Identification of serum microRNAs with differential expression between dogs with splenic masses and healthy dogs with histologically normal spleens

Janet A. Grimes Department of Small Animal Medicine and Surgery, College of Veterinary Medicine, University of Georgia, Athens, GA 30602.

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Kelsey R. Robinson Department of Small Animal Medicine and Surgery, College of Veterinary Medicine, University of Georgia, Athens, GA 30602.

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Anna-Claire M. Bullington Department of Small Animal Medicine and Surgery, College of Veterinary Medicine, University of Georgia, Athens, GA 30602.

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Jennifer M. Schmiedt Department of Small Animal Medicine and Surgery, College of Veterinary Medicine, University of Georgia, Athens, GA 30602.
Department of Population Health, College of Veterinary Medicine, University of Georgia, Athens, GA 30602.

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Abstract

OBJECTIVE

To identify differential microRNA (miRNA) expression in dogs with splenic hemangiosarcoma, splenic hematoma, and histologically normal spleens.

ANIMALS

Dogs with splenic hemangiosarcoma (n = 10), splenic hematoma (n = 5), and histologically normal spleens (n = 5).

PROCEDURES

Splenic tissue and serum samples were collected from dogs with splenic masses (ie, hemangiosarcoma or hematoma samples) and healthy control dogs (ie, control samples), and total RNA was extracted. Reverse transcription quantitative real-time PCR was performed with 28 miRNAs associated with hemangiosarcoma, angiosarcoma, or associated genes. Differential expression analysis was performed.

RESULTS

Control tissue and serum samples had similar miRNA expression patterns, and hemangiosarcoma tissue and serum samples did not. Hemangiosarcoma serum samples had higher expression than hemangiosarcoma tissue for 13 miRNAs and lower expression for 1 miRNA. Control tissue and hemangiosarcoma tissue had varying expressions for 12 miRNAs, with 10 more highly expressed in control samples and 2 more highly expressed in hemangiosarcoma samples. Five miRNAs (miR-214-3p, miR-452, miR-494-3p, miR-497-5p, miR-543) had significantly different expression in serum between dogs with splenic masses (ie, hemangiosarcoma or hematoma) and serum of dogs with histologically normal spleens, with higher expression in the serum of dogs with splenic masses for all 5 miRNAs.

CONCLUSIONS AND CLINICAL RELEVANCE

5 circulating miRNAs were identified that distinguished dogs with splenic hemangiosarcoma or hematoma from those with histologically normal spleens. These 5 miRNAs had higher expression in dogs with splenic masses, indicating upregulation of these circulating miRNAs occurs in these splenic disease states. These miRNAs may be useful as a noninvasive screening tool that uses serum to identify dogs with splenic masses.

Abstract

OBJECTIVE

To identify differential microRNA (miRNA) expression in dogs with splenic hemangiosarcoma, splenic hematoma, and histologically normal spleens.

ANIMALS

Dogs with splenic hemangiosarcoma (n = 10), splenic hematoma (n = 5), and histologically normal spleens (n = 5).

PROCEDURES

Splenic tissue and serum samples were collected from dogs with splenic masses (ie, hemangiosarcoma or hematoma samples) and healthy control dogs (ie, control samples), and total RNA was extracted. Reverse transcription quantitative real-time PCR was performed with 28 miRNAs associated with hemangiosarcoma, angiosarcoma, or associated genes. Differential expression analysis was performed.

RESULTS

Control tissue and serum samples had similar miRNA expression patterns, and hemangiosarcoma tissue and serum samples did not. Hemangiosarcoma serum samples had higher expression than hemangiosarcoma tissue for 13 miRNAs and lower expression for 1 miRNA. Control tissue and hemangiosarcoma tissue had varying expressions for 12 miRNAs, with 10 more highly expressed in control samples and 2 more highly expressed in hemangiosarcoma samples. Five miRNAs (miR-214-3p, miR-452, miR-494-3p, miR-497-5p, miR-543) had significantly different expression in serum between dogs with splenic masses (ie, hemangiosarcoma or hematoma) and serum of dogs with histologically normal spleens, with higher expression in the serum of dogs with splenic masses for all 5 miRNAs.

CONCLUSIONS AND CLINICAL RELEVANCE

5 circulating miRNAs were identified that distinguished dogs with splenic hemangiosarcoma or hematoma from those with histologically normal spleens. These 5 miRNAs had higher expression in dogs with splenic masses, indicating upregulation of these circulating miRNAs occurs in these splenic disease states. These miRNAs may be useful as a noninvasive screening tool that uses serum to identify dogs with splenic masses.

Introduction

Cancer is the leading cause of death in adult dogs, accounting for 20% of deaths in 79% of breeds evaluated.1 Among cancers in dogs, hemangiosarcoma, a tumor of vascular endothelial origin, accounts for 12% to 21% of all mesenchymal tumors, approximately 66% of all malignant splenic masses, and is overrepresented in large-breed dogs such as Golden Retrievers, Labrador Retrievers, and German Shepherd Dogs.2 The prognosis for splenic hemangiosarcoma is poor, with a mortality rate approaching 100% in a matter of months despite intensive multimodal treatment.2

