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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References
  9. Supporting Information

MYEOV and NEGR1 are novel candidate gene targets in neuroblastoma that were identified by chromosomal gain in 11q13 and loss in 1p31, respectively, through single nucleotide polymorphism array analysis. In the present study, to assess the involvement of MYEOV and NEGR1 in the pathogenesis of neuroblastoma, we analyzed their mutation status and/or expression profiles in a panel of 55 neuroblastoma samples, including 25 cell lines, followed by additional functional studies. No tumor-specific mutations of MYEOV or NEGR1 were identified in our case series. Expression of MYEOV was upregulated in 11 of 25 cell lines (44%) and in seven of 20 fresh tumors (35%). The siRNA-mediated knockdown of MYEOV in NB-19 cells, which exhibit high expression of MYEOV, resulted in a significant decrease in cell proliferation (= 0.0027). Conversely, expression studies of NEGR1 revealed significantly lower expression of this gene in neuroblastomas at an advanced stage of the disease. Exogenous NEGR1 expression in neuroblastoma cells induced significant inhibition of cell growth (= 0.019). The results of these studies provide supporting evidence for MYEOV and NEGR1 as gene targets of 11q13 gains and 1p31 deletions in a neuroblastoma subset. In addition, the findings suggest a possible prognostic value for NEGR1 in neuroblastoma. (Cancer Sci 2011; 102: 1645–1650)

Neuroblastoma is one of the most common forms of solid tumors in childhood and accounts for approximately 15% of all pediatric cancer deaths.(1) Despite recent advances in chemoradiotherapy, the prognosis for advanced neuroblastoma remains poor, with an approximate 40% 5-year survival, underscoring the importance of developing novel therapeutic modalities on the basis of an understanding of the pathogenesis of neuroblastoma.(1) Conversely, knowledge of the molecular pathogenesis of neuroblastoma is largely limited in terms of targets, except for the role of MYCN amplifications in advanced neuroblastoma.(2) Thus, the recent discovery of ALK mutations/amplifications in 6–8% of neuroblastomas(3–6) represents a major development in neuroblastoma research because it not only unravels a novel molecular mechanism involved in neuroblastoma development, but could also a basis for the development of molecular-targeted therapies using ALK inhibitors.(3–6) Similar to a number of novel genetic targets discovered recently in other human cancers, ALK mutations were identified thorough genome-wide analyses of copy numbers using high-throughput technologies, including high-density single nucleotide polymorphism (SNP) genotyping microarrays.(3–6) A number of recurrent copy number changes other than those of the ALK locus have been identified by genome-wide copy number analysis of neuroblastoma, including losses of 1p31, 3q13, 9p24, 15q11, and 16p13, and high-grade amplifications of 1p36, 7q21, 7q31, 11q13, and 15q13,(3) which may provide important clues for the identification of novel target genes. In fact, several candidate target genes of these common deletions and amplifications have been identified, including MYEOV as the target of gains/amplifications in 11q13(7) and NEGR1 as a candidate tumor suppressor in 1p31 deletions.(8) Previously, MYEOV was reported as a putative transforming gene within the 11q13 amplicons in multiple myeloma,(9) whereas NEGR1 was described as a member of the IgLON (limbic system-associated membrane protein [LAMP]/opioid-binding cell adhesion molecule [OBCAM]/neurotrimin subgroup of the immunoglobulin superfamily) family of cell adhesion molecules.(8) However, the involvement of these genes aberrations in the pathogenesis of neuroblastoma remains unknown. Therefore, in the present study we focused on the abnormalities in both genes and assessed their role, both genetically and functionally, in the pathogenesis of neuroblastoma.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References
  9. Supporting Information

