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MicroRNA expression in Epstein-Barr virus-associated post-transplant smooth muscle tumours is related to leiomyomatous phenotype

MicroRNA expression in Epstein-Barr virus-associated post-transplant smooth muscle tumours is... Epstein-Barr virus (EBV)-associated post-transplant smooth muscle tumours (PTSMT) are rare complications. In our previous molecular analysis, we have evaluated the expression of regulatory microRNA which are known to be EBV-related (miR-146a and miR-155) but found no deregulation in PTSMT. In this current analysis, we aimed to characterize the expression profiles of several hundred microRNA. Tissue samples from PTSMT and uterine leiomyomas were analysed by quantitative real-time PCR for the expression of 365 mature microRNA. PTSMT and leiomyomas share a highly similar microRNA profile, e.g. strong expression of miR-143/miR-145 cluster and low expression of miR-200c. Among EBV-related microRNA (miR-10b, miR-21, miR-29b, miR-34a, miR-127, miR-146a, miR-155, miR-200b, miR-203 and miR-429) only miR-10b and miR-203 were significantly deregulated. The expression pattern of microRNA in PTSMT is not associated with EBV infection but reflects the leiomyomatous differentiation of the tumour cells. Keywords: Epstein-Barr virus, EBV, Post-transplant smooth muscle tumour, PTSMT, MicroRNA Introduction analyses have addressed this issue. Ong et al. [3] have Epstein-Barr virus (EBV)-associated diseases are often analysed cell cycle factors, cytokines and gene promoter associated with acquired or congenital immunosuppres- methylation in PTSMT and found an activated mTOR/ sion or immunodeficiency, e.g. bone marrow and solid Akt cell cycle pathway by demonstrating phosphorylated organ-transplanted patients are at a higher risk. Up to mTOR in tumour cells. In our previous analysis, we have 10% of transplant recipients develop post-transplant evaluated the expression of EBV-associated human genes lymphoproliferative disorders (PTLD) while EBV-associated in PTSMT, including transcription, cell cycle and apoptosis post-transplant smooth muscle tumours (PTSMT) are rare factors and cytokines/cytokine receptors [2]. We found that complications (<1% of transplant patients) [1,2]. Neoplastic the transcription factor v-myc myelocytomatosis viral on- spindle cells in PTSMT express leiomyogenous marker cogene homolog (avian) (MYC) is significantly upregulated proteins such as smooth muscle actin and desmin, and the in PTSMT. In addition to mRNA, we have analysed micro- majority of tumour cells is positive for EBER. We could RNAs which are known to be expressed in an EBV-related previously show that PTSMT differ from conventional fashion (miR-146a and miR-155) but in PTSMT we found leiomyosarcomas by their lack of marked atypia, unusual low expression levels and no delimitable deregulation. sites of involvement (>50% in the recipient or donor liver) MicroRNAs are non-coding RNA molecules of 20–25 and defined EBV association [2]. nucleotides in length [4,5]. These small RNA molecules The molecular pathobiology of this rare neoplastic en- can bind semi-complementarily to the 3′-untranslated re- tity is not fully understood and only few experimental gion (3′-UTR) of target mRNAs and repress translation or target mRNA for degradation. The microRNA genes can be present as single gene or gene clusters (different * Correspondence: Hussein.Kais@MH-Hannover.de Institute of Pathology, Hannover Medical School, Carl-Neuberg-Str. 1, microRNA species are encoded on the same chromosome Hannover D-30625, Germany segment). Furthermore, microRNA families represent Full list of author information is available at the end of the article © 2013 Jonigk et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Jonigk et al. Clinical Sarcoma Research 2013, 3:9 Page 2 of 6 http://www.clinicalsarcomaresearch.com/content/3/1/9 different microRNA genes with different precursor forms Cluster analysis was performed with the Qlucore Omics but very similar mature microRNA with no or minor dif- Explorer 2.2 (Qlucore AB, Lund, Sweden). ferences in their nucleotide sequence. For target prediction, an open access bioinformatics Aberrant microRNA expression patterns have been platform was used (http://www.targetscan.org/). identified in several neoplasms and are known to con- tribute to the deregulated cell homeostasis in tumour Results cells, e.g. leiomyomas [6-17]. In this current analysis, we Similar microRNA expression profile in PTSMT and aimed to characterize the expression profiles of several leiomyomas hundred microRNA in PTSMT, in particular regarding Cluster analysis of the expression profile of 365 micro- an association with smooth muscle phenotype and EBV RNA revealed that PTSMT and leiomyomas share a highly infection. similar