Although overall prognosis with hemangiosarcoma is poor, dogs with less advanced stage disease have improved survival rates, compared with dogs with more advanced stage disease.3 However, in most dogs with hemangiosarcoma, the diagnosis is made late in their disease course, with many dogs presenting with tumor rupture, massive hemorrhage into the abdomen, and micrometastasis or gross metastasis to other organs because of the lack of screening tests to allow for early identification of affected patients.3,4 Splenic hematoma is another common cause of splenic masses that can also be a life-threatening disease in dogs as a result of sudden blood loss following rupture.5,6,7 As with splenic hemangiosarcoma, the recommended treatment for splenic hematoma is splenectomy, which is curative. Currently, no blood tests can screen dogs for splenic masses, and other diagnostic tests are unable to differentiate the etiology of splenic masses prior to surgical removal. Although the presence of hemoperitoneum is more common in dogs with hemangiosarcoma, this clinical finding is neither a sensitive nor a specific indicator of hemangiosarcoma, as other splenic diseases can also result in hemoperitoneum.5,6,7 Fine-needle aspiration is also unrewarding because of blood contamination and poor exfoliation of the tumor, in addition to the risk of rupture and seeding of the tumor.8 Various other methods have been evaluated for diagnosis of hemangiosarcoma, including comparison of mass to splenic volume and splenic weight to body weight, contrast harmonic ultrasonography, CT, MRI, and blood testing including multiparameter flow cytometry and measurement of plasma vascular endothelial growth factor concentrations, but none have proven sensitive or sufficiently specific for definitive preoperative diagnosis.8,9,10,11,12,13 A need exists to identify biomarkers to allow for identification of hemangiosarcoma earlier than is currently possible. Earlier identification will allow for diagnosis at less advanced disease stages, leading to increased survival times.

A class of potential biomarkers includes miRNAs, which are 22- to 26-nucleotide-long RNAs that are known to regulate cellular responses and host pathways including malignancy.14,15 MicroRNAs affect posttranscriptional regulation via binding of mRNAs, either preventing their translation into proteins or causing their degradation. Substantial alteration of miRNA expression exists in malignant cells, compared with their nonmalignant counterparts, with cancer tissues exhibiting a specific miRNA signature reflecting their malignant state and features of that particular cancer and its progression.16,17,18 In addition to having altered tissue expression levels, miRNAs also have altered expression levels in the circulation of patients with cancer, compared with patients without cancer.19,20,21 Dysregulation of host processes has been shown to contribute to the pathogenesis of canine hemangiosarcoma, with several genes being implicated, including PTEN and those involved in the retinoblastoma protein pathway and expression of vascular endothelial growth factor.22,23,24 A previous study25 found alteration of miRNA expression in the splenic tissue among dogs with histologically normal spleens, dogs with hemangiosarcoma, and those with splenic nodular hyperplasia.

The objective of the present study was to identify miRNAs that differ between dogs with histologically normal spleens, dogs with splenic hemangiosarcoma, and dogs with splenic hematoma and to evaluate both tissue and serum miRNA expression. The hypothesis was that miRNA expression profiles in both tissue and serum would differentiate dogs with hemangiosarcoma, splenic hematoma, and histologically normal spleens.

Materials and Methods

Sample collection from dogs with splenic masses

Approval by the clinical research committee was obtained prior to collection of samples from patients with splenic masses. Patients with splenic masses were prospectively enrolled, and informed client consent was obtained. Serum samples (ie, hemangiosarcoma or hematoma serum samples) were obtained from whole blood samples collected prior to splenectomy. Blood samples were allowed to clot and were centrifuged, and serum was stored at –80°C. Only samples grossly free of hemolysis were used. Tissue samples (ie, hemangiosarcoma or hematoma tissue samples) were collected from within the visible masses within 30 minutes of splenectomy, flash frozen in liquid nitrogen, and stored at –80°C. Tissue samples adjacent to the areas sampled for tumor collection were submitted for histo-logic examination to confirm the diagnosis in the sampled area.

Sample collection from control dogs

Institutional animal care and use committee approval was obtained prior to collection of samples from healthy control dogs. Control dogs were undergoing abdominal surgery for an unrelated study. Serum samples (ie, control serum samples) were collected prior to surgery as already described for dogs with splenic masses. Splenic tissue samples (ie, control tissue samples) were obtained and stored as also already described for dogs with splenic masses. On histologic examination, splenic tissues from control dogs were confirmed as normal.

RNA isolation

Total RNA was isolated per the manufacturer's directions from seruma and splenicb samples. Briefly, 200 μL of serum or approximately 25 mg of splenic tissue was used for extraction. Lysis reagent was added to the sample and either homogenized for splenic samplesc or mixed by gently vortexing serum samples. Chloroform was used for phase separation, and 100% ethanol was used to precipitate RNA. Samples were washed per instructions on the minispin column and eluted with 14 or 40 μL of RNase-free water for serum and spleen samples, respectively. Splenic samples were analyzed for RNA purity via spectrophotometry.d Serum samples were not evaluated via spectrophotometry because of poor purity and low yield.26

Complementary DNA synthesis

Isolated RNA from splenic samples was diluted to 1 ng/μL prior to cDNA synthesis. The cDNA was synthesized with a cDNA synthesis kite with 2 μL of eluted RNA from samples. Steps included the poly(A) tailing reaction, adaptor ligation reaction, reverse transcription reaction, and miRNA amplification, all of which were performed according to kit instructions with the exception of increasing the miRNA amplification cycles from 14 to 18. All steps were performed on a thermocycler.f Undiluted cDNA was stored at –20°C until RT-qPCR evaluation.

RT-qPCR assays

Reverse transcription quantitative real-time PCR assays were performed for 28 miRNAs. These miRNAs were identified from miRNAs previously found to be associated with canine hemangiosarcoma and human angiosarcoma,25,27 along with miRNAs that were identified through a search of databasesg,h for genes shown to drive the pathogenesis of hemangiosarcoma and angiosarcoma.28,29 Reactions were performed with predesigned and validated primers,i all of which were human equivalents with confirmation of identical sequence to the canine miRNA.30 The cDNA for each sample was diluted at a ratio of 1:10 in RNase-free water prior to amplification, and reactions were run in volumes of 20 μL comprising 10 μL of master mix,j 1 μL of miRNA assay, 5 μL of cDNA, and 4 μL of RNase-free water. A real-time PCR detection systemk was used with cycling conditions as follows: step 1, 50°C for 2 minutes; step 2, 95°C for 20 seconds; step 3, 95°C for 3 seconds; step 4, 60°C for 30 seconds; and step 5, repeat steps 2 to 4 for 40 cycles. All reactions were performed in triplicate on a 96-well platel with an adhesive seal.m A cutoff value of < 40 was used for the quantification cycle.