Specimens.  Primary neuroblastoma specimens were obtained at the time of surgery or biopsy from patients who had been diagnosed with neuroblastoma and had been admitted to Tokyo University Hospital, Saitama Children’s Medical Center, or various other hospitals between November 1993 and October 2006. Patients were staged according to the International Neuroblastoma Staging System,(10) with five patients classified as Stage 3 and 25 classified as Stage 4. The clinicopathological findings for all patients are listed in Table 1. Twenty-five neuroblastoma cell lines were also used in the present study (Table 2). The SCMC-N2 series was established in our laboratory;(11) the SJNB series and UTP-N-1(12) were generous gifts from Drs A.T. Look (Department of Pediatric Oncology, Dana Farber Cancer Institute, Harvard Medical School, Boston, USA) and A. Inoue (Department of Molecular Biology, Toho University School of Medicine, Tokyo, Japan), respectively; all other cell lines were obtained from the Japanese Cancer Resource Cell Bank (http://cellbank.nibio.go.jp/wwwjcrbj.htm, accessed 7 Sep 2008). All cells were maintained in RPMI 1640 medium (Gibco-BRL, Grand Island, NY, USA) supplemented with 10% fetal bovine serum in a humidified atmosphere containing 5% CO2 at 37°C.

Table 1.   Clinical data for the neuroblastoma cases in the present study
Case no.AgeStageDiagnosisHistologyMYCN amplificationOutcome
  1. C, clinical; MS, mass screening program; NBL, neuroblastoma; NBL poorly dif., poorly differentiated neuroblastoma; GNB, ganglioneuroblastoma; GNB well dif., well-differentiated ganglioneuroblastoma.

 14 years 2 months4CNBL poorly dif.+Alive
 22 years 4 months4CNBL poorly dif.+Alive
 34 years4CNBL poorly dif.+Alive
 43 years4CNBL poorly dif.Alive
 51 year 5 months4CNBL poorly dif.Alive
 610 day3CNBL dif.Alive
 74 years4CNBL poorly dif.Dead
 84 years 2 months4CNBL poorly dif.Alive
 92 years3CGNB well dif.Alive
1010 years4CNBLDead
114 years4CNBL+Dead
123 years3CNBL+Alive
1311 years 9 months4CNBL poorly dif.Alive
146 months3MSGNBAlive
157 months4MSNBL poorly dif.Dead
164 years4CNBL+Dead
174 years 9 months4CNBLDead
187 months4MSNBLAlive
192 years4CNBL poorly dif.+Alive
203 years4CNBL+Dead
218 years4CNBL poorly dif.Alive
222 years 3 months4CNBL+Alive
234 years4CNBL+Dead
245 months4CNBLAlive
255 years4CNBLDead
264 years 10 months4CNBLAlive
277 years4CNBL poorly dif.+Dead
281 year 6 months3CNBLAlive
291 year 8 months4CNBLAlive
308 months4CNBLAlive
Table 2.   Neuroblastoma cell lines used in the present study
Cell lineMYCN amplification
CHP-134
GOTO+
IMR-32+
LAN-1+
LAN-2+
LAN-5+
NB-1
NB-16+
NB-19+
NB-69
NH-12+
SCMC-N2+
SCMC-N4+
SCMC-N5+
SJNB-1
SJNB-2+
SJNB-3
SJNB-4+
SJNB-5+
SJNB-6+
SJNB-7+
SJNB-8+
SK-N-SH
TGW+
UTP-N-1+

Semi-quantitative RT-PCR.  Total RNA was extracted from the 25 cell lines and 20 frozen stocked tumors using Isogen reagent (Nippon Gene, Osaka, Japan) according to the manufacturer’s instructions and was subjected to reverse-transcription reactions to synthesize cDNA using the SuperScript Preamplification System for First Strand cDNA synthesis (Life Technologies, Rockville, MD, USA). Semi-quantitative RT-PCR analysis for MYEOV, CCND1, and NEGR1 gene expression was performed as described previously(13) using the primer sets listed in Table S1, available as an accessory publication to this paper. The concentration of the cDNA was normalized against that of β-actin, used as an internal control. The signal intensity of MYEOV and CCND1 expression was estimated using NIH Image 1.61 software (Wayne Rasband; National Institutes of Health, Bethesda, MD, USA).

Quantitative RT-PCR.  To quantify the expression levels of NEGR1, real-time PCR (RQ-PCR) analysis was performed using the QuantiTect SYBR Green PCR kit (Qiagen, Tokyo, Japan) with an iCycler iQ real-time PCR detection system (Bio-Rad Japan, Tokyo, Japan). The primer sets used for the RQ-PCR are listed in Table S1 and the PCR conditions were as described previously.(13) For the purpose of normalization, relative expression levels were calculated by dividing the expression level of the respective gene by that of β-actin.

