MicroRNA has been suspected to be generally involved in carcinogenesis since
their first description. A first study supported this assumption for canine
mammary tumors when miRNA expression was compared to normal gland. The present
study extends these results by comparing the expression of 16 microRNA (miRNA)
and 4 small nucleolar RNA (snoRNA) in tumors of different malignancy, for
example, adenomas, nonmetastasizing and metastasizing carcinomas as well as
lymph node metastases, with each other and with normal mammary gland. All
neoplastic tissues differed in their miR-210 expression levels from normal
gland. While metastatic cells differed in their expression of mir-29b,
miR-101, mir-125a, miR-143, and miR-145 from primary tumors, the comparison of
miRNA expression in primary tumors of different malignancy failed to reveal
significant differences except for a significant downregulation of mir-125a in
metastasizing carcinomas when compared to adenomas

Deetzen, Marie-Charlotte

Gruber, Achim Dieter

Klopfleisch, Robert

Schmeck, B. T.

ISRN Veterinary Science

PubMed

MicroRNA has been suspected to be generally involved in carcinogenesis since
their first description. A first study supported this assumption for canine
mammary tumors when miRNA expression was compared to normal gland. The present
study extends these results by comparing the expression of 16 microRNA (miRNA)
and 4 small nucleolar RNA (snoRNA) in tumors of different malignancy, for
example, adenomas, nonmetastasizing and metastasizing carcinomas as well as
lymph node metastases, with each other and with normal mammary gland. All
neoplastic tissues differed in their miR-210 expression levels from normal
gland. While metastatic cells differed in their expression of mir-29b,
miR-101, mir-125a, miR-143, and miR-145 from primary tumors, the comparison of
miRNA expression in primary tumors of different malignancy failed to reveal
significant differences except for a significant downregulation of mir-125a in
metastasizing carcinomas when compared to adenomas

