Experimental and Molecular Pathology 96 (2014) 261–266

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Experimental and Molecular Pathology journal homepage: www.elsevier.com/locate/yexmp

Low expression of miR-150 in pediatric intestinal Burkitt lymphoma Miao Wang a,⁎, Wenping Yang b, Min Li c, Yong Li a a b c

Department of Pathology, Basic Medical College, Capital Medical University, Beijing, China Department of Pathology, Jiangxi Children's Hospital, Jiangxi, China Department of Pathology, Basic Medical College, Peking University Health Science Center, Beijing, China

a r t i c l e

i n f o

Article history: Received 7 January 2014 and in revised form 24 February 2014 Available online 6 March 2014 Keywords: Burkitt lymphoma Reactive lymphoid hyperplasia miR-150 qRT-PCR ISH

a b s t r a c t Background: Burkitt lymphoma (BL) is a highly aggressive B-cell lymphoma with rapid proliferation. It has become evident that miRNAs are involved in hematopoietic malignancies. This study was undertaken to investigate the miRNA expression patterns of pediatric intestinal BL tissues. Methods: We collected 28 BL and 8 reactive lymphoid hyperplasia (RLH) samples. miRNA expression profiling was performed in BL and RLH tissues to identify BL-related miRNAs, which were further analyzed by qRT-PCR and miRNA-ISH. In addition, immunohistochemistry (IHC) and western blot were used to define the protein targets of the BL-related miRNAs. Furthermore, we evaluated cell growth status by using methylthiazolyldiphenyltetrazolium bromide (MTT) assay in Raji cell line, which was transected with the BL-related miRNA mimics or inhibitors. Results: miRNA expression profiling showed that miR-150 had extremely decreased expression levels in BL patients. In both ISH and qRT-PCR analyses, BL had reduced levels of miR-150 expression compared with RLH. However, there is no significant correlation of miR-150 expression and EBV status in BL. Moreover, IHC and western blotting defined that c-Myb and Survivin are the protein targets of miR-150. Re-expression of miR-150 reduced the proliferation of Raji cells. Conclusions: Deregulation of miR-150 may be useful as a diagnostic tool in BL, based on miRNA profile screening, qRT-PCR and miRNA-ISH. miR-150 plays an important role in BL by targeting c-Myb and Survivin. Re-expression of miR-150 reduced the proliferation of Raji cells, which suggests it to be a promising novel candidate for tumor treatment. © 2014 Elsevier Inc. All rights reserved.

Introduction Non-Hodgkin's lymphoma (NHL) is the most common intestinal malignancy in children, and Burkitt's lymphoma is the most frequently encountered histologic subtype. In pediatric patients, intestinal involvement of the lymphoma is a common finding (Fallon et al., 2013). Burkitt's lymphoma is a highly aggressive B-cell nonHodgkin lymphoma and is the fastest growing human tumor. The disease is associated with the Epstein–Barr virus and is a common childhood cancer (Molyneux et al., 2012). Outcome with intensive chemotherapy has improved, but the prognosis is still poor (Di et al., 2004; Li et al., 2010; Osier et al., 2006). There is an urgent need to find key carcinogenesis-associated molecules to provide new insights into novel targeted therapeutic strategies in BL.

⁎ Corresponding author at: Department of Pathology, Basic Medical College, Capital Medical University, No. 10 Xitoutiao, You An Men, Beijing 100069, China. Fax: +86 10 83911642. E-mail address: [email protected] (M. Wang).

http://dx.doi.org/10.1016/j.yexmp.2014.02.015 0014-4800/© 2014 Elsevier Inc. All rights reserved.