Mutational analysis of MYEOV and NEGR1 genes.  Genetic screening for MYEOV and NERG1 genes in 25 cell lines was performed by denaturing HPLC (DHPLC) using the WAVE System Model 4500 (Transgenomic, Omaha, NE, USA), as described previously.(14) The primer sets used in the present study are listed in Table S1.

Bisulfate modification and methylation-specific PCR.  Bisulfate modification of genomic DNA was performed as described previously.(15) For methylation-specific PCR (MSP), approximately 10 ng bisulfite-treated DNA was amplified with primers for both the methylated and unmethylated sequences. Reaction products were separated by electorophoresis on a 2.0% agarose gel. The primer sets for methylation-specific PCR analysis are listed in Table S1.

Knockdown of MYEOV using siRNA.  The functional roles of the MYEOV gene in neuroblastoma cells was assessed using gene knockdown with siRNA.(16) The siRNA was designed and synthesized for silencing MYEOV (Invitrogen, Carlsbad, CA, USA). The siRNA duplex had the following sequences: 1132 sense, 5′-UCA ACG CCC ACU CUA AAG GCU UCU C-3′; and 1132 antisense, 5′-GAG AAG CCU UUA GAG UGG GCG UUG A-3′. A chemically synthesized non-silencing siRNA duplex that had no known homology to any mammalian gene was used as a control for non-specific silencing events and had the following sequences: sense, 5′-UUC UCC GAA CGU GUC ACG UdT dT-3′; and antisense, 5′-ACG UGA CAC GUU CGG AGA AdT dT-3′. Gene knockdown was achieved in NB-19, CHP-134 and PF-SK-1 cells using HiPerFect transfection reagent (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions.

Transient transfection.  The expression vector (pME18S) containing the full-length EcoRI-Xba1 fragment of the NEGR1 cDNA was transfected into NB-19, SJNB-7, and PF-SK-1 cells using the lipofection method according to the manufacturer’s instructions (Qiagen).(11) Briefly, 1.5 × 105 cells were seeded in a six-well plate and incubated in 1.6 mL RPMI 1640 (Gibco-BRL) with 10 μL Effectance reagent (Qiagen), 3.2 μL Enhancer (1:8) (Qiagen), 10 μL Effectene (Qiagen), and 0.4 μg expression vector. Cells were counted 72 h after transfection.

Statistical analysis.  Expression of the NEGR1 gene was compared between favorable and unfavorable cases of neuroblastomas using the Mann–Whitney U-test. Exact 95% confidence intervals (CI) of the proportions were calculated on the basis of binomial distribution. The Kruskal–Wallis test was used to compare the functional effects of MYEOV inhibition and NEGR1 expression in neuroblastoma cells.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References
  9. Supporting Information

Gain and high-grade amplification of 11q13 involving the MYEOV locus in neuroblastoma.  In the present series, gains of chromosome 11q13 were detected in multiple neuroblastoma cases.(3) Within this gain, a high-grade amplification was found in a single case with Stage 4 disease (Case 22; Fig. 1a). The critical amplicon that had minimum overlapping amplification/gain was found in a 340-kb region exclusively containing MYEOV, located 360 kb upstream from the CCND1 locus(7) (Fig. 1a). Previously, MYEOV had been identified as a putative transforming gene based on the NIH/3T3 tumorigenicity assay(9) and was shown to be highly expressed in a subset of multiple myelomas harboring t(11;14)(q13;q32).(7) We further examined the expression patterns of MYEOV in a total of 45 neuroblastoma samples using semi-quantitative RT-PCR analysis, in which 11 of 25 cell lines (44%) and seven of 20 fresh tumors (35%) showed higher expression levels of MYEOV compared with the median expression level (MYEOV/β-actin signal intensity = 1.4; Fig. 1b). Although most tumors exhibited increased expression of both CCND1 and MYEOV, Case 22 showed high expression of MYEOV but not CCND1 (Fig. 1c). Mutational analysis of the coding region of MYEOV was also performed in 25 cell lines, but no tumor-specific mutations were detected.