profile (Additional file 2: Table S2); cluster analysis could not discriminate between the two tumours. In PTSMT, only 15/365 microRNA and in leiomyomas Material and methods 23/365 microRNA showed a mean relative expression level Tissue specimens of >1 (Table 1). The five microRNA with the highest All available PTSMT samples from our tissue archive were expression levels in PTSMT and leiomyomas were the evaluated; these PTSMT cases have been characterized miR-143/miR-145 cluster (both genes are encoded on earlier [2]. Five EBV PTSMT samples from four patients, chromosome region 5q32), miR-24 (two genes on segments including two tumours from one patient (#4) were analysed 9q22.32 and 19p13.13 encode for the mature miR-24 mol- (Additional file 1: Table S1). Controls: seven EBV benign ecule), let-7b (22q13.31) and miR-21 (17q23.1). In general, uterine leiomyomas. Formalin-fixed and paraffin-embedded most microRNA were expressed at low levels (<1 relative (FFPE) samples were retrieved from the archives of the Institute of Pathology (Hannover Medical School/MHH, Hannover, Germany). The retrospective analysis of the Table 1 MicroRNA with a mean relative expression level samples has been approved by the local ethics committee of >1 (p <0.05*; p <0.01**) of the Hannover Medical School (MHH). microRNA PTSMT (mean) Leiomyomas (mean) miR-145 36.82 36.01 Laser microdissection of the PTSMT compartment and miR-24 10.63 20.75 gene expression analysis miR-143 7.35 8.12 Tissue from FFPE blocks with >90% tumour cells were let-7b 2.61 2.79 cut and processed for further PCR analysis. In blocks with miR-223 1.98 0.43 <90% aberrant cells, the PTSMT compartments were miR-342-3p 1.96 1.65 laser microdissected using a SmartCutPlus-System (MMI, Glattbrugg, Switzerland), as previously described [2]. miR-133a 1.65 2.79 A set of 365 mature microRNA and corresponding en- miR-19b 1.63 2.23 dogenous controls were analysed by quantitative real-time miR-320 1.49 1.42 PCR (Pool A, Applied Biosystems, Carlsbad, CA, USA). In miR-21 1.34 5.40 brief, cells were digested in proteinase K and RNA was miR-191 1.33 2.38 extracted with phenol/chloroform [2,18,19]. Synthesis of miR-30c 1.33 1.23 cDNA from microRNA, subsequent pre-amplification of cDNA and real-time quantitative PCR with a 7900HT Fast miR-17 1.15 1.20 Real-Time PCR system were performed according to the miR-106a 1.14 1.18 manufacturers’ instructions (Applied Biosystems). miR-126 1.05 4.74** miR-222 0.75 7.20** Data analyses miR-29a 0.34 2.45* The sample- and detector-specific evaluation of amplifica- miR-16 0.68 1.98 tion curves was accomplished with the software RQ miR-214 0.11 1.73** Manager 1.2 (Applied Biosystems). C values established miR-193b 0.38 1.34* in this manner were converted into ΔC values and into -ΔCT 2 values (normalized to mean of endogenous control let-7e 0.19 1.25** genes). Statistical analysis was performed with Prism 5.0 miR-125b 0.01 1.21 (GraphPad Software, San Diego, California, USA) by ap- miR-199a-3p 0.09 1.06** plying the Mann–Whitney test for two-group comparison. miR-100 0.01 1.01** P values < 0.05 were considered as statistically significant. Jonigk et al. Clinical Sarcoma Research 2013, 3:9 Page 3 of 6 http://www.clinicalsarcomaresearch.com/content/3/1/9 expression level) in PTSMT as well as in leiomyomas, e.g. 200b, miR-203 and miR-429. Only two of these microRNA the leiomyomatous phenotype-associated miR-150 (mean were significantly down-regulated in EBV PTSMT: miR- relative level of 0.38 in leiomyomas versus 0.37 in PTSMT), 10b (mean relative expression of 0.45 in leiomyomas versus miR-221 (0.09 versus 0.01) and miR-200c (0.00 versus 0.01). 0.01 in PTSMT) and miR-203 (0.01 versus 0.00). Further- In PTSMT, 59/365 microRNA were significantly de- more, in leiomyomas, the level of miR-21 was higher than regulated compared with leiomyomas: a set of 51/59 in PTSMT but the difference did not reach statistical microRNA were down-regulated and 8/59 were up- significance (5.40 versus 1.34; p = 0.0876). As could be regulated (Figure 1). However, most of these signifi- expected from our previous experiments, miR-146a and cances in down-regulated microRNA are a result of very miR-155 were not deregulated while the miR-146a-homo- low expression in PTSMT versus low expression in log miR-146b was significantly down-regulated in PTSMT leiomyomas (<1 mean relative expression level), e.g. (mean 0.23 versus 0.73 in leiomyomas; p = 0.0101). Other let-7c (0.22 in leiomyomas versus 0.01 in PTSMT) and EBV-related microRNA (miR-29b, miR-34a, miR-200b and miR-221 (0.09 versus 0.01). Furthermore, significantly miR-429) were expressed at similarly low levels in both up-regulated microRNA in PTSMT were a result of very leiomyomas and PTSMT (not significantly deregulated). low expression levels in leiomyomas rather than