miRNAs are small endogenous non-coding RNAs that bind to partially complementary recognition sequences of target mRNAs, causing either degradation or prevention of their translation (He and Hannon, 2004). Accumulating evidence has shown that miRNAs have crucial functions in specific cellular processes such as differentiation, morphogenesis and tumorigenesis (Calin and Croce, 2006; Ferracin et al., 2011; He and Hannon, 2004; Lu et al., 2005). In human cancer, miRNAs can function as oncogenes or tumor suppressor genes during tumorigenesis (Calin and Croce, 2006). Gene expression profiling studies have demonstrated that miRNA expression is an excellent biomarker associated with specific tumor subtypes and clinical outcomes (Li et al., 2011; Lu et al., 2005; Lyng et al., 2012). However, it is unclear which miRNAs are potential diagnostic and therapeutic biomarkers in BL. Given the therapeutic potential for miRNAs as biomarkers in BL, we evaluated differentially expressed miRNAs from BL and reactive lymphoid hyperplasia (RLH) tissues to study their potential roles in tumor formation in BL. miR-150, which has shown decreasing expression levels in BL tissues, was chosen for further validation and analysis.

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Methods

qRT-PCR miRNA analysis

Tissue sections and EBV status analysis

qRT-PCR for mature miRNAs was performed as described by the manufacturer (Exiqon, Vedbaek, Denmark). The non-coding RNU6B (U6 control) was used for normalization (ΔCt = ΔCtmiRNA − ΔCtU6) and to check the quality of the samples, i.e. only cases with a cycle threshold (Ct) value lower than 35 were used resulting in a total of 28 cases. miR-17-5p, miR-34a and miR-150 were analyzed by qRT-PCR. Relative expression levels were determined by using the formula 2−ΔCt.

28 paraffin-embedded Burkitt lymphoma (BL) tissue samples were obtained from patients diagnosed in the Department of Pathology, Jiangxi Children's Hospital from 1984 to 2009. Eight reactive lymphoid hyperplasia (RLH) tissue samples were obtained from the Department of Pathology, Peking University Health Science Center. All protocols for obtaining and studying human tissues and cells were approved by the institution's review board for human subject research and diagnosis was in accordance with WHO classification guidelines (2013). EBV status analysis in BL was performed as reported previously (Lu et al., 2011).

Immunohistochemistry (IHC) The slides were deparaffinized and endogenous peroxidase was blocked by incubation with 3% H2O2 for 10 min. Antigen retrieval was performed according to various protocols of the manufacturers. Monoclonal and polyclonal antibodies to the following antigens were used for immunophenotyping of the cases: CD19, CD20, CD10, BCL2, BCL6 and Ki-67 (Maxin, Fuzhou, China); Survivin (Cell Signaling Technology, Boston, MA) and c-Myb (Abcam, Cambridge, UK). Signals were amplified by incubation with the appropriate horseradish peroxidaseconjugated antibodies for 60 min and the reactivity was visualized by diaminobenzidine. Immunoreactivity was defined as follows: negative (b 10% positively stained neoplastic lymphoid cells); weakly positive (10–30% positively stained neoplastic lymphoid cells); or positive (N30% positively stained neoplastic lymphoid cells). Ki-67 results were expressed as the percentage of positively stained nuclei of neoplastic lymphoid cells.

In situ hybridization analysis miRNA ISH was performed as reported previously (Ma et al., 2012). The miRCURY LNA miRNA detection probes for human miR-150, scramble, and U6 were purchased from Exiqon (Exiqon, Vedbaek, Denmark), and have high affinity and discrimination, enabling specific and sensitive detection of miR-150. In situ hybridization was performed with reference to the manufacturer's protocol for formalin-fixed paraffinembedded tissues (http://www.exiqon.com/ls/Documents/Scientific/ miRCURY-LNA-microRNA-ISH-Optimization-Kit-manual.pdf). Cell culture, transfection and western blotting Burkitt lymphoma (BL) cell line, Raji (Karpova et al., 2005) obtained from cell resource center (IBMS, CAMS/PUMC), was cultured in RPMI 1640 (GIBCO, Life Technologies) supplemented with 10% fetal calf serum (GIBCO), 1% L-glutamine, and 1% penicillin–streptomycin in a 5% CO2-humidified chamber. miRNA control mimics and miR-150 mimics were purchased from RiboBio Co., Ltd. (Guangzhou, China). For transfection, miRNA control mimics (50 nM final concentration), or miR-150 mimics (50 nM final concentration) were transfected into appropriate cells using Lipofectamine 2000 (Invitrogen). 24 h and 48 h after transfection cells were used for western blotting. Cells were lysed, separated in 10% SDS-polyacrylamide gel and immunoblotted for c-Myb, Foxp1 and Survivin with the same antibodies as used for IHC.