image

Figure 1.  Gains and high-grade amplification on chromosome 11q13 in neuroblastoma. (a) A common 340-kb region having copy number (CN) gains contains a single known gene, MYEOV. In addition, CCND1 is frequently contained in CN gains at 11q13, but mapped outside the minimum region of common CN gains. Red bars, gains (3 < CN < 5); green bars, losses (CN = 1); light red bar (circled), high-grade amplification (CN ≥ 5). (b) Representative results of MYEOV expression in fresh tumors and cell lines (RNA from normal muscle was used as a control). (c) Expression of MYEOV and CCND1 in Cases 22 and 23 (RNA from normal muscle was used as a control). The expression of MYEOV in Case 22 tended to be higher than that in Case 23. tel, telomere; centr, centromere.

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Homozygous deletion on 1p31 detected in neuroblastoma.  Detection of homozygous deletions was also of interest because they provide an important clue in pinpointing tumor suppressor loci. In an allele-specific copy number analyzer for GeneChip (CNAG) and allele-specific copy number analysis using anonymous references (AsCNAR), homozygous deletions could be identified as the loss of both parental alleles, even in the presence of significant components of normal tissues.(3) In the present study, 70 homozygous deletions were identified at 50 independent loci in the neuroblastoma samples. Unfortunately, we were not able to completely exclude the possibility that some may represent copy number variations (CNV) rather than real homozygous deletions, because paired DNA was available only in four primary neuroblastoma cases and many homozygous deletions were found in established neuroblastoma cell lines. Complete loss of genetic material at eight loci was confirmed by genomic PCR (data not shown).

Of the 70 homozygous deletions identified, we focused on a homozygous deletion involving a 370-kb region at 1p31 in NB-19. This region contains a part of NEGR (exon 1 and a part of intron 1), a unique candidate target gene, which was also disrupted by a translocation in another cell line, namely SJNB-6 (Fig. 2a,b). Because NEGR1 encodes a member of the IgLON family of cell adhesion molecule sand has been reported to be a putative tumor suppressor gene in ovarian cancer,(8) we examined its expression in neuroblastoma cases in the present study to evaluate the clinical impact of NEGR1 expression. As shown in Figure 2(c), NEGR1 expression was absent or very low in 10 of 25 (40%) cell lines, as determined by semi-quantitative RT-PCR (Fig. 2c). In quantitative RT-PCR analysis using fresh tumor samples (20 fresh advanced-stage tumors and an additional 20 cases of early stage tumors), the expression of the NEGR1 gene was significantly lower in advanced-stage tumors compared with early stage tumors (= 0.0041; Fig. 2c). Similarly, the expression of the NEGR1 gene was significantly lower in patients who died compared with patients who survived (= 0.018; Fig. S1). Mutation analysis was also performed in neuroblastoma cell lines, but no tumor-specific mutations were detected. Methylation analysis of the promoter region of NEGR1 using 10 neuroblastoma cell lines without NEGR1 expression did not reveal any tumor-specific methylation pattern in neuroblastoma cell lines or fresh neuroblastoma samples (data not shown).

