IJMM International Journal of Molecular Medicine 1107-3756 1791-244X D.A. Spandidos 10.3892/ijmm.2012.1008 ijmm-30-02-0365 Articles Enrichment of neural-related genes in human mesenchymal stem cells from neuroblastoma patients RODRÍGUEZ-MILLAMIGUEL ÁNGEL1 MIRONESISABEL1 MARIÑAS-PARDOLUIS1 MELENGUSTAVO J.2 CUBILLOISABEL1 RAMÍREZMANUEL2 GARCÍA-CASTROJAVIER1 Cellular Biotechnology Unit, Institute of Health Carlos III, Majadahonda, Madrid; Department of Oncohematology, Hospital Niño Jesús, Madrid, Spain Correspondence to: Dr Javier García-Castro, Institute of Health Carlos III, lab. 51-00-031, Ctra Majadahonda-Pozuelo, km 2, E-28220 Majadahonda, Madrid, Spain, E-mail: jgcastro@isciii.es 8 2012 22 05 2012 30 2 365 373 25 01 2012 22 03 2012 Copyright © 2012, Spandidos Publications 2012

This is an open-access article licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported License. The article may be redistributed, reproduced, and reused for non-commercial purposes, provided the original source is properly cited.

Neuroblastoma (NB) is one of the most common pediatric solid tumors and, like most human cancers, is characterized by a broad variety of genomic alterations. Although mesenchymal stem cells (MSCs) are known to interact with cancer cells, the relationship between MSCs and metastatic NB cancer cells in bone marrow (BM) is unknown. To obtain genetic evidence about this interaction, we isolated BM-derived MSCs from children with NB and compared their global expression patterns with MSCs obtained from normal pediatric donors, using the Agilent 44K microarrays. Significance analysis of microarray results with a false discovery rate (FDR) <5% identified 496 differentially expressed genes showing either a 2-fold upregulation or downregulation between both groups of samples. Comparison of gene ontology categories of differentially expressed genes revealed the upregulation of genes categorized as ‘neurological system process’, ‘cell adhesion’, ‘apoptosis’, ‘cell surface receptor linked signal transduction’, ‘intrinsic to membrane’ and ‘extracellular region’. Among the downregulated genes, several immunology-related terms were the most abundant. These findings provide preliminary genetic evidence of the interaction between MSCs and NB cancer cells in BM as well as identify relevant biological processes potentially altered in MSCs in response to NB.

 neuroblastoma metastasis bone marrow mesenchymal stem cells tumor stroma pediatric cancer Introduction

Neuroblastoma (NB), a poorly differentiated tumor derived from neural crest cells that affects mainly children, is the most common extracranial pediatric solid tumor. The origin of stroma in primary NB tumors, formed by Schwann cells, and whether the stroma is the cause or consequence of the maturation potential of tumor cells remain controversial. It has been hypothesized that crosstalk between Schwann cells and neuroblasts influences the biology and clinical behavior of NB tumors. However, little is known about the role of the NB microenvironment in metastasis localizations, especially in bone marrow (BM). Recent articles suggest that mesenchymal stem cells (MSCs) have a major role in maintaining stem cells niches in the BM (1), as well as in creating the tumor microenvironment (2). In this study we focused in BM-derived MSC from NB patients as a key factor in the development of metastasis.

During the past years several studies have used microarray-based high-throughput technologies to identify biological processes altered in NB cells. Hiyama et al (3) surveyed the differences in gene expression between unfavorable and maturing/regressing NB. Interestingly, in favorable NB, neuronal differentiation signals such as CD44, IGF2, NTRK1 and ANK1 were overexpressed in maturing tumors. Similarly, Kamei et al (4) identified genes that exhibited altered gene expression in NB tumors associated with a favorable outcome. More recently, Chen et al (5) performed parallel global protein and mRNA expression profiling on NB tumors and identified that cell adhesion, nervous system development and cell differentiation processes were downregulated in stage 4 MYCN-amplified NB tumors, suggesting a less mature neural and a more invasive phenotype of this type of cells. Finally, Abel et al (6), based on expression profiling, identified 4 molecular subgroups of NB that can be distinguished by a 6-gene signature. These studies show that microarray techniques are useful tools for gene expression profiling in NB tumors.