Statistical analysis

Reverse transcription quantitative real-time PCR assay outputs were normalized to the reference miRNAs with methods for normalization to > 1 reference gene.31 Data were normalized and log transformed. Data were tested for normality with a Shapiro-Wilk test. A paired t test was used to compare miRNA expression between control tissue and control serum and also hemangiosarcoma tissue and hemangiosarcoma serum samples. A Student t test was used to compare miRNA expression between control tissue and hemangiosarcoma tissue samples. Serum miRNA expression among the 3 groups (control, hemangiosarcoma, hematoma) was compared with an ANOVA with Tukey honestly significant difference test for multiple comparisons. Patients were then grouped into diseased (hemangiosarcoma or hematoma) and nondiseased (control) states, receiver operating characteristic curves were created, and the area under the curve was calculated for the significance of each miRNA identified as significant on ANOVA testing. The Youden J statistic was used to determine the maximum cutoff for fold change for each miRNA on the receiver operating characteristic curves. Values of P < 0.05 were considered significant.

Results

Samples were obtained from 5 healthy control dogs (paired tissue and serum samples, n = 5), 10 dogs with splenic hemangiosarcoma (paired tissue and serum samples, 5; serum samples only, 5), and 5 dogs with splenic hematoma (serum samples only, 5). Healthy control dogs had a median age of 4.4 years (range, 4.4 to 6.4 years), and all 5 were sexually intact female mixed-breed dogs. Dogs with splenic hemangiosarcoma had a median age of 10.9 years (range, 6.8 to 12.1 years) and were castrated male (n = 8) or spayed female (2) dogs. Types and breeds of dogs with splenic hemangiosarcoma were as follows: mixed breed (n = 4), Golden Retriever (3), Labrador Retriever (2), and Australian Cattle Dog (1). Dogs with splenic hematoma had a median age of 8.0 years (range, 6.1 to 10.5 years) and were castrated male (n = 2), spayed female (2), or sexually intact male (1) dogs. Types and breeds of dogs with splenic hematoma were 1 each of American Pit Bull Terrier, German Shepherd Dog, Labrador Retriever, mixed breed, and Siberian Husky.

For the 10 dogs with hemangiosarcoma, 6 dogs had hemoperitoneum and 4 did not. Seven dogs had a single mass within the spleen, and 3 dogs had multiple masses. The median largest mass dimension was 9 cm (range, 1.8 to 20 cm). Five dogs had no evidence of metastatic disease. One dog had omental metastasis, and another had metastasis to the liver. One dog had suspected metastasis to a local lymph node, and 1 dog had a single nodule identified on thoracic radio-graphs. One dog had a concurrent hemangiosarcoma of the third eyelid. Two dogs had no known comorbidities. Comorbidities in the 8 remaining dogs included orthopedic disease (n = 3), dermatologic disease (2), undefined heart murmur (2), myxomatous mitral valve dysplasia (1), epilepsy (1), and cystolithiasis (1) with some dogs having > 1 comorbidity.

For the 5 dogs with hematoma, 1 had hemoperitoneum and 4 did not. Four dogs had a single mass within the spleen, and 1 dog had multiple masses. The median largest mass dimension was 10 cm (range, 8 to 20 cm). No dogs with hematoma had evidence of metastasis. Three dogs had no known comorbidities, and the remaining 2 dogs had a testicular seminoma (n = 1) or pulmonary hypertension (1).

Of 25 miRNAs evaluated, 21 miRNAs were successfully amplified (Table 1), 4 were not identified in the samples (ie, miR-134-5p, miR-136-5p, miR-758-3p, and miR-876-5p), and all 3 reference miRNAs (miR-103a-3p, miR-16-5p, and miR-93-5p) were successfully amplified (Supplementary Table S1, available at: avmajournals.avma.org/doi/suppl/10.2460/javma.82.8.659). Control tissue and serum samples had similar expression patterns (Table 2). Hemangiosarcoma tissue and serum samples had varying expression values for 14 of 21 miRNAs. Of these, 13 were more highly expressed in serum, compared with tissue, and 1 was more highly expressed in tissue, compared with serum. Control tissue and hemangiosarcoma tissue samples had varying expression values for 12 of 21 miRNAs. Ten were more highly expressed in control tissue than hemangiosarcoma tissue, and 2 were more highly expressed in hemangiosarcoma tissue than control tissue.