bio- logically relevant increased expression in PTSMT. In Discussion these 8/59 microRNA, the highest relative expression levels No EBV-related microRNA deregulation in + - were less than 0.5 (miR-181a, miR-34c, miR-142-3p; EBV /LMP1 PTSMT Figure 1) or even less than 0.05 in PTSMT and leiomyomas In our first molecular analysis of PTSMT, we analysed (miR-138, miR-181c, miR-190, miR-330-3p/-5p, miR-504). the expression of miR-146a and miR-155 because these microRNA are known to be involved in the pathobiology Minor association of microRNA expression with EBV of EBV infection of B cells [2,34,35]. In our current ana- infection in PTSMT lysis, we could confirm our previous results and found The following microRNA are known to be related to EBV no significant deregulation of these two microRNA using infection in solid tumours and haematopoietic malignan- a microarray technique. Furthermore, it is remarkable cies (Additional file 3: Table S3) [20-33]: miR-10b, miR-21, that several EBV-related microRNA, other than miR- miR-29b, miR-34a, miR-127, miR-146a, miR-155, miR- 146a and miR-155, are not deregulated in PTSMT. In Figure 1 Representative selection of significantly (p <0.01) de-regulated microRNA expression in PTSMT. A-C) Examples of down-regulated microRNA. D-F) Up-regulated microRNA in PTSMT show relative expression levels below 0.5. Jonigk et al. Clinical Sarcoma Research 2013, 3:9 Page 4 of 6 http://www.clinicalsarcomaresearch.com/content/3/1/9 particular, miR-10b, miR-21, miR-29b, miR-34a and PTSMT but also in leiomyomas. Because PTSMT can be miR-127, which are increased in EBV nasopharyngeal found next to vessels (e.g. manifestation in cerebral sinus), carcinoma and high grade B cell lymphomas/Burkitt it is thought that the aberrant founder cells might be lymphomas [20-33], were expressed at low or very low derived from a vessel wall [2,41]. However, due to very levels in PTSMT. In non-PTSMT EBV carcinomas and similar miR-143/miR-145 expression patterns in PTSMT, lymphomas miR-200b, miR-203 and miR-429 are gener- uterus wall-derived leiomyomas and pulmonary vessels, ally expressed at low levels [20-33]. In PTSMT, but also the high expression of these two microRNA does not in EBV leiomyomas, these microRNAs were also prove a vessel wall origin of PTSMT but reflects the expressed at low levels, indicating no specific EBV- smooth muscle differentiation. related decreased expression in smooth muscle tumours. In PTSMT and leiomyomas, many microRNA are It has to be taken into account that different cell and expressed at low or very low levels, which makes it likely tumour types can react in different manners to EBV that protein translation of potential target mRNA types infection. Furthermore, depending on the latency type, is not inhibited. The problem for target prediction, but EBV induces expression of different virus proteins, simultaneously the hallmark of microRNA/mRNA bio- which interfere in the host cell cycle. Latent membrane logy, is the characteristic semi-complementary binding protein 1 (LMP1) is one of these EBV proteins and it of the seven nucleotides at the 3′-end of the mature has been shown that LMP1 alone can induce altered microRNA (so-called seed sequence) to corresponding microRNA expression in nasopharyngeal carcinomas mRNA-nucleotides of the 5′-UTR [4,5]. This semi- and lymphomas [20-33]. We and others have found complementary binding is sufficient to induce a biological previously that EBV PTSMT express EBNA2 and effect, the inhibition of mRNA/protein translation. As a EBNA3 while LMP1 expression is weak or not detect- result, one microRNA can bind to several 5′-UTR-mRNA able [2,3,36,37]. Thus, despite EBV infection in PTSMT, and vice versa one 5′-UTR-mRNA can be targeted by our finding of no major changes in the microRNA ex- several microRNA. In our previous analysis, we have pression profile is likely to be related to lack of LMP1 evaluated the expression of several mRNA transcripts in expression. PTSMT and leiomyomas, including MYC, vascular endo- thelial growth factor A (VEGFA), nuclear factor of kappa Leiomyomatous phenotype-associated microRNA light polypeptide gene enhancer in B-cells 1 (NFKB1), expression in PTSMT tumour protein p53 (TP53), transforming growth factor, MicroRNA expression analyses in uterine leiomyomas beta receptor II (TGFBR2) and transforming growth fac- and leiomyosarcomas were introduced only a few years tor, beta 1 (TGFB1) [2]. ago (Additional file 4: Table S4) [6-17]. The majority of Among several EBV-associated human factors, we studies have evaluated patient-derived leiomyomas as a found only MYC to be significantly increased in PTSMT model disease of neoplastic smooth muscle proliferation. [2]. The mRNA of this transcription factor can be regu- Among different studies and different analytical