of circulating microRNAs independent of vesicles in human plasma. Proc Natl Acad Sci U S A. 2011, 108: 5003-5008. 10.1073/pnas.1019055108.PubMed CentralView ArticlePubMedGoogle Scholar
- Creemers EE, Tijsen AJ, Pinto YM: Circulating microRNAs: novel biomarkers and extracellular communicators in cardiovascular disease?. Circ Res. 2012, 110: 483-495. 10.1161/CIRCRESAHA.111.247452.View ArticlePubMedGoogle Scholar
- Valadi H, Ekström K, Bossios A, Sjöstrand M, Lee JJ, Lötvall JO: Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat Cell Biol. 2007, 9: 654-659. 10.1038/ncb1596.View ArticlePubMedGoogle Scholar
- Vickers KC, Palmisano BT, Shoucri BM, Shamburek RD, Remaley AT: MicroRNAs are transported in plasma and delivered to recipient cells by high-density lipoproteins. Nat Cell Biol. 2011, 13: 423-433. 10.1038/ncb2210.PubMed CentralView ArticlePubMedGoogle Scholar
- Zernecke A, Bidzhekov K, Noels H, Shagdarsuren E, Gan L, Denecke B, Hristov M, Köppel T, Jahantigh MN, Lutgens E, Wang S, Olson EN, Schober A, Weber C: Delivery of microRNA-126 by apoptotic bodies induces CXCL12-dependent vascular protection. Sci Signal. 2009, 2: ra81-10.1126/scisignal.2000610.View ArticlePubMedGoogle Scholar
- van Schooneveld E, Wouters MC, Van der Auwera I, Peeters DJ, Wildiers H, Van Dam PA, Vergote I, Vermeulen PB, Dirix LY, Van Laere SJ: Expression profiling of cancerous and normal breast tissues identifies microRNAs that are differentially expressed in serum from patients with (metastatic) breast cancer and healthy volunteers. Breast Cancer Res. 2012, 14: R34-10.1186/bcr3127.PubMed CentralView ArticlePubMedGoogle Scholar
- Tang D, Shen Y, Wang M, Yang R, Wang Z, Sui A, Jiao W, Wang Y: Identification of plasma microRNAs as novel noninvasive biomarkers for early detection of lung cancer. Eur J Cancer Prev. 2013, 22: 540-548. 10.1097/CEJ.0b013e32835f3be9.View ArticlePubMedGoogle Scholar
- Weber DG, Johnen G, Bryk O, Jöckel KH, Brüning T: Identification of miRNA-103 in the cellular fraction of human peripheral blood as a potential biomarker for malignant mesothelioma–a pilot study. PLoS One. 2012, 7: e30221-10.1371/journal.pone.0030221.PubMed CentralView ArticlePubMedGoogle Scholar
- Zheng D, Haddadin S, Wang Y, Gu LQ, Perry MC, Freter CE, Wang MX: Plasma microRNAs as novel biomarkers for early detection of lung cancer. Int J Clin Exp Pathol. 2011, 4: 575-586.PubMed CentralPubMedGoogle Scholar
- Corsten MF, Dennert R, Jochems S, Kuznetsova T, Devaux Y, Hofstra L, Wagner DR, Staessen JA, Heymans S, Schroen B: Circulating MicroRNA-208b and MicroRNA-499 reflect myocardial damage in cardiovascular disease. Circ Cardiovasc Genet. 2010, 3: 499-506. 10.1161/CIRCGENETICS.110.957415.View ArticlePubMedGoogle Scholar
- Fukushima Y, Nakanishi M, Nonogi H, Goto Y, Iwai N: Assessment of plasma miRNAs in congestive heart failure. Circ J. 2011, 75: 336-340. 10.1253/circj.CJ-10-0457.View ArticlePubMedGoogle Scholar
- Wang GK, Zhu JQ, Zhang JT, Li Q, Li Y, He J, Qin YW, Jing Q: Circulating microRNA: a novel potential biomarker for early diagnosis of acute myocardial infarction in humans. Eur Heart J. 2010, 31: 659-666. 10.1093/eurheartj/ehq013.View ArticlePubMedGoogle Scholar
- Atkins C, Bonagura J, Ettinger S, Fox P, Gordon S, Haggstrom J, Hamlin R, Keene B, Luis-Fuentes V, Stepien R: Guidelines for the diagnosis and treatment of canine chronic valvular heart disease. J Vet Intern Med. 2009, 23: 1142-1150. 10.1111/j.1939-1676.2009.0392.x.View ArticlePubMedGoogle Scholar