Table 1

Mean ± SD quantification cycle values for 21 successfully amplified miRNAs from splenic tissue and serum samples from healthy control dogs (n = 5 tissue, 5 serum), dogs with splenic hemangiosarcoma (5 tissue, 10 serum), and dogs with splenic hematoma (5 serum).

miRNA Control (Cq values) HSA (Cq values) HTA (Cq values)
Splenic tissue Serum Splenic tissue Serum Serum
126 20.4 ± 1.1 20.2 ± 1.1 21.2 ± 0.9 22.5 ± 1.2 21.2 ± 0.2
150—5p 20.2 ± 0.9 19.7 ± 1.0 21.5 ± 2.1 21.8 ± 1.3 20.6 ± 1.4
185–5p 24.7 ± 0.7 24.0 ± 0.6 25.2 ± 1.2 26.6 ± 2.3 24.2 ± 0.7
193a 25.8 ± 0.9 23.4 ± 1.0 25.5 ± 0.8 23.9 ± 1.5 23.2 ± 0.6
2l4—3p 22.2 ± 0.9 26.3 ± 0.5 22.4 ± 1.4 24.8 ± 1.8 23.1 ± 1.2
22 19.3 ± 0.8 19.5 ± 0.7 18.5 ± 0.8 19.6 ± 1.7 18.5 ± 1.3
26a-5p 17.4 ± 1.2 20.8 ± 1.1 19.2 ± 1.5 22.9 ± 2.3 20.1 ± 0.5
30lb-3p 36.8 ± 3.0 27.7 ± 1.8 30.5 ± 3.1 31.8 ± 2.7 28.5 ± 1.2
30e-3p 26.6 ± 0.6 26.9 ± 1.1 28.3 ± 2.6 29.8 ± 2.2 26.6 ± 0.6
326 24.2 ± 0.4 21.0 ± 1.4 24.9 ± 1.2 24.6 ± 2.3 22.5 ± 0.4
33b-5p 28.2 ± 0.8 30.1 ± 3.0 27.9 ± 1.2 30.5 ± 1 .8 30.2 ± 1.3
365–3p 22.7 ± 0.9 21.3 ± 1.6 23.2 ± 0.9 23.1 ± 1.7 21.5 ± 0.5
423–5p 21.2 ± 0.8 18.1 ± 0.8 20.9 ± 0.6 19.3 ± 1.5 18.2 ± 0.6
424–3p 28.1 ± 0.8 25.8 ± 0.8 28.0 ± 1.0 27.1 ± 1.5 25.8 ± 1.0
448 28.2 ± 1.5 27.3 ± 1.0 27.3 ± 1.9 28.2 ± 1.6 27.1 ± 1.1
452 27.0 ± 1.1 30.6 ± 1.1 29.1 ± 5.5 29.8 ± 2.6 27.6 ± 1.4
494–3p 35.5 ± 0.7 31.6 ± 1.1 29.4 ± 4.7 30.2 ± 0.9 28.0 ± 1.5
497–5p 19.2 ± 0.8 25.8 ± 1.1 20.5 ± 1.2 24.9 ± 1.9 23.5 ± 0.9
505–5p 26.5 ± 0.6 26.1 ± 1.1 28.3 ± 1.4 27.9 ± 1.9 26.0 ± 0.5
542–3p 35.2 ± 3.5 34.9 ± 3.1 31.2 ± 1.7 33.4 ± 2.2 33.0 ± 4.4
543 35.8 ± 2.6 34.0 ± 1.4 29.3 ± 1.8 33.2 ± 2.2 29.9 ± 1.2

Cq = Quantification cycle. HSA = Hemangiosarcoma. HTA = Hematoma.

Table 2

Mean ± SD relative gene expression values and comparison statistics for target miRNAs (n = 21) in tissue and serum samples from healthy control dogs (5) and dogs with splenic hemangiosarcoma (5 dogs with paired serum and tissue samples).

miRNA Control (RGE values) HSA (RGE values) P values*
Control (RGE values) HSA (RGE values) Control vs HSA (RGE values)
Splenic tissue Serum Splenic tissue Serum Tissue vs serum Tissue vs serum Tissue vs tissue
126 4.4 ± 0.5 4.4 ± 0.6 2.5 ± 1.5 4.7 ± 0.8 1.000 0.017 0.049
150–5p 3.7 ± 0.6 3.7 ± 0.5 1.4 ± 1.7 3.5 ± 1.1 1.000 0.065 0.030
185–5p 2.5 ± 0.3 2.5 ± 0.4 0.9 ± 0.9 2.5 ± 0.4 1.000 0.015 0.015
193a 1.7 ± 0.5 1.7 ± 0.4 1.0 ± 1.0 3.8 ± 0.8 1.000 0.002 0.187
2l4–3p 1.6 ± 0.2 1.6 ± 0.9 0.4 ± 0.4 5.6 ± 2.3 1.000 0.011 0.001
22 1.7 ± 0.5 1.7 ± 0.7 1.4 ± 0.8 4.6 ± 0.9 1.000 0.002 0.448
26a-5p 3.6 ± 0.9 3.6 ± 0.5 0.7 ± 0.6 3.9 ± 1.0 1.000 0.004 0.001
30lb-3p 7.0 ± 3.1 7.1 ± 1.0 12.4 ± 2.0 5.7 ± 1.6 0.915 0.016 0.029
30e-3p 5.5 ± 0.3 5.5 ± 0.4 2.7 ± 1.7 5.1 ± 1.8 1.000 0.165 0.018
326 3.2 ± 0.3 3.2 ± 0.7 1.4 ± 0.7 2.5 ± 1.5 1.000 0.284 0.003
33b-5p 5.2 ± 0.5 5.2 ± 3.7 4.4 ± 0.8 8.1 ± 0.6 0.939 0.028 0.105
365–3p 2.3 ± 0.5 2.3 ± 0.9 0.8 ± 0.9 3.2 ± 0.8 1.000 0.002 0.015
423–5p 2.2 ± 0.3 2.2 ± 0.2 1.4 ± 1.2 3.4 ± 0.5 1.000 0.017 0.213
424–3p 3.3 ± 0.6 3.3 ± 1.5 2.3 ± 1.9 4.7 ± 0.9 1.000 0.046 0.313
448 2.9 ± 0.8 2.9 ± 1.0 2.8 ± 2.9 4.5 ± 1.7 1.000 0.138 0.955
452 11.4 ± 0.9 11.4 ± 1.4 8.3 ± 5.2 15.5 ± 2.9 1.000 0.043 0.254
494–3p 2.2 ± 1.0 2.4 ± 1.2 7.5 ± 5.1 5.6 ± 1.8 0.753 0.491 0.080
497–5p 4.5 ± 0.3 4.5 ± 1.3 2.2 ± 1.4 7.9 ± 1.7 1.000 0.001 0.019
505–5p 3.3 ± 0.3 3.3 ± 0.4 0.4 ± 0.5 3.9 ± 1.0 1.000 0.003 < 0.001
542–3p 5.1 ± 3.5 5.1 ± 3.2 8.1 ± 2.0 9.1 ± 1.3 1.000 0.130 0.148
543 4.3 ± 3.2 4.3 ± 1.8 9.8 ± 2.1 7.5 ± 1.4 0.920 0.290 0.034

Values of P < 0.05 are considered significant.