methods, lated by miR-150, miR-143 and miR-145 [42,43] but no microRNA expression patterns of patient-derived tumour PTSMT-specific inverse correlation was found. samples showed a set of microRNA which are recurrently VEGFA can be negatively regulated by miR-200c and deregulated in comparison to normal uterus wall cells. other microRNA. In leiomyomatous cell lines, miR-200c Similar to PTSMT, decreased expression of miR-150, interaction with VEGFA has been shown [7] and accord- miR-200c and miR-221 and increased expression of miR- ingly, in leiomyomas as well as in PTSMT, very low levels 21 and let-7 family members have become particularly evi- of miR-200c correlate with increased levels of VEGFA [2]. dent in leiomyomas [6-17]. It has been demonstrated that In many different tumour types, miR-21 is aberrantly mesenchymal cells which differentiate into smooth muscle expressed, because this microRNA can target several signal networks, either directly by binding to different cells in vitro change their microRNA expression patterns, e.g. down-regulation of 13q31.3-clustered miR-17/miR- types of mRNA from similar signal cascades or indirectly 18a/miR-20a and up-regulation of miR-181a/miR-181c via deregulation of factors down/up-stream to the fac- tors directly suppressed by miR-21 [44,45]. In particular, paralogs [16]. Furthermore, in smooth muscle cells, it has been shown that the 5q32-encoded miR-143/miR-145 miR-21 is a negative regulator of TP53 signalling and cluster is co-expressed [38,39]. Both microRNA target a simultaneously a promoter of NFKB1 signalling [44,45]. Increased expression of miR-21 has been previously network of factors to promote vascular smooth muscle cell differentiation and repress proliferation [38,39]. We demonstrated in leiomyomas [6,10,15,16] which we could previously demonstrate high expression of miR-143 could confirm in our analysis. In smooth muscle cells, miR-21 is involved in regulation of apoptosis and and even higher expression of miR-145 in pulmonary vessel wall cells [40]. A similar expression pattern of high TGFBR2/TGFB1 signalling [6,10,13]. TGFBR2-3′UTR miR-143 and higher miR-145 could also be found in has an miR-21 binding site and can therefore directly be Jonigk et al. Clinical Sarcoma Research 2013, 3:9 Page 5 of 6 http://www.clinicalsarcomaresearch.com/content/3/1/9 regulated by miR-21 in smooth muscle cells [10]. In vitro Funding DJ, FL: “Integriertes Forschungs- und Behandlungszentrum Transplantation” studies also suggested an indirect regulatory interaction (IFB-Tx, German Federal Ministry of Education, reference number: 01EO0802). between miR-21 and TGFB1; of note, TGFB1 is not a BMK: German Children’s Cancer Fund and IFB-Tx. direct target of miR-21 [10]. Furthermore, inhibition of Author details miR-21 expression in smooth muscle cells indirectly Institute of Pathology, Hannover Medical School, Carl-Neuberg-Str. 1, increases caspase 3 and caspase 7 activity in vitro; both Hannover D-30625, Germany. Department of Paediatric Haematology and caspases have no miR-21 binding site [6,13]. The miR-21 Oncology, Hannover Medical School, Hannover, Germany. Integrated Research and Treatment Center Transplantation (IFB-Tx), Hannover, Germany. expression was lower in PTSMT than leiomyomas but the difference was not significant. In addition, in our pre- Received: 25 April 2013 Accepted: 25 June 2013 vious analysis we found no differences in the expression of Published: 6 July 2013 miR-21-related NFKB1, TP53, TGFBR2 or TGFB1 between PTSMTand leiomyomas [2]. Therefore, our in situ-derived References 1. Shroff R, Rees L: The post-transplant lymphoproliferative disorder - a results do not reveal a PTSMT-specific deregulated miR-21 literature review. Pediatr Nephrol 2004, 19:369–377. signal cascade, but an expression pattern related to smooth 2. Jonigk D, Laenger F, Maegel L, et al: Molecular and clinicopathological muscle phenotype. analysis of Epstein-Barr virus-associated posttransplant smooth muscle tumors. Am J Transplant 2012, 12:1908–1917. Members of the let-7 family are increased in leiomyomas 3. Ong KW, Teo M, Lee V, et al: Expression of EBV latent antigens, [15] and smooth muscle cell lines, e.g. let-7b [13,16]. 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Jiang X, Huang H, Li Z, et al: Blockade of miR-150 Maturation by • Inclusion in PubMed, CAS, Scopus and Google Scholar MLL-Fusion/MYC/LIN-28 Is Required for MLL-Associated Leukemia. Cancer Cell 2012, 22:524–535. • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Clinical Sarcoma Research Springer Journals

MicroRNA expression in Epstein-Barr virus-associated post-transplant smooth muscle tumours is related to leiomyomatous phenotype

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Copyright © 2013 by Jonigk et al.; licensee BioMed Central Ltd.