It is well known that the BM is a common site for metastasis in patients with high-risk NB. Although little is known about the control of NB tumor growth by the BM microenvironment, there has been an increasing interest in the role of MSCs and their BM niches in cancer (7,8). Some reports implicate MSCs having tumor-promoting effects whereas others show inhibition of tumor growth. Regarding the relationship between MSCs and NB, Ma et al (9) reported that MSCs in BM may enhance metastasis of NB via SDF-1/CXCR4 and SDF-1/CXCR7 signaling. Moreover, CXCR5 may be involved in the attraction of human metastatic NB cells to the BM (10). Despite these findings, the relationship between MSCs and NB cancer cells is still unknown, and research in this area would add new scientific knowledge and provides new therapeutic ideas and targets. Thus, we isolated MSCs from BM of NB patients and control donors and compared their global expression patterns using microarrays. Our findings provide preliminary genetic evidence of the interaction between MSCs and NB cancer cells in BM as well as identify relevant biological processes potentially altered in MSCs in response to NB.

 Materials and methods Mesenchymal stem cell isolation, culture and characterization

MSCs were isolated from 4 NB pediatric patients (with no amplification of the N-Myc gene) and 4 healthy donors. The study protocol was approved by the Ethics Committee of the Hospital. All patients and volunteers were informed about the purpose of the study and provided written consent, by the parents or legal guardians, regarding their participation in the study. BM-derived MSCs were obtained by adherence to plastic. Mononucleated cells were obtained after centrifugation using a Ficoll-Paque gradient. Cells were cultured at 37°C with 5% CO2 in DMEM (Lonza) supplemented with 10% fetal bovine serum (PAN-Biotech GmbH). MSCs cultures were characterized according the International Society for Cellular Therapy criteria (11).

 RNA isolation and cRNA labeling

MSCs were stabilized in PrepProtect™ (Miltenyi Biotec) and total-RNA was isolated using standard RNA extraction protocols (NucleoSpin® RNA II, Macherey-Nagel). RNA integrity and overall quality was checked via the Agilent 2100 Bioanalyzer expert software (Agilent Technologies). All RNA samples revealed an RNA Integrity Number (RIN) between 7.3 and 10. For the linear T7-based amplification step, 1 μg of each total-RNA sample was used. To produce Cy3-labeled cRNA, the RNA samples were amplified and labeled using the Agilent Low RNA Input Linear Amp kit (Agilent Technologies) following the manufacturer’s protocol. Yields of cRNA and the dye-incorporation rate were measured with the ND-1000 Spectrophotometer (NanoDrop Technologies).

 Microarray hybridization

The hybridization procedure was performed according to the Agilent 60-mer oligo microarray processing protocol using the Agilent Gene Expression Hybridization kit (Agilent Technologies). Briefly, 1.65 μg Cy3-labeled fragmented cRNA in hybridization buffer was hybridized overnight (17 h, 65°C) to Agilent Whole Human Genome Oligo Microarrays 4×44K using Agilent’s recommended hybridization chamber and oven. Finally, the microarrays were washed once with 6X SSPE buffer containing 0.005% N-lauroylsarcosine for 1 min at room temperature followed by a second wash with preheated 0.06X SSPE buffer (37°C) containing 0.005% N-lauroylsarcosine for 1 min. The last washing step was performed with acetonitrile for 30 sec. Fluorescence signals of the hybridized Agilent microarrays were detected using Agilent’s Microarray Scanner System (Agilent Technologies). The Agilent Feature Extraction Software (FES) was used to read out and process the microarray image files.