RGE = Relative gene expression (normalized, log transformed).

See Table 1 for remainder of key.

Five miRNAs had significantly different expression in serum among the 3 groups (Table 3), with all 5 having lower expression in control serum than serum from dogs with either hemangiosarcoma or hematoma.

Table 3

Mean ± SD relative gene expression values and significant results for ANOVA analysis of serum samples from healthy control dogs (n = 5), dogs with splenic hemangiosarcoma (10), and dogs with splenic hematoma (5).

miRNA Control (RGE values) HSA (RGE values) HTA (RGE values) P value* AUC Cutoff (RGE values) Sensitivity Specificity
2l4–3p 1.6 ± 0.9 5.3 ± 1.8 5.4 ± 1.8 0.001 0.973 3.9 0.87 0.87
452 11.4 ± 1.4 14.6 ± 2.6 15.0 ± 1.8 0.033 0.907 12.1 0.87 0.67
494–3p 2.4 ± 1.2 5.6 ± 1.6 6.7 ± 1.4 < 0.001 0.971 4.5 0.86 0.86
497–5p 4.5 ± 1.3 7.3 ± 2.0 7.4 ± 1.6 0.023 0.908 6.6 0.69 0.69
543 4.3 ± 1.8 7.2 ± 1.8 9.0 ± 1.5 0.002 0.954 7.3 0.85 0.85

AUC = Area under the curve.

See Tables 1 and 2 for remainder of key.

Discussion

Circulating miRNAs have been successfully used in human medicine to differentiate healthy from disease states,19,20,21 and the objective of the present study was to determine whether similar methods could be used to differentiate dogs with histologically normal spleens, dogs with splenic hemangiosarcoma, and dogs with splenic hematoma. Five circulating miRNAs (miR-214-3p, miR-452, miR-494-3p, miR-497-5p, and miR-543) were identified that distinguished healthy control dogs from dogs with splenic masses. These 5 miRNAs had higher expression in dogs with either hemangiosarcoma or hematoma than healthy control dogs, indicating upregulation of these circulating miRNAs occurs in these splenic disease states. Of the 5 miRNAs that had differential expression between control dogs and dogs with hemangiosarcoma or hematoma, 2 have been studied in canine hemangiosarcoma previously.

In cell lines and clinical tissue samples of dogs with hemangiosarcoma in 1 study,32 miR-214 was found to be downregulated. Another study found that miR-214 was upregulated in microvesicles from cell lines of hemangiosarcoma and was also upregulated in the plasma of dogs with hemangiosarcoma.27 This is consistent with the findings of the present study, in which miR-214 expression was significantly higher in the serum than in the tissues of dogs with hemangiosarcoma. In 1 study,33 miR-214 was shown to be anti-angiogenic in mice through its targeting of quaking, a protein required for vascular development. It has also been shown to act as a tumor suppressor in canine hemangiosarcoma by targeting p53 through modulation of COP1 protein expression.32 Other studies have demonstrated oncogenic properties of miR-214. One group found that blockade of miR-214 reduced tumor progression and metastasis in mice.34 Another demonstrated that overexpression of miR-214 led to down-regulation of PTEN in mice.35 Inactivation of PTEN has been demonstrated in canine hemangiosarcoma, with loss of the PTEN protein leading to increased cell proliferation and reduced apoptosis through the phosphoinositide 3-kinase/Akt pathway.22 These conflicting roles identified for miR-214 may relate to its location, being either intra- or extracellular,27 and further work needs to be done to determine the specific action of miR-214 in canine splenic masses.

An miRNA-sequencing study found that miR-452 was associated with splenic hemangiosarcoma, consistent with the findings in the present study.25 Although miR-452 has not been investigated thoroughly in canine splenic masses, it has been shown to promote tumor progression in human hepatocellular carcinoma through increased expression of epidermal growth factor receptor and Akt.36 Akt is a prosurvival protein and is an end product in the phosphoinositide-3-kinase pathway. Reduction of PTEN protein leads to phosphorylation of Akt and a prosurvival state. In canine hemangiosarcoma cell lines, epidermal growth factor receptor has been shown to support anchorage-independent growth, which is important for metastasis.37 Additional investigation needs to be performed to determine whether miR-452 acts through these same pathways in canine splenic masses.

In humans, miR-497-5p is associated with malignancy in angiosarcoma and has been shown to directly target KCa3.1, a calcium-activated potassium channel.38 Such potassium channels have been previously shown to regulate activity of cancer cells, increasing cell proliferation and metastasis. Downregulation of miR-497-5p was found in human angiosarcoma tissue samples, compared with capillary hemangioma samples.38 Similarly, expression of this miRNA in the present study was lower in hemangiosarcoma tissues, compared with control tissues; however, miR-497-5p had higher expression in the serum of dogs with hemangiosarcoma, compared with tissue samples, indicating that diseased tissue expression and serum levels of miRNAs may not always correspond.