Fluorescence in situ hybridization (FISH) MTT assay C-MYC, BCL2 and BCL6 gene rearrangements were investigated by interphase FISH on paraffin sections as previously described (Wang et al., 2013). Probes used in this study included C-MYC, BCL2 and BCL6 (Dual Color Break Apart Rearrangement Probe) (Vysis, Abbott Laboratories, Abbott Park, IL). The slides were examined by using a fluorescence microscope (BX51; Olympus, Tokyo, Japan) by two investigators independently (M Wang and M Li). Slides with known structural or numerical abnormality for the above probes were used as positive controls, and a case of reactive hyperplasia of the tonsil was used as a negative control. In each case, around 100–200 nuclei from at least 5–8 areas were examined. Nuclei with apparent overlapping or truncation were excluded from the analysis. The results were defined as follows: normal: the nucleus in the presence of two fusion signals (orange); and gene break apart: the nucleus in the presence of one fusion signal (orange),one green signal and one red signal.

MTT assay was performed as reported previously (Wang et al., 2009). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma) was added to cells and incubated for 4 h at 37 °C. The cells were centrifuged and the supernatant was removed. DMSO (Sigma) was added and absorption was measured at 540 nm in an ELISA reader. Statistical analysis The correlation of miRNA expression levels with EBV status was analyzed with the unpaired t-test. All p values were two-sided and p b 0.05 was considered to be significant. SPSS 14.0 software was used for the analysis. Results

RNA isolation, miRNA profiling and clustering analysis

Clinical and pathological features

Total RNA was extracted from paraffin-embedded tissues using RecoverAll™ Total Nucleic Acid Isolation Kit (Catalog Number: AM1975; Applied Biosystems, Foster City, CA, USA) as reported previously (Ma et al., 2012). Total RNA was quantified by microfluidic analysis. qRT-PCR profiling for 183 mature miRNAs (Applied Biosystems, Foster City, USA) was carried out according to manufacturers' protocols as reported previously (Tan et al., 2009). Mean Ct values were used for normalization of miRNA expression (ΔCt = CtmiRNA − Ctmean). Relative expression levels were determined with the formula 2− ΔCt. U6 was used for normalization.

In this study, we collected 28 pediatric intestinal BL tissues. There was a male predominance in the disease group, with the male to female ratio being 5:2. The median age at diagnosis was 6.1 years. All tumors occurred in the intestinal region. Tumor stage (St. Jude staging system) was obtained in 22 cases of BL. 11 (50.0%) patient tumors are classified as St. Jude stage II and 11 (50.0%) patient tumors are classified as St. Jude stages III–IV. Two patients with St. Jude stages III–IV died one month after surgery. Histologically, BL consists of sheets of a monotonous population of medium sized lymphoid cells with high proliferative activity and