Deetzen, Marie-Charlotte von

Institutional Repository of the Freie Universität Berlin

Research ArticleMalignancy Associated MicroRNA Expression Changes in CanineMammary Cancer of Different MalignanciesMarie-Charlotte von Deetzen,1 Bernd T. Schmeck,2Achim D. Gruber,1 and Robert Klopfleisch11 Institute of Veterinary Pathology, Freie Universita¨t Berlin, Robert-von-Ostertag-Straße 15, 14163 Berlin, Germany2Molecular Pulmonology, German Center for Lung Research, Philipps Universita¨t Marburg, Hans-Meerwein-Straße 2,35043 Marburg, GermanyCorrespondence should be addressed to Robert Klopfleisch; robert.klopfleisch@fu-berlin.deReceived 11 February 2014; Accepted 18 March 2014; Published 2 April 2014Academic Editors: M. H. Kogut and A. ShamayCopyright © 2014 Marie-Charlotte von Deetzen et al. This is an open access article distributed under the Creative CommonsAttribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work isproperly cited.MicroRNA has been suspected to be generally involved in carcinogenesis since their first description. A first study supported thisassumption for canine mammary tumors when miRNA expression was compared to normal gland. The present study extendsthese results by comparing the expression of 16 microRNA (miRNA) and 4 small nucleolar RNA (snoRNA) in tumors of differentmalignancy, for example, adenomas, nonmetastasizing and metastasizing carcinomas as well as lymph node metastases, with eachother and with normal mammary gland. All neoplastic tissues differed in their miR-210 expression levels from normal gland.While metastatic cells differed in their expression of mir-29b, miR-101, mir-125a, miR-143, and miR-145 from primary tumors,the comparison of miRNA expression in primary tumors of different malignancy failed to reveal significant differences except fora significant downregulation of mir-125a in metastasizing carcinomas when compared to adenomas.1. IntroductionMicroRNA (miRNA) is an evolutionarily conserved, non-coding, but regulatory RNA species of approximately 22nucleotides in length. It plays a crucial role in variousphysiological and pathological processes by regulating geneexpression posttranscriptionally. miRNA binds to messengerRNA (mRNA) and thereby induces a sequence-dependingmRNA degradation or translational repression [1–3]. Aderegulation of miRNA is associated with a wide variety ofpathologic states including carcinogenesis [4]. Nevertheless,in many cases the specific function of individual miRNAspecies is still unknown. For instance, miR-10b has beenidentified as a tumor suppressor which prevents humanbreast cancer development but also as an oncogene whichinitiates breast cancer invasion and metastasis [5]. SeveralmiRNA species have been identified to be involved in humanbreast cancer development including miR-21, miR-145, andmiR-210 [6–8]. In veterinary medicine, only a single study isavailable on miRNA expression in canine mammary tumors.Boggs et al. [7] compared the expression levels of ten miRNAspecies in malignant mammary tumors and normal caninemammary gland and found a significant deregulation ofmiR-21, miR-29b, let-7f, miR-15a, and miR-16 in the tumors.In the present study, we expand these recent findingson the impact of miRNA deregulation on canine mammarytumors by asking for differences in expression levels of foursmall nucleolar RNA (snoRNA) and 16 canine miRNA withknown relevance for human and canine mammary tumordevelopment in tissue samples of normal mammary gland,adenomas, metastasizing, and nonmetastasizing caninemammary carcinomas as well as lymph node metastases.2. Materials and MethodsMammary gland tissues including regional lymph nodesfrom 30 dogs submitted to the Department of VeterinaryPathology of the Freie Universita¨t Berlin were included inthe study. Clinical data of the dogs included breed, age, andHindawi Publishing CorporationISRN Veterinary ScienceVolume 2014, Article ID 148597, 5 pageshttp://dx.doi.org/10.1155/2014/1485972 ISRN Veterinary Sciencelocation of tumors. All tissue samples were freshly frozennot later than 30min after surgical excision and stored at−80∘C until further use. Directly adjacent tissue sampleswere also fixed in 4% neutral buffered formalin, routinelyprocessed stained with haematoxylin and eosin (HE) forhistologic examination. Ten simple mammary adenomas,ten simple carcinomas without evidence of tumor cells inthe regional lymph node, and ten simple carcinomas withregional lymph node metastases as well as normal mammarygland tissues of ten dogs were selected and macrodissectedfor this study to assure a tumor content of more than 80%.The fifth sample group contained laser microdissected lymphnode metastases from five dogs with simple, metastasizingmammary carcinoma.The study was approved by the local animal welfareofficer. Surgical resection of the tumors was part of thetherapy according to the welfare of the animals and to thestate of the art of medical science under full anesthesia. Thestudy therefore had no influence on the common diagnosticor therapeutic measures usually applied on animals withmammary gland tumors and inclusion into the study didnot induce any additional treatments, pain, or discomfort-inducing manipulations during the entire study.Total RNA was extracted as previously described [9,10]. The lymph node metastases were microdissected fromcryostat sections by laser capture microdissection as previ-ously described [11]. All miRNA and snoRNA species wereelongated prior to cDNA synthesis by universal poly-A-tailing and amplified by quantitative reverse transcriptionPCR as previously described [9]. Housekeeping genes wereselected from the complete set of genes analyzed usingboth geNorm (version 3.4) and NormFinder (version 20)algorithms [12, 13]. The most stably expressed RNA specieswere determined by calculating the gene expression stabilitymeasure value (𝑀) using the geNorm tool as previouslydescribed [12]. Stepwise exclusion of the least stable genesidentified miR-155, let-7f, and miR-181b as the three moststably expressed genes. In a second approach NormFinderanalysis was performed [13] and identified miR-181b, U44,andU48 as the threemost stable reference genes. Consideringboth the results of the geNorm analysis and the NormFinderalgorithm miR-181b, miR-155, and U44 were used for datanormalization. Consequently, relative expression levels of themiRNA and snoRNA species were determined using the2−ΔΔCt method [14]. Due to the different efficiencies of thePCR assays, the actual efficiencies were used as the base of theexponential amplification function for calculation. Statisticalsignificance of differences inmiRNA and snoRNA expressionlevels was evaluated using the IBM SPSS Statistic 20 software.The results in the different tissue groups were statisticallycompared using the parametric ANOVA analysis. A 𝑃 ≤ 0.05was considered statistically significant.3. ResultsComparison of the expression levels of 16 miRNA and foursnoRNA leads to the identification of statistically differentexpression levels of ninemiRNAand one snoRNA in 27 of the400 comparisons. Of these, microdissected tumormetastasesdiffered most often from other tissue types. miR-101, miR-143, and miR-145 were differently expressed in metastaseswhen compared to all other tissues. miR-29b differed in itsexpression level between metastases and all tumor tissuesamples except normal mammary gland. miR-125a expres-sion only differed between metastases and metastasizingcarcinomas primary sites (Table 1, Figure 1). The comparisonof the primary tumors of different malignancies identifieda difference in miR-125a expression between metastasizingcarcinoma and adenoma as the only significant difference(Table 1, Figure 1). All other differences in expression wererestricted to comparisons between normal mammary glandand neoplastic tissues. miR-21, miR-143, miR-194, miR-203,miR-210, and the snoRNA U24 differed in their expressionlevels between normal gland and neoplastic tissues (Table 1,Figure 