image

Figure 2. NEGR1 as a candidate tumor suppressor gene in neuroblastoma. (a) Deletion mapping of 1p31.1 disclosed a homozygous deletion spanning a 370-kb region in the NB-19 cell line, which contains part of NEGR1 as the only structural gene. The NEGR1 gene is also disrupted in intron 1 by the breakpoint of a segmental duplication at 1p31.1 in another neuroblastoma cell line (SJNB-6). For each panel, total copy numbers (tCN; red dots), moving averages of tCN for five consecutive single nucleotide polymorphisms (SNP; blue line), an ideogram of the relevant chromosome, the location of heterozygous SNP calls (green bars), and allele-specific copy numbers (AsCN) averaged for five consecutive SNP (red and green lines for larger and smaller alleles, respectively) are plotted. Note that the CN are expressed in terms of “observed” signal ratios between tumor and reference samples, where the baseline is adjusted to 2 for tCN plots and to 1 for AsCN. (b) Summary of CN abnormalities of 1p31.1 in neuroblastoma. Red bars, gains (3 < CN < 5); green bars, losses (CN = 1); light green bar (circled), homozygous deletion (CN = 0). A homozygous deletion detected in NB-19 and a chromosomal rearrangement detected in SJNB-6 are indicated by the red arrows. The location of NEGR1 is shown by the blue line. (c) NEGR1 expression in neuroblastoma. Top panel: representative result of NEGR1 expression in neuroblastoma cell lines showing frequently reduced expression levels in a subset of neuroblastoma cell lines. Bottom graph: expression of the NEGR1 gene as measured by quantitative PCR was significantly lower in tumors with an unfavorable outcome than in tumors with a favorable outcome (= 0.0041, Mann–Whitney U-test). tel, telomere; centr, centromere.

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Functional analyses of MYEOV and NEGR1 in neuroblastoma cell lines.  We further evaluated the oncogenic potential of MYEOV using siRNA-mediated gene knockdown in the NB-19 cell line, which highly expresses MYEOV. As shown in Figure 3(a,b). When MYEOV expression was suppressed by siRNA, NB-19 cells exhibited retarded growth compared with the growth of control cells (= 0.0027), indicating that MYEOV positively regulates cell proliferation (Fig. 3a,b). Similar results were obtained in CHP-134 and PF-SK-1 cells (Fig. S2). To assess the tumor suppressor function of NEGR1 in neuroblastoma cells, we generated an NEGR1 expression vector that was transiently transfected into NB-19 cells, in which NEGR1 is homozygously deleted. Expression of NEGR1 significantly suppressed the proliferation of NB-19 cells compared with mock transfection (= 0.019; Fig. 3c,d). In addition, the NEGR1 expression vector was transiently transfected into SJNB-7 and PF-SK-1 cells, in which NEGR1 expression is absent. Following transfection into these cell lines, profound inhibition of cell proliferation was observed for both SJNB-7 and PF-SK-1 cells expressing NEGR1 (Fig. S3).

image

Figure 3.  Effect of MYEOV inhibition by siRNA on cell growth and effect of NEGR1 on cell growth in neuroblastoma cells. (a) Confirmation of siRNA-mediated gene knockdown using semi-quantitative RT-PCR analysis. Following siRNA treatment, MYEOV mRNA was absent in treated cells; however, abundant MYEOV expression was detected in wild-type and control cells. (b) Effect of MYEOV inhibition by siRNA transfected into NB-19 cells on cell growth. Cell growth was impaired cell growth in siRNA-transfected cells compared with that of control cells (*= 0.0027, Kruskal–Wallis test). (c) Analysis (RT-PCR) of NB-19 cells transfected with the pME18S vector. Mock-transfected and wild-type cells were used as controls. (d) The growth of cells transiently expressing NEGR1 was impaired compared with that of mock-transfected and wild-type cells (†= 0.019, Kruskal–Wallis test).

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References
  9. Supporting Information

In the present study, we showed that MYEOV and NEGR1 are candidate gene targets of 11q13 gain and 1p31 deletion, respectively, in a neuroblastoma subset. To our knowledge, this is the first report to describe aberrations of MYEOV and NEGR1 in neuroblastoma.

Initially, MYEOV was reported as a gene that was possibly co-overexpressed with CCND1 in some cases of multiple myeloma with t(11;14)(q13;q32); later, it was shown to be co-amplified and co-overexpressed with CCND1 in a subset of esophageal squamous cell carcinomas, breast cancers, gastric cancers, and colorectal cancers.(7,17–19) Although the major genetic targets of these rearrangements and amplifications have been shown to be CCND1, in some breast cancer cases the 11q13 amplicon exclusively contained MYEOV and not CCDN1, suggesting a CCDN1-independent oncogenic role for MYEOV.(7) The oncogenic role of MYEOV has also been investigated in functional studies, showing that in vitro siRNA-mediated knockdown of MYEOV resulted in inhibition of proliferation, invasion, and migration of colorectal cancer cell lines.(19) In our neuroblastoma cases, MYEOV was overexpressed in approximately 30% of primary neuroblastoma cases, with seven cases showing gain/amplification of MYEOV. We also confirmed that MYEOV was the only gene found in the common gain/amplicon at 11q13 and that proliferation of neuroblastoma cell lines was inhibited by siRNA-mediated MYEOV knockdown, supporting an oncogenic role for MYEOV in some neuroblastoma cases. Although several studies have revealed that MYEOV amplification is associated with poor prognosis in multiple myeloma, esophageal squamous cell carcinoma, and breast cancer,(7,18,20) the clinical impact of MYEOV gain/amplification or overexpression in neuroblastoma is unclear and requires further evaluation.