- Goren Y, Kushnir M, Zafrir B, Tabak S, Lewis BS, Amir O: Serum levels of microRNAs in patients with heart failure. Eur J Heart Fail. 2012, 14: 147-154. 10.1093/eurjhf/hfr155.View ArticlePubMedGoogle Scholar
- Satoh M, Minami Y, Takahashi Y, Tabuchi T, Nakamura M: A cellular microRNA, let-7i, is a novel biomarker for clinical outcome in patients with dilated cardiomyopathy. J Card Fail. 2011, 17: 923-929. 10.1016/j.cardfail.2011.07.012.View ArticlePubMedGoogle Scholar
- van Rooij E, Sutherland LB, Thatcher JE, DiMaio JM, Naseem RH, Marshall WS, Hill JA, Olson EN: Dysregulation of microRNAs after myocardial infarction reveals a role of miR-29 in cardiac fibrosis. Proc Natl Acad Sci U S A. 2008, 105: 13027-13032. 10.1073/pnas.0805038105.PubMed CentralView ArticlePubMedGoogle Scholar
- Fichtlscherer S, De Rosa S, Fox H, Schwietz T, Fischer A, Liebetrau C, Weber M, Hamm CW, Röxe T, Müller-Ardogan M, Bonauer A, Zeiher AM, Dimmeler S: Circulating microRNAs in patients with coronary artery disease. Circ Res. 2010, 107: 677-684. 10.1161/CIRCRESAHA.109.215566.View ArticlePubMedGoogle Scholar
- Voellenkle C, van Rooij J, Cappuzzello C, Greco S, Arcelli D, Di Vito L, Melillo G, Rigolini R, Costa E, Crea F, Capogrossi MC, Napolitano M, Martelli F: MicroRNA signatures in peripheral blood mononuclear cells of chronic heart failure patients. Physiol Genomics. 2010, 42: 420-426. 10.1152/physiolgenomics.00211.2009.View ArticlePubMedGoogle Scholar
- Tijsen AJ, Creemers EE, Moerland PD, de Windt LJ, van der Wal AC, Kok WE, Pinto YM: MiR423-5p as a circulating biomarker for heart failure. Circ Res. 2010, 106: 1035-1039. 10.1161/CIRCRESAHA.110.218297.View ArticlePubMedGoogle Scholar
- Ji X, Takahashi R, Hiura Y, Hirokawa G, Fukushima Y, Iwai N: Plasma miR-208 as a biomarker of myocardial injury. Clin Chem. 2009, 55: 1944-1949. 10.1373/clinchem.2009.125310.View ArticlePubMedGoogle Scholar
- Bostjancic E, Zidar N, Stajer D, Glavac D: MicroRNAs miR-1, miR-133a, miR-133b and miR-208 are dysregulated in human myocardial infarction. Cardiology. 2010, 115: 163-169. 10.1159/000268088.View ArticlePubMedGoogle Scholar
- Steudemann C, Bauersachs S, Weber K, Wess G: Detection and comparison of microRNA expression in the serum of Doberman Pinschers with dilated cardiomyopathy and healthy controls. BMC Vet Res. 2013, 9: 12-10.1186/1746-6148-9-12.PubMed CentralView ArticlePubMedGoogle Scholar
- Mizuno H, Nakamura A, Aoki Y, Ito N, Kishi S, Yamamoto K, Sekiguchi M, Takeda S, Hashido K: Identification of muscle-specific microRNAs in serum of muscular dystrophy animal models: promising novel blood-based markers for muscular dystrophy. PLoS One. 2011, 6: e18388-10.1371/journal.pone.0018388.PubMed CentralView ArticlePubMedGoogle Scholar
- Gioia G, Mortarino M, Gelain ME, Albonico F, Ciusani E, Forno I, Marconato L, Martini V, Comazzi S: Immunophenotype-related microRNA expression in canine chronic lymphocytic leukemia. Vet Immunol Immunopathol. 2011, 142: 228-235. 10.1016/j.vetimm.2011.05.020.View ArticlePubMedGoogle Scholar
- Jensen SG, Lamy P, Rasmussen MH, Ostenfeld MS, Dyrskjot L, Orntoft TF, Andersen CL: Evaluation of two commercial global miRNA expression profiling platforms for detection of less abundant miRNAs. BMC Genomics. 2011, 12: 435-10.1186/1471-2164-12-435.PubMed CentralView ArticlePubMedGoogle Scholar