Although miR-494-3p and miR-543 have not been previously associated with hemangiosarcoma in dogs or angiosarcoma in humans, they have been associated with other cancer types. In human glioma and prostate cancer, miR-494-3p is overexpressed.20,39 In human non–small cell lung cancer and colorectal cancer, miR-543 has been shown to promote angio-genesis and tumorigenesis.40,41 Although their specific mechanism of action in canine splenic masses is unknown, both miR-494 and miR-543 have been shown to target PTEN directly, which is known to be downregulated in canine hemangiosarcoma.22,41,42 The results of the present study indicated that the up-regulation of these miRNAs is also present in dogs with splenic masses.

These 5 miRNAs were able to differentiate splenic hemangiosarcoma and hematoma from histologically normal spleens with high specificity and sensitivity. None of the miRNAs were differentially expressed between the serum of animals with splenic hemangiosarcoma and hematoma. These are 2 of the most common diagnoses for splenic masses, and although prognosis is substantially different between them, the initial treatment of splenectomy is the same. These miRNAs may be a useful tool for screening dogs to identify splenic masses at an earlier stage of disease, thereby decreasing morbidity and mortality rates by preventing splenic rupture and hemoperitoneum for both conditions and improving treatment outcomes for dogs with hemangiosarcoma with earlier diagnosis.

When comparing control tissue to hemangiosarcoma tissue, differential expression for 12 of 21 miRNAs evaluated was found, with control tissue having higher expression of 10 of 12 miRNAs. Further examination into the significance of these differences in the healthy versus diseased state may provide more insight into the role of these miRNAs in hemangiosarcoma. No differences were found in miRNA expression between control tissue and control serum, indicating that for the 21 miRNAs evaluated, expression between the spleen and the serum was stable.

For dogs with hemangiosarcoma, 14 of 21 miRNAs had differential expression between tissue and serum, with 13 being upregulated in the serum, compared with the tissues. This is interesting in that miRNAs present in serum may be regulated by more complex biological processes. Hemangiosarcoma is a tumor of the vascular endothelium and is histologically characterized by irregular vascular channels, papillary structures that bulge into these irregular vascular channels, and frequent anaplasia.43 These vascular abnormalities may explain why these miRNAs are increased in the serum, as they may be more likely to be shed into circulation either from circulating tumor cells or as cell-free miRNAs.44 Another explanation lies in the export of miRNAs from cells, either in extracellular vesicles or associated with Ago proteins.45,46,47 Extracellular vesicles and miRNA-Ago protein complexes are exported from cells into circulation from both active cells and those undergoing apoptosis or necrosis. Extracellular vesicles have been shown to promote cancer metastasis through multiple mechanisms, including roles in angiogenesis,45 and it is possible that the miRNAs identified here may have a therapeutic potential in future treatment of canine splenic masses.

Four miRNAs were not successfully amplified in the samples from the present study. Two of these miRNAs were found via a database search,g and the other 2 were identified in an miRNA-sequencing study.25,28 Despite a 100% sequence match between the human and canine miRNAs, it is possible that cross-binding or technical issues with the RT-qPCR assay occurred. Further work should be done to confirm that these sequences match between humans and dogs and to determine whether these miRNAs exist in the canine spleen.

Although the present study is limited by the small number of dogs included, 5 miRNAs were identified with higher expression in the serum of animals with splenic masses with high sensitivity and specificity. Larger sample size may lead to significant differences between expression patterns of miRNAs between hemangiosarcoma and hematoma samples. Future studies may also be used to determine whether other differences, including tumor rupture and hemoperitoneum or the receipt of a blood transfusion, affect miRNA expression. It is possible that the splenic tissue samples obtained were not reflective of hemangiosarcoma as a result of sampling location, although tissue adjacent to the samples taken for the study were submitted for histologic examination to ensure collection of hemangiosarcoma tumor cells to control for this variable. Additionally, influence of age, sex, breed, comorbidities, and other clinical factors on the results of differential miRNA expression is unknown. Other miRNAs that were not assessed in the present study may also be involved in the pathogenesis of splenic masses. Use of other methods of analysis such as microarray or RNA sequencing may be useful to identify additional miRNA that may be involved in the pathogenesis of canine splenic masses.

The 5 miRNAs identified in the present study distinguished healthy dogs from those with other splenic masses via serum evaluation, which has not been previously done. Validation testing of these 5 miRNAs to detect splenic masses is the next step in developing a minimally invasive screening test and is a future goal of this work with a larger number of dogs included. Such a minimally invasive diagnostic test is needed for the diagnosis of splenic masses in dogs. Abdominal ultra-sonography is not routinely used to screen older dogs unless symptoms are present pertaining to abdominal disease, and smaller lesions may be missed, whereas a blood test could be more easily incorporated into regular screening. Earlier intervention would be helpful to prevent tumor rupture, hemoperitoneum, and subsequent hypovolemic shock and the need for emergency surgery in dogs with hemangiosarcoma or hematoma. In the case of hemangiosarcoma, earlier intervention may also improve prognosis. With additional testing, a blood test with a panel of miRNA such as those identified here could be added to routine blood testing of older dogs to detect the presence of splenic masses earlier than may otherwise be possible.

Acknowledgments

Funding for this work was supported by a New Faculty Research Grant from the University of Georgia College of Veterinary Medicine Office of Research and Faculty and Graduate Affairs. Funding sources did not have any involvement in the study design, data analysis and interpretation, or writing and publication of the manuscript.

Abbreviations

miRNA

MicroRNA

PTEN

Phosphatase and tensin homolog

RT-qPCR

Reverse transcription quantitative real-time PCR

Footnotes

a.

miRNeasy serum/plasma kit, Qiagen, Hilden, Germany.

b.

miRNeasy kit, Qiagen, Hilden, Germany.

c.