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apoptotic activity. The “starry sky” appearance can be seen in all cases. Tumor cells strongly express markers of B cell differentiation (CD20, CD19) as well as CD10, and BCL6. Tumor cells are negative for BCL2 and mean Ki-67 proliferation index (PI) is over 90%. All cases are characterized by C-MYC gene rearrangement but not BCL2 or BCL6 rearrangement. miRNA profiling for BL and RLH To search for potential miRNAs in the course of the development of BL malignancy, we globally analyzed the miRNA expression profiles of 1 RLH sample (RNA from 3 different patients) and 1 BL sample (RNA from 3 different patients). Out of 183 miRNAs assessed, 86 miRNAs were expressed (Ct b 35) in BL and RLH. We focused on the 15 most abundant miRNAs in BL (Fig. 1). Nine of these 15 miRNAs, namely miR-150, miR-142-3p, miR-342, miR-25, miR-15b, miR-92, miR-16, miR-21 and miR-93, showed decreased expression levels in BL while 6 miRNAs, miR-127, miR-30c, miR-17-5p, miR-181b, miR-146 and miR34a, showed a slightly higher expression level in BL (Fig. 1). Compared with RLH, the expression of miR-150 was obviously decreased in BL, whereas the expression of miR-34a and miR-17-5p was increased in BL. In addition, miR-17-5p is a member of the miR-1792 polycistron (miR-17-3p, miR-17-5p, miR-18a, miR-19a, miR-20a, miR-19b, miR-92a), which is a potential human oncogene that plays a causative role in lymphomagenesis by coordinating multiple oncogenic pathways (He et al., 2005). Therefore, miR-17-5p, miR-34a and miR150 were chosen for further validation and analysis. qRT-PCR for BL and RLH To confirm the changes in expression of miR-17-5p, miR-34a and miR-150, validation experiments were carried out by qRT-PCR in 28 BL cases and 8 RLH cases. Analysis showed that there was no significant upregulation in miR-17-5p or miR-34a expression in BL compared with RLH (p N 0.05, Figs. 2A–B). There was a significant reduction in miR-150 expression in BL compared with RLH (p b 0.001, Fig. 2C). No correlation of the expression levels of miR-17, miR-34a and miR-150 with EBV status Among 28 BL cases, 13 (46.4%) patients are EBV+. We did not find a significant correlation between the expression level of any of these 3 miRNAs (miR-17, miR-34a and miR-150) and EBV status (Figs. 2D–F). Although not significant, these 3 miRNA expression levels were higher in EBV− BL samples than in EBV+ BL.

Relative expression

2.5

2.0

RLH BL

1.5

1.0

0.5

0.0

50 -3p 342 -25 15b -17 -92 -16 -93 -21 30c 81b 146 127 34a - -1 iR iR iR iR iR iR 42 Rm miR m m m m m miR iR miR miR miR -1 mi iR m

-1

iR

m

m

Fig. 1. The 15 most abundant miRNA expression levels (relative to U6) in BL and their respective expressions in RLH by miRNA profiling. BL: Burkitt lymphoma; RLH: reactive lymphoid hyperplasia.