 Microarray data analysis

The microarray raw data have been deposited at the NCBI Gene Expression Omnibus under the accession number GSE35133 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=rdszfaigqkmgqdw&acc=GSE35133). Intensity values of flagged spots below background were filtered out, data were normalized using the array median and the mean values of 3 replicates for each biological sample were calculated. Statistical analysis of microarray significance (SAM) was performed to identify genes with significant changes in expression, and permutations were used to estimate the false discovery rate (FDR) (12). Genes were selected if they showed a 2-fold regulation and a FDR <0.05%. These statistical analyses were performed using TMEV and default parameters were used unless specified (13).

 Gene ontology (GO) analysis

To better characterize the functionally-related genes which showed at least a 2-fold regulation in the microarray analysis, the genes were assigned to Gene Ontologies using a database for annotation, visualization and integrated discovery (DAVID) (14). We used the 3 ontologies produced by the GO consortium, namely ‘biological process’, ‘cellular component’ and ‘molecular function’. GO terms were collected, redundant terms were excluded, and P-values were used to evaluate the significance of the terms.

 Results

We isolated MSCs from NB patients (NB-MSCs) as well as from normal donors. All MSC cultures were characterized according to the International Society for Cellular Therapy criteria: morphology, positive/negative markers and differentiation properties (data not shown) (11). We analyzed the expression profiles of RNA from NB-MSCs compared to those from normal donors, using the Agilent Whole Human Genome Oligo Microarrays. The results from the SAM analysis (≥2-fold regulation and FDR <0.05%) revealed that 454 genes had transcript levels significantly higher in NB-MSCs, whereas 42 genes had transcript levels significantly lower in NB-MSCs (Fig. 1). A list of the genes showing the strongest upregulation in NB-MSCs is shown in Table I whereas the downregulated genes are shown in Table II.

To illustrate the differences between NB and normal samples the genes whose expression was induced or repressed by at least 2.5-fold in NB samples are shown as heat map in the Fig. 2, where NB and normal samples are clearly differentiated. Interestingly, several of the genes are known to play roles in NB (ANXA10, ITGA2, COL4A5 and SHC3) or other types of cancer (TMEFF2, TSPAN8, DCBLD2, PSCDBP and BCAN) (Table I). Table V lists the genes involved in neuronal processes that were >2.0-fold upregulated.

To further examine the differences in the expression profiles between NB-MSCs and normal donors, the 496 significantly up or downregulated genes were analyzed with the DAVID software and classified into the 3 main GO domains. For the upregulated genes, in the gene ontology ‘biological process’ we identified 138 terms, in ‘cellular component’ 23 terms, and in ‘molecular function’ 17 terms. The highest ranked terms are shown in Table III. For the downregulated genes, in the gene ontology ‘biological process’ we identified 24 terms, in ‘cellular component’ 1 term, and in ‘molecular function’ 9 terms. The highest ranked terms are shown in Table IV.

 Discussion

The role of MSCs in tumor progression is unclear as it has been suggested that MSCs may promote or suppress tumor growth (8). Therefore, identifying potential genes regulated in BM-derived MSCs by NB cancer cells would be of great importance to assess their role in determining disease outcome. Previously, Hahn et al (15) studied the effect of conditioned medium of BM cultures in NB cell growth in vitro (15). They showed that BM cultures may stimulate the proliferation and differentiation suppression of NB cells. In this model, monocytes seem to be the mediators of these effects. However, there is no data on how NB cells modify the characteristics of BM-resident cell populations. In this study, we analyzed for the first time the expression profiles of BM-derived MSCs from NB patients and report the identification of 496 genes with more than a 2-fold increase or decrease transcript levels. Our findings suggest that NB cancer cells may have an impact on several processes of MSCs localized in BM.

Interestingly, our microarray analysis revealed that some of the top ranked upregulated genes in NB-MSCs (ANXA10, ITGA2, COL4A5 and SHC3) have been previously reported to have a potential role in NB (Table I). For instance, Annexin A10 (ANXA10) has been identified in a microarray analysis of human NB stem cells as a gene associated with malignancy (16). Similarly, integrin upregulation has been reported as a marker of NB cell differentiation (17). The same study identified the overexpression of COL4A5 in unfavorable NB. Finally, a distinct role of ShcC (SHC3) docking protein in the differentiation of NB has been proposed (18). In addition, we identified several genes reported in other studies to be regulated in different types of cancer: TMEFF2 (19,20), TSPAN8 (21,22), DCBLD2 (23,24). Taken together, these observations suggest that the interaction between MSCs and NB cancer cells in the BM microenvironment induces changes in the expression of cancer-related genes in the MSCs. An unlikely explanation would be the cellular fusion of NB cells and MSCs, in a similar manner to Rizvanov et al (25) who observed rare in vitro cell fusion in co-cultures of NB tumor cells and MSCs.