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Biomedicine; Cancer Research; Oncology; Surgical Oncology
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2045-3329
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10.1186/2045-3329-3-9
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23830214
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Abstract

Epstein-Barr virus (EBV)-associated post-transplant smooth muscle tumours (PTSMT) are rare complications. In our previous molecular analysis, we have evaluated the expression of regulatory microRNA which are known to be EBV-related (miR-146a and miR-155) but found no deregulation in PTSMT. In this current analysis, we aimed to characterize the expression profiles of several hundred microRNA. Tissue samples from PTSMT and uterine leiomyomas were analysed by quantitative real-time PCR for the expression of 365 mature microRNA. PTSMT and leiomyomas share a highly similar microRNA profile, e.g. strong expression of miR-143/miR-145 cluster and low expression of miR-200c. Among EBV-related microRNA (miR-10b, miR-21, miR-29b, miR-34a, miR-127, miR-146a, miR-155, miR-200b, miR-203 and miR-429) only miR-10b and miR-203 were significantly deregulated. The expression pattern of microRNA in PTSMT is not associated with EBV infection but reflects the leiomyomatous differentiation of the tumour cells. Keywords: Epstein-Barr virus, EBV, Post-transplant smooth muscle tumour, PTSMT, MicroRNA Introduction analyses have addressed this issue. Ong et al. [3] have Epstein-Barr virus (EBV)-associated diseases are often analysed cell cycle factors, cytokines and gene promoter associated with acquired or congenital immunosuppres- methylation in PTSMT and found an activated mTOR/ sion or immunodeficiency, e.g. bone marrow and solid Akt cell cycle pathway by demonstrating phosphorylated organ-transplanted patients are at a higher risk. Up to mTOR in tumour cells. In our previous analysis, we have 10% of transplant recipients develop post-transplant evaluated the expression of EBV-associated human genes lymphoproliferative disorders (PTLD) while EBV-associated in PTSMT, including transcription, cell cycle and apoptosis post-transplant smooth muscle tumours (PTSMT) are rare factors and cytokines/cytokine receptors [2]. We found that complications (<1% of transplant patients) [1,2]. Neoplastic the transcription factor v-myc myelocytomatosis viral on- spindle cells in PTSMT express leiomyogenous marker cogene homolog (avian) (MYC) is significantly upregulated proteins such as smooth muscle actin and desmin, and the in PTSMT. In addition to mRNA, we have analysed micro- majority of tumour cells is positive for EBER. We could RNAs which are known to be expressed in an EBV-related previously show that PTSMT differ from conventional fashion (miR-146a and miR-155) but in PTSMT we found leiomyosarcomas by their lack of marked atypia, unusual low expression levels and no delimitable deregulation. sites of involvement (>50% in the recipient or donor liver) MicroRNAs are non-coding RNA molecules of 20–25 and defined EBV association [2]. nucleotides in length [4,5]. These small RNA molecules The molecular pathobiology of this rare neoplastic en- can bind semi-complementarily to the 3′-untranslated re- tity is not fully understood and only few experimental gion (3′-UTR) of target mRNAs and repress translation or target mRNA for degradation. The microRNA genes can be present as single gene or gene clusters (different * Correspondence: Hussein.Kais@MH-Hannover.de Institute of Pathology, Hannover Medical School, Carl-Neuberg-Str. 1, microRNA species are encoded on the same chromosome Hannover D-30625, Germany segment). Furthermore, microRNA families represent Full list of author information is available at the end of the article © 2013 Jonigk et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Jonigk et al. Clinical Sarcoma Research 2013, 3:9 Page 2 of 6 http://www.clinicalsarcomaresearch.com/content/3/1/9 different microRNA genes with different precursor forms Cluster analysis was performed with the Qlucore Omics but very similar mature microRNA with no or minor dif- Explorer 2.2 (Qlucore AB, Lund, Sweden). ferences in their nucleotide sequence. For target prediction, an open access bioinformatics Aberrant microRNA expression patterns have been platform was used (http://www.targetscan.org/). identified in several neoplasms and are known to con- tribute to the deregulated cell homeostasis in tumour Results cells, e.g. leiomyomas [6-17]. In this current analysis, we Similar microRNA expression profile in PTSMT and aimed to characterize the expression profiles of several leiomyomas hundred microRNA in PTSMT, in particular regarding Cluster