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miR-150 ISH demonstrated an expression pattern in BL and RLH In view of the differential miRNA expression in BL and RLH, miR-150 was chosen for miRNA ISH. The scramble control probe shows no significant staining in BL and RLH, as expected (Figs. 3A–B). The snRNA U6 ISH signal is mainly expressed in the cytoplasm and nucleus in all cells of (Fig. 3C) RLH tissue and (Fig. 3D) BL tissue. For miR-150, relatively strong and homogeneous staining was observed for the cells outside the germinal centers (GCs), whereas only few cells within the GCs stained positive in RLH tissue (Fig. 3E). The miR-150 ISH signal is weakly expressed in BL cells (Fig. 3F). This pattern was observed in all 8 RLH cases and 8 BL cases that could be analyzed. Possible role of miR-150 in BL In view of the dynamic expression patterns of miR-150 throughout the germinal center reaction and its absence in proliferation centers of CLL cases, we investigated the possible role of miR-150 in the regulation of BL. Two proven targets for miR-150, c-Myb and Survivin were examined by immunohistochemical staining. The c-Myb and Survivin staining patterns were inversely correlated with miR-150 ISH staining patterns in RLH and BL (Figs. 4A–B), supporting the predicted targeting by miR-150. Considering the dynamic and differential expression patterns of miR-150 during the B-cell development and its low expression or absence in human lymphoma cell lines, we explored the potential role of miR-150 in BL in vitro. By transfection with miR-150 mimics, expression of miR-150 was induced in Raji (confirmed by qRT-PCR, data not shown). MTT analysis showed that re-expression of miR-150 significantly reduced the proliferation of Raji cells (p b 0.05) (Fig. 5A). Especially after 24 h, the proliferation of Raji cells with miR-150 mimics' transfection can reduce about 50% of cell growth. Western blot analysis of cell lysates showed repression of c-Myb and Survivin in the cells transfected with miR-150 (Fig. 5B). Discussion In this study we compared the miRNA profiles in BL and RLH tissues using miRNA expression profiling, which was also validated by qRT-PCR and miRNA-ISH. miR-150 showed obviously decreased expression levels in BL, which suggests that changes in miRNA expression patterns may occur during tumorigenesis of BL. The expression of miR-150 was significantly downregulated in BL tumor samples compared with RLH non-tumor tissues. Chang et al. showed that miR-150 is downregulated by C-MYC and injection of mouse lymphoma cell lines into mice expressing miR-150 produced fewer tumor cells in vivo (Chang et al., 2008). The molecular feature of BL is the high expression of C-MYC, which maybe the cause of downregulation of miR-150 expression. Robertus et al. also found that miR150 was significantly downregulated in BL compared with other types of NHL (Robertus et al., 2010). Downregulation of miR-150 expression acts as an anti-apoptotic factor in human colorectal cancer and NK/T cell lymphoma (Ma et al., 2012; Watanabe et al., 2011). Our data are consistent with these published studies that provide evidence for changes in miR-150 expression promoting tumor formation. Taken together, these findings also support a causal role for changes in miR-150 expression during tumorigenesis. In our studies, 46.4% (13/28) patients are EBV+, which is in accordance to previous studies (Chuang et al., 2007; Lu et al., 2011). Although epidemiological evidence clearly points to a role of the virus in African cases, the role of EBV in the pathogenesis of BL has remained largely elusive (Bornkamm, 2009). Recently, Chen et al. (2013) found that reexpression of miR-150 reduced the proliferation of Daudi and Raji cells, which are of EBV-positive germinal center B-cell origin. However, no significant changes were observed in BJAB or Ramos cells, which are of EBV-negative germinal center B-cell origin. They concluded that

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Fig. 2. qRT-PCR analysis of specific miRNA expression levels (relative to U6). (A–C) Differential expression of specific miRNAs in BL and RLH: significant downregulation of miR-150 in BL (p b 0.05). (D–F) Differential expression of specific miRNAs in EBV + BL and EBV− BL: lower expression of specific miRNAs (miR-17, miR-34a and miR-150) in EBV + BL than in EBV − BL. However, there is no significant difference of miRNA (miR-17, miR-34a and miR-150) expression in EBV+ BL and EBV− BL (p N 0.05). BL: Burkitt lymphoma; EBV+: EBV positive; EBV−: EBV negative.

miR-150 can induce EBV-positive BL differentiation (Chen et al., 2013). However, our results showed that there was no correlation of the expression levels of miR-17, miR-34a and miR-150 with EBV status in BL. The controversies on the correlation between miR-150 expression and EBV status in BL may account for different samples, which need to be further studied. Our group has previously demonstrated that low miR-150 expression levels were found in GC B-cells sorted from normal tonsil (Tan

et al., 2009). BL cells are considered to be of memory B-cell origin. Our RNA-ISH also indicated that miR-150 is weakly expressed in BL cells. In RLH, relatively strong and homogeneous staining of miR-150 was observed for the cells outside the germinal centers (GCs), whereas only few cells within the GCs stained positive. These data appear to be consistent with the qRT-PCR data of BL samples that show a significant lower expression level of miR-150 compared with RLH samples. These results also suggest that miR-150 might play a role in the GCs of RLH. Recently,

Fig. 3. ISH analysis of miR-150 expression patterns in BL and RLH. Tissue sections were incubated with a full length DIG-labeled LNA probes to scramble, snRNA U6 and miR-150. The positive staining of cells was expressed as blue-violet. A, B: The scramble control probe shows no significant staining in BL and RLH. C, D: The snRNA U6 ISH signal is mainly expressed in the cytoplasm and nucleus in all cells of RLH tissue (C) and BL tissue (D). E, F: The miR-150 ISH signal is strongly expressed in the cytoplasm, especially outside of germinal center cells in RLH (E), and is weakly expressed in BL cells (F). 20× magnification for all samples. ISH: in situ hybridization. BL: Burkitt lymphoma. RLH: reactive lymphoid hyperplasia.