The NEGR1 gene is a single gene found in one of the recurrent deletions at 1p31. Although the NEGR1 locus is known as one of the most common CNV regions,(21) we also identified a neuroblastoma cell line in which NEGR1 was disrupted in by gene rearrangement, supporting the fact that NEGR1 is one of the target genes in neuroblastoma. In ovarian cancer, NEGR1 is a putative tumor suppressor gene encoding one of the IgLON cell adhesion family members, namely OPCML, and it plays a central role in the establishment and remodeling of the central nervous system.(22) Notably, OPCML has been shown to exhibit functional characteristics of a tumor suppressor gene in epithelial ovarian cancer.(23) In our analysis, expression of NEGR1 was substantially reduced in 43% of advanced-stage tumors without 1p31 deletions/rearrangement. In addition, re-expression of NEGR1 in the NB-19 cell line with homozygous deletion of NEGR1, as well as in other neuroblastoma cell lines that did not express NEGR1, resulted in the inhibition of cell growth, suggesting that NEGR1 is a candidate tumor suppressor gene in neuroblastoma and may have possible prognostic value. Although expression of OPCML in ovarian cancers is suppressed or reduced mainly through epigenetic mechanisms,(23) tumor-specific methylation was not detected in neuroblastoma cells in the present study. The mechanisms for the absence of NEGR1 in the tumors without homozygous deletion, mutation, and methylation were not clear in the present study. We cannot role out the possibility that mutations are harbored in the promoter region of NEGR1 with consequent gene inactivation. Furthermore, NEGR1 was often heterozygously deleted, but not mutated or methylated, in neuroblastoma; most deletions occur in tumors at advanced stages, suggesting that NEGR1 has haploinsufficient effects on advanced disease in neuroblastoma.

In conclusion, the results of the present study suggest that MYEOV at 11q13 and NEGR1 at 1p31 are functional gene targets in a subset of neuroblastoma. Further studies on both genes will expand these pathways and provide insights into the progression of neuroblastoma, as well as possibly enabling the development of novel therapeutics based on targeting MYEOV and NEGR1 in neuroblastoma.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References
  9. Supporting Information

The authors thank Mrs Matsumura M, Mrs Hoshino N, Mrs Yin Y and Mrs Saito F for their excellent technical assistance. The authors also express their appreciation to Drs A.T. Look (Harvard Medical University, Boston, MA, USA) and Dr. A. Inoue (St. Jude Children’s Research Hospital, Memphis, TN, USA), for their generous gifts of neuroblastoma cell lines. This work was supported by Research on Measures for Intractable Diseases, Health, and Labor Sciences Research Grants; the Ministry of Health, Labor and Welfare via a grant for Research on Health Sciences focusing on Drug Innovation; by the Japan Health Sciences Foundation; and by the Core Research for Evolutional Science and Technology, Japan Science and Technology Agency.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References
  9. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References
  9. Supporting Information

Fig. S1.NEGR1 expression in 30 neuroblastoma cases.

Fig. S2. Effect of siRNA inhibition of MYEOV on cell growth in CHP-134 and PF-SK-1 cells.

Fig. S3. Effect of NEGR1 on cell growth in the neuroblastoma cell lines PF-SK-1 and SJNB-7.

Table S1. Primer sequences used in the present study.

FilenameFormatSizeDescription
CAS_1995_sm_fS1-3-and-tS1.pdf287KSupporting info item

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