The GO functional classification analysis through DAVID showed a number of mainly affected categories further suggesting that NB-MSCs are altered (Table III). Overall, we noted that in the ‘cellular component’ domain, NB upregulated a large number of genes encoding proteins ‘intrinsic to membrane’ (n=98) and localized to the ‘extracellular region’ (n=54), suggesting that NB cancer cells may exert a large repertoire of changes in these MSCs compartments. It is likely that functional relationships between NB and MSCs are mostly mediated through these proteins. Analysis of the category ‘biological process’ revealed effects on MSCs in terms previously described in NB cancer cells in the literature. Most importantly, we remark on the upregulation of genes in the term ‘neurological system processes’. Despite of the wide variety of proteins encoded by these genes, they may provide insights into potential neurological functions altered in NB-MSCs. Interestingly, regulation of neural-related genes has also been shown in previous microarray analysis of NB tumors. Thus, Hiyama et al (3) reported that in favorable NB neuronal differentiation signals were overexpressed in maturing tumors whereas Chen et al (5) found that in NB tumors of stage 4+, proteins with functions in nervous system development were downregulated, suggesting a less mature neural and a more invasive phenotype of these tumors.

The absence of NB markers in our MSC cultures pants to the absence of a tumor cell contamination. Then, it is tempting to speculate whether our findings reflect that MSCs in the BM microenvironment of NB patients redirect toward neuronal lineage. Therefore, MSCs have been proposed to adopt neural cell phenotypes, although this occurs at a very low frequency (26). In this sense, it has been proposed that NB cells would induce MSCs differentiation into Schwann cells (27). However in our data we did not observe an increase of classical Schwann-markers such as S100, Egr-1 or Egr-2 in NB-MSCs. On the other hand, MSCs would suffer a dedifferentiation process since a neuroectodernal origin of fetal MSCs localised in BM has been proposed (28).

The term ‘cell surface receptor linked signal transduction’ included several members of the G protein-coupled receptor family (GPR128, GPR135, GPR132, GPR56 and GPR34). Indeed, GPR128 was the strongest upregulated gene in our microarray analysis (Table I). The G protein-coupled receptor (GPCR) superfamily has long been proposed to have vital dual roles in cellular adhesion and signaling (29). One of the best described GPCRs is GPR56. In addition to its role in neural progenitor cell migration (30), a role in suppression of tumor growth by the microenvironment have been investigated (31). By interacting with an extracellular matrix ligand, TG2 (transglutaminase 2), GPR56 seems to suppress tumor growth and metastasis in vivo; conversely, reduced expression is associated with tumor progression. In addition, it is overexpressed in many human glioblastomas and functions in tumor cell adhesion (32). SHC3, another component of this GO term, has been described in the literature. Miyake et al (18) observed a significantly higher level of ShcC protein in NBs with poor prognostic factors and indicated that the expression of ShcC potentially has a function in inhibiting the differentiation of NB cells (18).

Upregulation of ‘cell adhesion’ genes supports the hypothesis that NB-MSCs may undergo changes in their extracellular matrix and cell adhesion properties. Previous research has shown the modulation of NB cell differentiation by the extra-cellular matrix (33). In this study, the authors showed how extracellular matrix rigidity potentiates NB cell differentiation and decreases cell proliferation; and, as we mentioned above, the receptors of extracellular matrix molecules have been reported as markers of NB cell differentiation (17). Similarly, Chen et al (5) reported that suppression of cell adhesion proteins in NB tumors of stage 4+ indicates the metastatic nature of this kind of NB tumors.