1). Of note, only miR-125a and the snoRNA U24showed a decreased expression in the primary tumors whencompared to normal gland or adenomas while all othersignificantly regulatedmiRNAhad an upregulation in benignandmalignant primary tumors (Table 1).This was in contrastto the observation in metastases which, except for miR-125aand miR-210, had a general downregulation of miRNA whencompared to all other tissues (Table 1).miR-210 was the only miRNA which allowed the dif-ferentiation of normal mammary gland from all neoplastictissues. In addition, miR-210 was the only miRNA with acontinuous significant increase in expression levels betweennormal mammary gland and all tumor groups and themetastases with expression differences of 7.01-fold betweenadenomas and normal gland, 10.41-fold between nonmetas-tasizing carcinomas and normal gland, 10.72-fold betweenmetastasizing carcinomas and normal gland, and 19.63-foldbetween metastases and normal gland (Table 1).U24 was the only snoRNA with a significant difference inexpression. The expression difference was 0.38-fold betweenthemetastasizing carcinoma and the normalmammary glandbut was not significantly altered in any other comparison.miR-9, miR-10b, miR-15a, miR-16, miR-125b, miR-136, andlet-7f as well as the snoRNA U66, Z30, and U48 had nosignificant difference in expression between any of the tissuesanalyzed.4. DiscussionThe aim of the present study was the identification of malig-nancy associated miRNA expression to discover potentialmalignancy marker and to further elucidate aspects of themolecular carcinogenesis and gene expression associatedwith metastatic behavior of canine mammary tumors [15].To this end, the expression of 16 miRNA and four snoRNAwas compared between normal mammary gland, adeno-mas, carcinomas, and metastases. Only nine of the miRNAspecies and one snoRNA tested were significantly differentlyexpressed between the diverse tissues analyzed.Interestingly, miR-210 was significantly overexpressed inall neoplastic tissues when compared to normal mammarygland. Previous work described miR-210 as a “hypoxamir”which is upregulated in hypoxic tissues by HIF-1 action andmediates ametabolic adaptation to anaerobic conditions [16].ISRN Veterinary Science 3Table 1: miRNA and snoRNA expression differences between mammary tissues at different stages of malignancy and normal gland.Normal Adenoma NonmetastasizingcarcinomaMetastasizingcarcinoma Metastasis RegulationNormalmiR-203 (4.37)miR-210 (7.01)miR-143 (2.70)miR-21 (3.18)miR-210 (10.41)miR-194 (4.44)miR-21 (5.05)miR-210 (10.72) miR-210 (19.63) UpU24 (0.38)miR-143 (0.04),miR-145 (0.02),miR-101 (0.12)DownAdenomaUpmiR-125a (0.40)miR-143 (0.02),miR-145 (0.02),miR-101 (0.05),miR-29b (0.14)DownNonmetastasizing carcinomaUpmiR-143 (0.02),miR-145 (0.01),miR-101 (0.08),miR-29b (0.18)DownMetastasizing carcinomamiR-125a (4.91) UpmiR-143 (0.06),miR-145 (0.04),miR-101 (0.13),miR-29b (0.18)DownMetastasis UpDownIn brackets (): fold change in expression difference.miRNA and snoRNA expression in canine mammary tumorsmirR-136miR-143let-7fU24miR-29bU48miR-145miR-9miR-10bmiR-203miR-125bmiR-15amiR-16miR-21miR-101miR-210miR-194miR-125aU66Z30Average group Ct-value121086420−2−4−6−8−10Normal mammary glandAdenomaNonmetastasizing carcinomaMetastasizing carcinomaMetastasisFigure 1: miRNA and snoRNA expression levels in normal gland, adenoma, nonmetastasizing carcinoma, metastasizing carcinoma, andlymph node metastasis. Ct-values are average values of expression levels of the tissue groups normalized to three housekeeping genes.4 ISRN Veterinary ScienceThe continuous upregulation of miR-210 over the courseof malignant progression may therefore be caused by anincreasing hypoxia in the developing tumor mass. miR-210overexpression has also been associated with the formationof capillary-like structures [17]. It can thus be hypothesizedthatmiR-210may indirectly promotemetastasis by triggeringangiogenesis in neighboring cells [18]. Another four miRNA,miR-21, miR-143, miR-194, and miR-203, were significantlyincreased in at least one mammary tumor group comparedto normalmammary gland and thereforematched the defini-tion of an oncogenic miRNA. While this disease associationis new formiR-143 andmiR-194, a similar oncogenic functionhas been suggested for miR-203 [19] in mammary cancer andfor miR-21 in several other tumors including human breastcancer but has to be determined yet [20].The general lack of differences in miRNA expressionbetween primary tumors at different stages of malignancywas intriguing. Only miR-125a was significantly differentlyexpressed between metastasizing carcinoma and adenoma.This was in contrast to the numerous miRNA differentiallyexpressed in metastatic tumor cells in the lymph nodes. Thisdifference may be caused by the different preparation ofmetastatic tumor cells by laser microdissection technologydue to the otherwise high contamination of metastases tissuesamples by lymphatic cells. The macrodissected samplesof normal gland and the primary tumors in contrast alsocontained, although at a minimal portion, cells of the tumorstroma. These cells may have contributed to the recordeddownregulation of the four miRNA inmicrodissectedmetas-tases when compared to macrodissected primary tumors andnormal mammary gland, respectively.5. ConclusionsIn conclusion, canine mammary tumors at different stagesof malignancy significantly differed from normal gland inthe expression of seven miRNA and one snoRNA species.miRNA and snoRNA expression however failed in mostcases to discriminate primary tumors at different stages ofmalignancy.Conflict of InterestsNone of the authors of this paper has a financial or personalrelationship with other people or organizations that couldinappropriately influence or bias the content of the paper.AcknowledgmentsThe authors acknowledge Alexander Weiss for the supportand Monika Schaerig for excellent technical assistance. Thestudy has been supported by the DFG KL 2240/1-1 and theDahlemResearch School and is part of a PhD thesis ofMarie-Charlotte von Deetzen.References[1] D. P. 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Mo, “MicroRNA-21 targetsthe tumor suppressor gene tropomyosin 1 (TPM1),”The Journalof Biological Chemistry, vol. 282, no. 19, pp. 14328–14336, 2007.Submit your manuscripts athttp://www.hindawi.comVeterinary MedicineJournal ofHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014 Veterinary Medicine InternationalHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014Hindawi Publishing Corporationhttp://www.hindawi.com Volume 2014International Journal ofMicrobiologyHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014AnimalsJournal ofEcologyInternational Journal ofHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014PsycheHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014Evolutionary BiologyInternational Journal ofHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014Hindawi Publishing Corporationhttp://www.hindawi.comApplied &EnvironmentalSoil ScienceVolume 2014Biotechnology Research InternationalHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014AgronomyHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014International Journal ofHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014Journal of Parasitology ResearchHindawi Publishing Corporation http://www.hindawi.com International Journal ofVolume 2014ZoologyGenomicsInternational Journal ofHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014InsectsJournal ofHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014The Scientific World JournalHindawi Publishing Corporation http://www.hindawi.com Volume 2014Hindawi Publishing Corporationhttp://www.hindawi.com Volume 2014VirusesJournal ofScientificaHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014Cell BiologyInternational Journal ofHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014Hindawi Publishing Corporationhttp://www.hindawi.com Volume 2014Case Reports in Veterinary Medicine