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Fig. 4. miR-150 and protein target expression patterns in BL and RLH. A, B: Higher magnifications show that miR-150 is strongly expressed in RLH, especially outside of germinal center cells (A), but is weakly expressed in BL cells showing cytoplasm staining (B). C, D: IHC shows that c-Myb is especially expressed in germinal center cells of RLH (C), but is diffusely expressed in BL cells showing nuclear staining (D). E, F: IHC shows that Survivin is especially expressed in germinal center cells of RLH (E), but is diffusely expressed in BL cells showing nuclear staining (F). 40× magnification for all samples. ISH: in situ hybridization. BL: Burkitt lymphoma. RLH: reactive lymphoid hyperplasia. IHC: immunohistochemistry.

Xiao et al. demonstrated that ectopic miR-150 expression in pro-B cells resulted in an increase in cell death (Xiao et al., 2007). It was concluded that miR-150 exerts this effect by suppression of its target, c-Myb, which plays an important role during B-cell development, maintenance of proliferation as well as cell cycle control of hematopoietic cells (Nakata et al., 2007). This coincides with our findings, where in GCs and BL, expression of c-Myb indeed was strong while expression of miR-150 that negatively regulates c-Myb was found to be relatively lower. Similarly, in GCs and BL sections, Survivin showed an inverse staining pattern with miR-150. According to our western blotting results, induction of miR-150 resulted in downregulation of c-Myb and Survivin, supporting the targeting by miR-150. Nonetheless, we cannot exclude the possibility of an indirect regulation pathway for downregulation of Survivin and Foxp1. Our results showed that re-expression of miR-150 reduced the proliferation of Raji cells, which suggest that miR-150 could provide new insights into novel targeted therapeutic strategies in BL. By transducing miR-150 into NK/T-cell lymphoma cells, Watanabe et al. (2011) showed that overexpression of miR-150 increased the incidence of apoptosis and reduced cell proliferation. Moreover, Ma et al. (2012) also demonstrated

that low miR-150 expression in colorectal cancer was associated with an unfavorable response to chemotherapy. Thus, promoting miR-150 activity may be helpful in BL treatment, especially elderly adults with the prognosis of intensive chemotherapy. In summary, deregulation of miR-150 may be useful as a diagnostic tool in BL, based on miRNA profile screening, qRT-PCR and miRNAISH. miR-150 plays a pivotal role in BL by targeting c-Myb and Survivin. Re-expression of miR-150 reduced the proliferation of Raji cells, which suggests it to be a promising novel candidate for tumor treatment.

Funding resources This study was financially supported by the grants from National Natural Science Foundation of China (81001054).

Conflict of interest statement There is no conflict of interest statement in the manuscript.

Fig. 5. The possible role of miR-150 in B-cell growth/survival. A: MTT analysis of Raji cell line growth status after control mimic or miR-150 mimic transfection. Results were expressed as percentage of cell growth in cells not transfected with any mimics. Each bar represents the mean value for three times experiment results ± SD. *p b 0.05 is significantly different from control. B: Down regulation of c-Myb and Survivin upon miR-150 overexpression in Raji cell line.