analysis of the expression profile of 365 micro- an association with smooth muscle phenotype and EBV RNA revealed that PTSMT and leiomyomas share a highly infection. similar profile (Additional file 2: Table S2); cluster analysis could not discriminate between the two tumours. In PTSMT, only 15/365 microRNA and in leiomyomas Material and methods 23/365 microRNA showed a mean relative expression level Tissue specimens of >1 (Table 1). The five microRNA with the highest All available PTSMT samples from our tissue archive were expression levels in PTSMT and leiomyomas were the evaluated; these PTSMT cases have been characterized miR-143/miR-145 cluster (both genes are encoded on earlier [2]. Five EBV PTSMT samples from four patients, chromosome region 5q32), miR-24 (two genes on segments including two tumours from one patient (#4) were analysed 9q22.32 and 19p13.13 encode for the mature miR-24 mol- (Additional file 1: Table S1). Controls: seven EBV benign ecule), let-7b (22q13.31) and miR-21 (17q23.1). In general, uterine leiomyomas. Formalin-fixed and paraffin-embedded most microRNA were expressed at low levels (<1 relative (FFPE) samples were retrieved from the archives of the Institute of Pathology (Hannover Medical School/MHH, Hannover, Germany). The retrospective analysis of the Table 1 MicroRNA with a mean relative expression level samples has been approved by the local ethics committee of >1 (p <0.05*; p <0.01**) of the Hannover Medical School (MHH). microRNA PTSMT (mean) Leiomyomas (mean) miR-145 36.82 36.01 Laser microdissection of the PTSMT compartment and miR-24 10.63 20.75 gene expression analysis miR-143 7.35 8.12 Tissue from FFPE blocks with >90% tumour cells were let-7b 2.61 2.79 cut and processed for further PCR analysis. In blocks with miR-223 1.98 0.43 <90% aberrant cells, the PTSMT compartments were miR-342-3p 1.96 1.65 laser microdissected using a SmartCutPlus-System (MMI, Glattbrugg, Switzerland), as previously described [2]. miR-133a 1.65 2.79 A set of 365 mature microRNA and corresponding en- miR-19b 1.63 2.23 dogenous controls were analysed by quantitative real-time miR-320 1.49 1.42 PCR (Pool A, Applied Biosystems, Carlsbad, CA, USA). In miR-21 1.34 5.40 brief, cells were digested in proteinase K and RNA was miR-191 1.33 2.38 extracted with phenol/chloroform [2,18,19]. Synthesis of miR-30c 1.33 1.23 cDNA from microRNA, subsequent pre-amplification of cDNA and real-time quantitative PCR with a 7900HT Fast miR-17 1.15 1.20 Real-Time PCR system were performed according to the miR-106a 1.14 1.18 manufacturers’ instructions (Applied Biosystems). miR-126 1.05 4.74** miR-222 0.75 7.20** Data analyses miR-29a 0.34 2.45* The sample- and detector-specific evaluation of amplifica- miR-16 0.68 1.98 tion curves was accomplished with the software RQ miR-214 0.11 1.73** Manager 1.2 (Applied Biosystems). C values established miR-193b 0.38 1.34* in this manner were converted into ΔC values and into -ΔCT 2 values (normalized to mean of endogenous control let-7e 0.19 1.25** genes). Statistical analysis was performed with Prism 5.0 miR-125b 0.01 1.21 (GraphPad Software, San Diego, California, USA) by ap- miR-199a-3p 0.09 1.06** plying the Mann–Whitney test for two-group comparison. miR-100 0.01 1.01** P values < 0.05 were considered as statistically significant. Jonigk et al. Clinical Sarcoma Research 2013, 3:9 Page 3 of 6 http://www.clinicalsarcomaresearch.com/content/3/1/9 expression level) in PTSMT as well as in leiomyomas, e.g. 200b, miR-203 and miR-429. Only two of these microRNA the leiomyomatous phenotype-associated miR-150 (mean were significantly down-regulated in EBV PTSMT: miR- relative level of 0.38 in leiomyomas versus 0.37 in PTSMT), 10b (mean relative expression of 0.45 in leiomyomas versus miR-221 (0.09 versus 0.01) and miR-200c (0.00 versus 0.01). 0.01 in PTSMT) and miR-203 (0.01 versus 0.00). Further- In PTSMT, 59/365 microRNA were significantly de- more, in leiomyomas, the level of miR-21 was higher than regulated compared with leiomyomas: a set of 51/59 in PTSMT but the difference did not reach statistical microRNA were down-regulated and 8/59 were up- significance (5.40 versus 1.34; p = 0.0876). As could be regulated (Figure 1). However, most of these signifi- expected from our previous experiments, miR-146a and cances in down-regulated microRNA are a result of very miR-155 were not deregulated while the miR-146a-homo- low expression in PTSMT versus low expression in log miR-146b was significantly down-regulated in PTSMT leiomyomas (<1 mean relative expression level), e.g. (mean 0.23 versus 0.73 in leiomyomas; p = 0.0101). Other let-7c (0.22 in leiomyomas versus 0.01 in PTSMT) and EBV-related microRNA (miR-29b, miR-34a, miR-200b and miR-221 (0.09 versus 0.01). Furthermore, significantly miR-429) were expressed at similarly low levels in both up-regulated microRNA in PTSMT were a result of very leiomyomas and PTSMT (not significantly deregulated). low expression levels in leiomyomas rather than bio- logically relevant increased expression in PTSMT. In Discussion these 8/59 microRNA, the highest relative expression levels No EBV-related microRNA deregulation in + - were less than 0.5 (miR-181a, miR-34c, miR-142-3p; EBV /LMP1 PTSMT Figure 1) or even less than 0.05 in PTSMT and leiomyomas In our first molecular analysis of PTSMT, we analysed (miR-138, miR-181c, miR-190, miR-330-3p/-5p, miR-504). the expression of miR-146a and miR-155 because these microRNA are known to be involved in the pathobiology Minor association of microRNA expression with EBV of EBV infection of B cells [2,34,35]. In our current ana- infection in PTSMT lysis, we could confirm our previous results and found The following microRNA are known to be related to EBV no significant deregulation of these two microRNA using infection in solid tumours and haematopoietic malignan- a microarray technique. Furthermore, it is remarkable cies (Additional file 3: Table S3) [20-33]: miR-10b, miR-21, that several EBV-related microRNA, other than miR- miR-29b, miR-34a, miR-127, miR-146a, miR-155, miR- 146a and miR-155, are not deregulated in PTSMT. In Figure 1 Representative selection of significantly (p <0.01) de-regulated microRNA expression in PTSMT. A-C) Examples of down-regulated microRNA. D-F) Up-regulated microRNA in PTSMT show relative expression levels below 0.5. Jonigk et al. Clinical Sarcoma Research 2013, 3:9 Page 4 of 6 http://www.clinicalsarcomaresearch.com/content/3/1/9 particular, miR-10b, miR-21, miR-29b, miR-34a and PTSMT but also in leiomyomas. Because PTSMT can be miR-127, which are increased in EBV nasopharyngeal found next to vessels (e.g. manifestation in cerebral sinus), carcinoma and high grade B cell lymphomas/Burkitt it is thought that the aberrant founder cells might be lymphomas [20-33], were expressed at low or very low derived from a vessel wall [2,41]. However, due to very levels in PTSMT. In non-PTSMT EBV carcinomas and similar miR-143/miR-145 expression patterns in PTSMT, lymphomas miR-200b, miR-203 and miR-429 are gener- uterus wall-derived leiomyomas and pulmonary vessels, ally expressed at low levels [20-33]. In PTSMT, but also the high expression of these two microRNA does not in EBV leiomyomas, these microRNAs were also prove a vessel wall origin of PTSMT but reflects the expressed at low levels, indicating no specific EBV- smooth muscle differentiation. related decreased expression in smooth muscle tumours. In PTSMT and leiomyomas, many microRNA are It has to be taken into account that different cell and expressed at low or very low levels, which makes it likely tumour types can react in different manners to EBV that protein translation of potential target mRNA types infection. Furthermore, depending on the latency type, is not inhibited. The problem for target prediction, but EBV induces expression of different virus proteins, simultaneously the hallmark of microRNA/mRNA bio- which interfere in the host cell cycle. Latent membrane logy, is the characteristic semi-complementary binding protein 1 (LMP1) is one of these EBV proteins and it of the seven nucleotides at the 3′-end of the mature has been shown that LMP1 alone can induce altered microRNA (so-called seed sequence) to corresponding microRNA expression in nasopharyngeal carcinomas mRNA-nucleotides of the 5′-UTR [4,5]. This semi- and lymphomas [20-33]. We and others have found complementary binding is sufficient to induce a biological previously that EBV PTSMT express EBNA2 and effect, the inhibition of mRNA/protein translation. As a EBNA3 while LMP1 expression is weak or not detect- result, one microRNA can bind to several 5′-UTR-mRNA able [2,3,36,37]. Thus, despite EBV infection in PTSMT, and vice versa one 5′-UTR-mRNA can be targeted by our finding of no major changes in the microRNA ex- several microRNA. In our previous analysis, we have pression profile is likely to be related to lack of LMP1 evaluated the expression of several mRNA transcripts in expression. PTSMT and leiomyomas, including MYC, vascular endo- thelial growth factor A (VEGFA), nuclear factor of kappa Leiomyomatous phenotype-associated microRNA light polypeptide gene enhancer in B-cells 1 (NFKB1), expression in PTSMT tumour protein p53 (TP53), transforming growth factor, MicroRNA expression analyses in uterine leiomyomas beta receptor II (TGFBR2) and transforming growth fac- and leiomyosarcomas were introduced only a few years tor, beta 1 (TGFB1) [2]. ago (Additional file 4: Table