Omni Tissue Homogenizer, Omni International, Kennesaw, Ga.

d.

Nanodrop 2000, Thermo Fisher Scientific, Waltham, Mass.

e.

TaqMan Advanced miRNA cDNA, Applied Biosystems, Thermo Fisher Scientific, Waltham, Mass.

f.

MJ Research PTC-200 Thermal Cycler, Bio-Rad, Hercules, Calif.

g.

TargetScan 7.2 [database online]. Cambridge, Mass: Whitehead Institute for Biomedical Research, 2018. Available at: targetscan.org. Accessed Mar 6, 2018.

h.

miRWalk [database online]. Heidelberg, Baden-Württemberg, Germany: University of Heidelberg, 2018. Available at: mirwalk.umm.uni-heidelberg.de. Accessed Mar 6, 2018.

i.

TaqMan Advanced MicroRNA Assays, Applied Biosystems, Thermo Fisher Scientific, Waltham, Mass.

j.

TaqMan Fast Advanced Master Mix, Applied Biosystems, Thermo Fisher Scientific, Waltham, Mass.

k.

CFX96 Touch Real-Time PCR Detection System, Bio-Rad, Calif.

l.

Hard-Shell PCR Plate, Bio-Rad, Hercules, Calif.

m.

Microseal B adhesive seal, Bio-Rad, Hercules, Calif.

References

  • 1.

    Fleming JM, Creevy KE, Promislow DE. Mortality in North American dogs from 1984 to 2004: an investigation into age-, size-, and breed-related causes of death. J Vet Intern Med 2011;25:187198.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 2.

    Thamm DH. Miscellaneous tumors: hemangiosarcoma. In: Withrow SJ, Vail DM, Page RL, eds. Withrow and MacEwen's small animal clinical oncology. 5th ed. St Louis: Elsevier/Saunders, 2013;679687.

    • Search Google Scholar
    • Export Citation
  • 3.

    Sorenmo KU, Baez JL, Clifford CA, et al. Efficacy and toxicity of a dose-intensified doxorubicin protocol in canine hemangiosarcoma. J Vet Intern Med 2004;18:209213.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 4.

    Ogilvie GK, Powers BE, Mallinckrodt CH, et al. Surgery and doxorubicin in dogs with hemangiosarcoma. J Vet Intern Med 1996;10:379384.

  • 5.

    Aronsohn MG, Dubiel B, Roberts B, et al. Prognosis for acute nontraumatic hemoperitoneum in the dog: a retrospective analysis of 60 cases (2003–2006). J Am Anim Hosp Assoc 2009;45:7277.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 6.

    Hammond TN, Pesillo-Crosby SA. Prevalence of hemangiosarcoma in anemic dogs with a splenic mass and hemoperitoneum requiring a transfusion: 71 cases (2003–2005). J Am Vet Med Assoc 2008;232:553558.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 7.

    Prymak C, McKee LJ, Goldschmidt MH, et al. Epidemiologic, clinical, pathologic, and prognostic characteristics of splenic hemangiosarcoma and splenic hematoma in dogs: 217 cases (1985). J Am Vet Med Assoc 1988;193:706712.

    • Search Google Scholar
    • Export Citation
  • 8.

    Mallinckrodt MJ, Gottfried SD. Mass-to-splenic volume ratio and splenic weight as a percentage of body weight in dogs with malignant and benign splenic masses: 65 cases (2007– 2008). J Am Vet Med Assoc 2011;239:13251327.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 9.

    O'Brien RT, Iani M, Matheson J, et al. Contrast harmonic ultrasound of spontaneous liver nodules in 32 dogs. Vet Radiol Ultrasound 2004;45:547553.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 10.

    Fife WD, Samii VF, Drost WT, et al. Comparison between malignant and nonmalignant splenic masses in dogs using contrast-enhanced computed tomography. Vet Radiol Ultra-sound 2004;45:289297.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 11.

    Clifford CA, Pretorius ES, Weisse C, et al. Magnetic resonance imaging of focal splenic and hepatic lesions in the dog. J Vet Intern Med 2004;18:330338.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 12.

    Lamerato-Kozicki AR, Helm K, Modiano JF. Early detection of hemangiosarcoma, in Proceedings. 23rd Annu Forum Am Coll Vet Intern Med 2005. Available at: vin.com Accessed Jan 4 2013.

    • Search Google Scholar
    • Export Citation
  • 13.

    Clifford CA, Hughes D, Beal MW, et al. Plasma vascular endothelial growth factor concentrations in healthy dogs and dogs with hemangiosarcoma. J Vet Intern Med 2001;15:131 135.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 14.

    Esquela-Kerscher A, Slack FJ. Oncomirs—microRNAs with a role in cancer. Nat Rev Cancer 2006;6:259269.

  • 15.

    Fazi F, Nervi C. MicroRNA: basic mechanisms and transcriptional regulatory networks for cell fate determination. Cardiovasc Res 2008;79:553561.

  • 16.

    Lu J, Getz G, Miska EA, et al. MicroRNA expression profiles classify human cancers. Nature 2005;435:834838.

  • 17.

    Rosenfeld N, Aharonov R, Meiri E, et al. MicroRNAs accurately identify cancer tissue origin. Nat Biotechnol 2008;26:462 469.

  • 18.

    Wijnhoven BP, Michael MZ, Watson DI. MicroRNAs and cancer. Br J Surg 2007;94:2330.

  • 19.

    Zöller M. Pancreatic cancer diagnosis by free and exosomal miRNA. World J Gastrointest Pathophysiol 2013;4:7490.

  • 20.

    Cai B, Peng JH. Increased expression of miR-494 in serum of patients with prostate cancer and its potential diagnostic value. Clin Lab 2019;65.