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References Bornkamm, G.W., 2009. Epstein–Barr virus and the pathogenesis of Burkitt's lymphoma: more questions than answers. Int. J. Cancer 124, 1745–1755. Calin, G.A., Croce, C.M., 2006. MicroRNA signatures in human cancers. Nat. Rev. Cancer 6, 857–866. Chang, T.C., Yu, D., Lee, Y.S., Wentzel, E.A., Arking, D.E., West, K.M., Dang, C.V., ThomasTikhonenko, A., Mendell, J.T., 2008. Widespread microRNA repression by Myc contributes to tumorigenesis. Nat. Genet. 40, 43–50. Chen, S., Wang, Z., Dai, X., Pan, J., Ge, J., Han, X., Wu, Z., Zhou, X., Zhao, T., 2013. Re-expression of microRNA-150 induces EBV-positive Burkitt lymphoma differentiation by modulating c-Myb in vitro. Cancer Sci. 104 (2013), 826–834. Chuang, S.S., Ye, H., Du, M.Q., Lu, C.L., Dogan, A., Hsieh, P.P., Huang, W.T., Jung, Y.C., 2007. Histopathology and immunohistochemistry in distinguishing Burkitt lymphoma from diffuse large B-cell lymphoma with very high proliferation index and with or without a starry-sky pattern: a comparative study with EBER and FISH. Am. J. Clin. Pathol. 128, 558–564. Di, N.M., Carlo-Stella, C., Mariotti, J., Devizzi, L., Massimino, M., Cabras, A., Magni, M., Matteucci, P., Guidetti, A., Gandola, L., Gianni, A.M., 2004. High response rate and manageable toxicity with an intensive, short-term chemotherapy programme for Burkitt's lymphoma in adults. Br. J. Haematol. 126, 815–820. Fallon, S.C., Redell, M.S., El-Bietar, J., Lopez, M.E., Vasudevan, S.A., Brandt, M.L., 2013. Intestinal perforation after treatment of Burkitt's lymphoma: case report and review of the literature. J. Pediatr. Surg. 48, 436–440. Ferracin, M., Pedriali, M., Veronese, A., Zagatti, B., Gafa, R., Magri, E., Lunardi, M., Munerato, G., Querzoli, G., Maestri, I., Ulazzi, L., Nenci, I., Croce, C.M., Lanza, G., Querzoli, P., Negrini, M., 2011. MicroRNA profiling for the identification of cancers with unknown primary tissue-of-origin. J. Pathol. 225, 43–53. He, L., Hannon, G.J., 2004. MicroRNAs: small RNAs with a big role in gene regulation. Nat. Rev. Genet. 5, 522–531. He, L., Thomson, J.M., Hemann, M.T., Hernando-Monge, E., Mu, D., Goodson, S., Powers, S., Cordon-Cardo, C., Lowe, S.W., Hannon, G.J., Hammond, S.M., 2005. A microRNA polycistron as a potential human oncogene. Nature 435, 828–833. Karpova, M.B., Schoumans, J., Ernberg, I., Henter, J.I., Nordenskjold, M., Fadeel, B., 2005. Raji revisited: cytogenetics of the original Burkitt's lymphoma cell line. Leukemia 19, 159–161. Li, Y., Huang, S., Wang, X., Zhou, D., Huang, K., Guo, H., Fang, J., Chen, C., Liu, Q., 2010. Rituximab combined with autologous peripheral blood stem cell transplantation improve therapeutic effects of chemotherapy in pediatric patients with Burkitt's lymphoma. J. Trop. Pediatr. 56, 337–341. Li, X., Chen, J., Hu, X., Huang, Y., Li, Z., Zhou, L., Tian, Z., Ma, H., Wu, Z., Chen, M., Han, Z., Peng, Z., Zhao, X., Liang, C., Wang, Y., Sun, L., Chen, J., Zhao, J., Jiang, B., Yang, H., Gui, Y., Cai, Z., Zhang, X., 2011. Comparative mRNA and microRNA expression profiling of three genitourinary cancers reveals common hallmarks and cancer-specific molecular events. PLoS One 6, e22570.