Malignancy Associated MicroRNA Expression Changes in Canine Mammary Cancer of
Different Malignancies

MicroRNA has been suspected to be generally involved in carcinogenesis since their first description. A first study supported this assumption for canine mammary tumors when miRNA expression was compared to normal gland. The present study extends these results by comparing the expression of 16 microRNA (miRNA) and 4 small nucleolar RNA (snoRNA) in tumors of different malignancy, for example, adenomas, nonmetastasizing and metastasizing carcinomas as well as lymph node metastases, with each other and with normal mammary gland. All neoplastic tissues differed in their miR-210 expression levels from normal gland. While metastatic cells differed in their expression of mir-29b, miR-101, mir-125a, miR-143, and miR-145 from primary tumors, the comparison of miRNA expression in primary tumors of different malignancy failed to reveal significant differences except for a significant downregulation of mir-125a in metastasizing carcinomas when compared to adenomas.</jats:p

Marie-Charlotte von Deetzen

Bernd T. Schmeck

Achim D. Gruber

Robert Klopfleisch

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Malignancy Associated MicroRNA Expression Changes in Canine Mammary Cancer of Different Malignancies

Malignancy Associated MicroRNA Expression Changes in Canine Mammary Cancer of
Different Malignancies

Malignancy Associated MicroRNA Expression Changes in Canine Mammary Cancer of Different Malignancies

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