S4) [6-17]. The majority of Among several EBV-associated human factors, we studies have evaluated patient-derived leiomyomas as a found only MYC to be significantly increased in PTSMT model disease of neoplastic smooth muscle proliferation. [2]. The mRNA of this transcription factor can be regu- Among different studies and different analytical methods, lated by miR-150, miR-143 and miR-145 [42,43] but no microRNA expression patterns of patient-derived tumour PTSMT-specific inverse correlation was found. samples showed a set of microRNA which are recurrently VEGFA can be negatively regulated by miR-200c and deregulated in comparison to normal uterus wall cells. other microRNA. In leiomyomatous cell lines, miR-200c Similar to PTSMT, decreased expression of miR-150, interaction with VEGFA has been shown [7] and accord- miR-200c and miR-221 and increased expression of miR- ingly, in leiomyomas as well as in PTSMT, very low levels 21 and let-7 family members have become particularly evi- of miR-200c correlate with increased levels of VEGFA [2]. dent in leiomyomas [6-17]. It has been demonstrated that In many different tumour types, miR-21 is aberrantly mesenchymal cells which differentiate into smooth muscle expressed, because this microRNA can target several signal networks, either directly by binding to different cells in vitro change their microRNA expression patterns, e.g. down-regulation of 13q31.3-clustered miR-17/miR- types of mRNA from similar signal cascades or indirectly 18a/miR-20a and up-regulation of miR-181a/miR-181c via deregulation of factors down/up-stream to the fac- tors directly suppressed by miR-21 [44,45]. In particular, paralogs [16]. Furthermore, in smooth muscle cells, it has been shown that the 5q32-encoded miR-143/miR-145 miR-21 is a negative regulator of TP53 signalling and cluster is co-expressed [38,39]. Both microRNA target a simultaneously a promoter of NFKB1 signalling [44,45]. Increased expression of miR-21 has been previously network of factors to promote vascular smooth muscle cell differentiation and repress proliferation [38,39]. We demonstrated in leiomyomas [6,10,15,16] which we could previously demonstrate high expression of miR-143 could confirm in our analysis. In smooth muscle cells, miR-21 is involved in regulation of apoptosis and and even higher expression of miR-145 in pulmonary vessel wall cells [40]. A similar expression pattern of high TGFBR2/TGFB1 signalling [6,10,13]. TGFBR2-3′UTR miR-143 and higher miR-145 could also be found in has an miR-21 binding site and can therefore directly be Jonigk et al. Clinical Sarcoma Research 2013, 3:9 Page 5 of 6 http://www.clinicalsarcomaresearch.com/content/3/1/9 regulated by miR-21 in smooth muscle cells [10]. In vitro Funding DJ, FL: “Integriertes Forschungs- und Behandlungszentrum Transplantation” studies also suggested an indirect regulatory interaction (IFB-Tx, German Federal Ministry of Education, reference number: 01EO0802). between miR-21 and TGFB1; of note, TGFB1 is not a BMK: German Children’s Cancer Fund and IFB-Tx. direct target of miR-21 [10]. Furthermore, inhibition of Author details miR-21 expression in smooth muscle cells indirectly Institute of Pathology, Hannover Medical School, Carl-Neuberg-Str. 1, increases caspase 3 and caspase 7 activity in vitro; both Hannover D-30625, Germany. Department of Paediatric Haematology and caspases have no miR-21 binding site [6,13]. The miR-21 Oncology, Hannover Medical School, Hannover, Germany. Integrated Research and Treatment Center Transplantation (IFB-Tx), Hannover, Germany. expression was lower in PTSMT than leiomyomas but the difference was not significant. In addition, in our pre- Received: 25 April 2013 Accepted: 25 June 2013 vious analysis we found no differences in the expression of Published: 6 July 2013 miR-21-related NFKB1, TP53, TGFBR2 or TGFB1 between PTSMTand leiomyomas [2]. Therefore, our in situ-derived References 1. Shroff R, Rees L: The post-transplant lymphoproliferative disorder - a results do not reveal a PTSMT-specific deregulated miR-21 literature review. Pediatr Nephrol 2004, 19:369–377. signal cascade, but an expression pattern related to smooth 2. Jonigk D, Laenger F, Maegel L, et al: Molecular and clinicopathological muscle phenotype. analysis of Epstein-Barr virus-associated posttransplant smooth muscle tumors. Am J Transplant 2012, 12:1908–1917. Members of the let-7 family are increased in leiomyomas 3. Ong KW, Teo M, Lee V, et al: Expression of EBV latent antigens, [15] and smooth muscle cell lines, e.g. let-7b [13,16]. 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Clinical Sarcoma Research – Springer Journals

Published: Jul 6, 2013

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