    • Search Google Scholar
    • Export Citation
  • 21.

    Mitchell PS, Parkin RK, Kroh EM, et al. Circulating microRNAs as stable blood-based markers for cancer detection. Proc Natl Acad Sci U S A 2008;105:1051310518.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 22.

    Dickerson EB, Thomas R, Fosmire SP, et al. Mutations of phosphatase and tensin homolog deleted from chromosome 10 in canine hemangiosarcoma. Vet Pathol 2005;42:618632.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 23.

    Yonemaru K, Sakai H, Murakami M, et al. The significance of p53 and retinoblastoma pathways in canine hemangiosarcoma. J Vet Med Sci 2007;69:271278.

  • 24.

    Yonemaru K, Sakai H, Murakami M, et al. Expression of vascular endothelial growth factor, basic fibroblast growth factor, and their receptors (flt-1, flk-1, and flg-1) in canine vascular tumors. Vet Pathol 2006;43:971980.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 25.

    Grimes JA, Prasad N, Levy S, et al. A comparison of microRNA expression profiles from splenic hemangiosarcoma, splenic nodular hyperplasia, and normal spleens of dogs. BMC Vet Res 2016;12:272.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 26.

    Qiagen. Guidelines for profiling biofluid miRNAs. Available at: go.qiagen.com/Guidelines-for-Profiling-Biofluid-miRNAs. Accessed Sep 18, 2020.

    • Search Google Scholar
    • Export Citation
  • 27.

    Heishima K, Mori T, Ichikawa Y, et al. MicroRNA-214 and microRNA-126 are potential biomarkers for malignant endothelial proliferative diseases. Int J Mol Sci 2015;16:25377 25391.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 28.

    Agarwal V, Bell GW, Nam JW, et al. Predicting effective microRNA target sites in mammalian mRNAs. eLife 2015;4:e05005.

  • 29.

    Dweep H, Gretz N. miRWalk2.0: a comprehensive atlas of microRNA-target interactions. Nat Methods 2015;12:697.

  • 30.

    Kozomara A, Birgaoanu M, Griffiths-Jones S. miRBase: from microRNA sequences to function. Nucleic Acids Res 2019;47:D155D162.

  • 31.

    Vandesompele J, De Preter K, Pattyn F, et al. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 2002;3:RESEARCH0034.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 32.

    Heishima K, Mori T, Sakai H, et al. MicroRNA-214 promotes apoptosis in canine hemangiosarcoma by targeting the COP1-p53 axis. PLoS One 2015;10:e0137361.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 33.

    van Mil A, Grundmann S, Goumans MJ, et al. MicroRNA-214 inhibits angiogenesis by targeting Quaking and reducing angiogenic growth factor release. Cardiovasc Res 2012;93:655665.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 34.

    Dettori D, Orso F, Penna E, et al. Therapeutic silencing of miR-214 inhibits tumor progression in multiple mouse models. Mol Ther 2018;26:20082018.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 35.

    Zhao C, Sun W, Zhang P, et al. miR-214 promotes osteoclastogenesis by targeting Pten/PI3k/Akt pathway. RNA Biol 2015;12:343353.

  • 36.

    Tang H, Zhang J, Yu Z, et al. Mir-452–3p: a potential tumor promoter that targets the CPEB3/EGFR axis in human hepatocellular carcinoma. Technol Cancer Res Treat 2017;16:11361149.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 37.

    Thamm DH, Dickerson EB, Akhtar N, et al. Biological and molecular characterization of a canine hemangiosarcoma-derived cell line. Res Vet Sci 2006;81:7686.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 38.

    Chen Y, Kuang D, Zhao X, et al. miR-497–5p inhibits cell proliferation and invasion by targeting KCa3.1 in angiosarcoma. Oncotarget 2016;7:5814858161.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 39.

    Zheng D, Chen D, Lin F, et al. LncRNA NNT-AS1 promote glioma cell proliferation and metastases through miR-494–3p/PRMT1 axis. Cell Cycle 2020;19:16211631.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 40.

    Wang D, Cai L, Tian X, et al. MiR-543 promotes tumorigenesis and angiogenesis in non-small cell lung cancer via modulating metastasis associated protein 1. Mol Med 2020;26:44.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 41.

    Liu J, Ke F, Chen T, et al. MicroRNAs that regulate PTEN as potential biomarkers in colorectal cancer: a systematic review. J Cancer Res Clin Oncol 2020;146:809820.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 42.

    Su S, Luo D, Liu X, et al. miR-494 up-regulates the PI3K/Akt pathway via targeting PTEN and attenuates hepatic ischemia/reperfusion injury in a rat model. Biosci Rep 2017;37:BSR20170798.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 43.

    Gamlem H, Nordstoga K. Canine vascular neoplasia—histo-logic classification and inmunohistochemical analysis of 221 tumours and tumour-like lesions. APMIS Suppl 2008;125:19 40.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 44.

    Mostert B, Sieuwerts AM, Martens JW, et al. Diagnostic applications of cell-free and circulating tumor cell-associated miRNAs in cancer patients. Expert Rev Mol Diagn 2011;11:259275.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 45.

    Kogure A, Yoshioka Y, Ochiya T. Extracellular vesicles in cancer metastasis: potential as therapeutic targets and materials. Int J Mol Sci 2020;21:4463.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 46.

    Tiberio P, Callari M, Angeloni V, et al. Challenges in using circulating miRNAs as cancer biomarkers. BioMed Res Int 2015;2015:731479.

  • 47.

    Turchinovich A, Weiz L, Langheinz A, et al. Characterization of extracellular circulating microRNA. Nucleic Acids Res 2011;39:72237233.

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