Lu, J., Getz, G., Miska, E.A., Alvarez-Saavedra, E., Lamb, J., Peck, D., Sweet-Cordero, A., Ebert, B.L., Mak, R.H., Ferrando, A.A., Downing, J.R., Jacks, T., Horvitz, H.R., Golub, T.R., 2005. MicroRNA expression profiles classify human cancers. Nature 435, 834–838. Lu, B., Zhou, C., Yang, W., Huang, H., Gao, Z., He, Y., Liu, H., Zhou, X., Gong, L., 2011. Morphological, immunophenotypic and molecular characterization of mature aggressive B-cell lymphomas in Chinese pediatric patients. Leuk. Lymphoma 52, 2356–2364. Lyng, M.B., Laenkholm, A.V., Sokilde, R., Gravgaard, K.H., Litman, T., Ditzel, H.J., 2012. Global microRNA expression profiling of high-risk ER+ breast cancers from patients receiving adjuvant tamoxifen mono-therapy: a DBCG study. PLoS One 7, e36170. Ma, Y., Zhang, P., Wang, F., Zhang, H., Yang, J., Peng, J., Liu, W., Qin, H., 2012. miR-150 as a potential biomarker associated with prognosis and therapeutic outcome in colorectal cancer. Gut 61, 1447–1453. Molyneux, E.M., Rochford, R., Griffin, B., Newton, R., Jackson, G., Menon, G., Harrison, C.J., Israels, T., Bailey, S., 2012. Burkitt's lymphoma. Lancet 379, 1234–1244. Nakata, Y., Shetzline, S., Sakashita, C., Kalota, A., Rallapalli, R., Rudnick, S.I., Zhang, Y., Emerson, S.G., Gewirtz, A.M., 2007. c-Myb contributes to G2/M cell cycle transition in human hematopoietic cells by direct regulation of cyclin B1 expression. Mol. Cell Biol. 27, 2048–2058. Osier, F.H., Berkley, J.A., Newton, C.R., 2006. Life-threatening hyponatraemia and neurotoxicity during chemotherapy for Burkitt's lymphoma. Trop. Dr. 36, 177–178. Robertus, J.L., Kluiver, J., Weggemans, C., Harms, G., Reijmers, R.M., Swart, Y., Kok, K., Rosati, S., Schuuring, E., van, I.G., Pals, S.T., Kluin, P., van den Berg, A., 2010. MiRNA profiling in B non-Hodgkin lymphoma: a MYC-related miRNA profile characterizes Burkitt lymphoma. Br. J. Haematol. 149, 896–899. Tan, L.P., Wang, M., Robertus, J.L., Schakel, R.N., Gibcus, J.H., Diepstra, A., Harms, G., Peh, S.C., Reijmers, R.M., Pals, S.T., Kroesen, B.J., Kluin, P.M., Poppema, S., van den Berg, A., 2009. miRNA profiling of B-cell subsets: specific miRNA profile for germinal center B cells with variation between centroblasts and centrocytes. Lab. Invest. 89, 708–716. Wang, M., Atayar, C., Rosati, S., Bosga-Bouwer, A., Kluin, P., Visser, L., 2009. JNK is constitutively active in mantle cell lymphoma: cell cycle deregulation and polyploidy by JNK inhibitor SP600125. J. Pathol. 218, 95–103. Wang, M., Zhou, C., Sun, Q., Cai, R., Li, Y., Wang, D., Gong, L., 2013. ALK amplification and protein expression predict inferior prognosis in neuroblastomas. Exp. Mol. Pathol. 95, 124–130. Watanabe, A., Tagawa, H., Yamashita, J., Teshima, K., Nara, M., Iwamoto, K., Kume, M., Kameoka, Y., Takahashi, N., Nakagawa, T., Shimizu, N., Sawada, K., 2011. The role of microRNA-150 as a tumor suppressor in malignant lymphoma. Leukemia 25, 1324–1334. Xiao, C., Calado, D.P., Galler, G., Thai, T.H., Patterson, H.C., Wang, J., Rajewsky, N., Bender, T. P., Rajewsky, K., 2007. MiR-150 controls B cell differentiation by targeting the transcription factor c-Myb. Cell 131, 146–159.