- Duisters RF, Tijsen AJ, Schroen B, Leenders JJ, Lentink V, van der Made I, Herias V, van Leeuwen RE, Schellings MW, Barenbrug P, Maessen JG, Heymans S, Pinto YM, Creemers EE: miR-133 and miR-30 regulate connective tissue growth factor: implications for a role of microRNAs in myocardial matrix remodeling. Circ Res. 2009, 104: 170-178. 10.1161/CIRCRESAHA.108.182535.View ArticlePubMedGoogle Scholar
- Chen M, Ma G, Yue Y, Wei Y, Li Q, Tong Z, Zhang L, Miao G, Zhang J: Downregulation of the miR-30 family microRNAs contributes to endoplasmic reticulum stress in cardiac muscle and vascular smooth muscle cells. Int J Cardiol. 2014, 173: 65-73. 10.1016/j.ijcard.2014.02.007.View ArticlePubMedGoogle Scholar
- Widera C, Gupta SK, Lorenzen JM, Bang C, Bauersachs J, Bethmann K, Kempf T, Wollert KC, Thum T: Diagnostic and prognostic impact of six circulating microRNAs in acute coronary syndrome. J Mol Cell Cardiol. 2011, 51: 872-875. 10.1016/j.yjmcc.2011.07.011.View ArticlePubMedGoogle Scholar
- Disatian S, Lacerda C, Orton EC: Tryptophan hydroxylase 1 expression is increased in phenotype-altered canine and human degenerative myxomatous mitral valves. J Heart Valve Dis. 2010, 19: 71-78.PubMedGoogle Scholar
- Zhao Y, Sheng Z, Huang J: A systematic analysis of heart transcriptome highlights divergent cardiovascular disease pathways between animal models and humans. Mol Biosyst. 2012, 8: 504-510. 10.1039/c1mb05415e.View ArticlePubMedGoogle Scholar
- Tutarel O, Dangwal S, Bretthauer J, Westhoff-Bleck M, Roentgen P, Anker SD, Bauersachs J, Thum T: Circulating miR-423_5p fails as a biomarker for systemic ventricular function in adults after atrial repair for transposition of the great arteries. Int J Cardiol. 2013, 167: 63-66. 10.1016/j.ijcard.2011.11.082.View ArticlePubMedGoogle Scholar
- McGregor RA, Poppitt SD, Cameron-Smith D: Role of microRNAs in the age-related changes in skeletal muscle and diet or exercise interventions to promote healthy aging in humans. Ageing Res Rev. 2014, S1568–1637 (14): 00051-00058.Google Scholar
- McDonald JS, Milosevic D, Reddi HV, Grebe SK, Algeciras-Schimnich A: Analysis of circulating microRNA: preanalytical and analytical challenges. Clin Chem. 2011, 57: 833-840. 10.1373/clinchem.2010.157198.View ArticlePubMedGoogle Scholar
- Cheng HH, Yi HS, Kim Y, Kroh EM, Chien JW, Eaton KD, Goodman MT, Tait JF, Tewari M, Pritchard CC: Plasma processing conditions substantially influence circulating microRNA biomarker levels. PLoS One. 2013, 8: e64795-10.1371/journal.pone.0064795.PubMed CentralView ArticlePubMedGoogle Scholar
- Kroh EM, Parkin RK, Mitchell PS, Tewari M: Analysis of circulating microRNA biomarkers in plasma and serum using quantitative reverse transcription-PCR (qRT-PCR). Methods. 2010, 50: 298-301. 10.1016/j.ymeth.2010.01.032.PubMed CentralView ArticlePubMedGoogle Scholar
- Xiao J, Jing ZC, Ellinor PT, Liang D, Zhang H, Liu Y, Chen X, Pan L, Lyon R, Liu Y, Peng LY, Liang X, Sun Y, Popescu LM, Condorelli G, Chen YH: MicroRNA-134 as a potential plasma biomarker for the diagnosis of acute pulmonary embolism. J Transl Med. 2011, 9: 159-10.1186/1479-5876-9-159.PubMed CentralView ArticlePubMedGoogle Scholar
- Kirschner MB, Edelman JJ, Kao SC, Vallely MP, van Zandwijk N, Reid G: The impact of hemolysis on cell-free microRNA biomarkers. Front Genet. 2013, 4: 94.PubMed CentralPubMedGoogle Scholar
- Livak KJ, Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2 − ΔΔCT method. Methods. 2001, 25: 402-408. 10.1006/meth.2001.1262.View ArticlePubMedGoogle Scholar
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