In addition, increased transcript levels of a number of relevant genes involved in cell-cycle regulation or apoptosis (e.g., CCND1, CDKN2A (p14/p16), RET and GDNF) suggest that there may be alterations in these processes in NB-MSCs. Similarly, the number of upregulated genes in the ‘calcium ion binding’ category may indicate that intracellular calcium is likely involved in the response of MSCs to NB. ANXA10 belongs to the annexin super-family of closely related calcium and membrane-binding proteins, and many studies have shown their potential role in tumor development and progression (34). An alternative explanation would be based in the evidence that NB cells stimulate osteoclasts to generate osteolytic lesions and set free calcium, in which interactions of NB cells with BM-derived MSCs play a critical role (35).

Finally, most of the downregulated GO terms contained genes encoding immune-related proteins. However, our results also showed upregulation of HLA-DRB5 and HLA-DOA1, which encode MHC class II molecules. In this sense, Johann et al (36) showed that NK cell cytotoxicity was significantly impaired after co-culturing NB cells with NB-MSCs, compared with MSCs of normal donors. Further study is needed to assess the impact of NB cancer cells on the immune response of MSCs.

In summary, we present initial data of a genome-wide analysis of MSCs from NB patients. Our data suggest that the microarray approach is a useful tool to identify deregulated genes in cultured MSCs isolated from NB patients. We provide preliminary genetic evidence of the interaction between MSCs and NB cancer cells in BM. Furthermore, we identifed relevant biological processes potentially altered in MSCs in response to NB. Future studies are necessary to connect these and other differentially expressed genes into their biological roles.

This study was supported by grants from the Fondo de Investigaciones Sanitarias (FIS; PI05/2217 and PI08/0029 to J.G.C.), MICINN (PLE2009-0115) and the Madrid Regional Government (S-BIO-0204-2006 and P2010/BMD-2420) in Spain. The experiments were approved by the appropriate committees.

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Significance analysis of microarrays (SAM) plot from the comparison of NB wih control. The plot identifies genes which are significantly different across the groups (2-fold upregulation or downregulation and FDR <0.05%). Spots above the upper dotted line indicate significantly upregulated genes and spots below the lower dotted line indicate significantly downregulated genes.

Heat map analysis of microarray data by SAM. Only genes undergoing a 2.5-fold (up or down) differential expression between control (C) and neuroblastoma (NB) samples (FDR <0.05%) are shown.

List of upregulated genes in hMSCs from NB patients.

Gene symbol Gene description GenBank accession no. Fold-change Q-value
GPR128 G protein-coupled receptor 128 NM_032787 44.269 4.5173
TMEFF2 Transmembrane protein with EGF-like and two follistatin-like domains 2 NM_016192 18.655 0.7810
ANXA10 Annexin A10 NM_007193 13.700 1.2968
ITGA2 Integrin, α 2 (CD49B, α 2 subunit of VLA-2 receptor) NM_002203 11.435 0.0000
A_32_P208076 Unknown A_32_P208076 10.707 0.0000
A_24_P365349 Unknown A_24_P365349 9.664 1.6333
THC2402993 Unknown THC2402993 9.011 0.0000
NEF3 Neurofilament 3 (150 kDa medium) NM_005382 8.468 1.0034
IGFBP1 Insulin-like growth factor binding protein 1, transcript variant 1 NM_000596 8.255 4.6632
WDR69 WD repeat domain 69 NM_178821 8.132 3.9950
C1orf110 Chromosome 1 open reading frame 110 BC040018 7.958 3.6213
TREM1 Triggering receptor expressed on myeloid cells 1 NM_018643 7.931 4.5173
FLJ37228 cDNA FLJ37228 fis, clone BRAMY2000411 AK094547 7.177 0.0000
KCTD4 Potassium channel tetramerisation domain containing 4 NM_198404 7.162 4.2453
CNIH3 Cornichon homolog 3 NM_152495 6.984 3.9107
ENST00000379108 Unknown ENST00000379108 6.882 3.3557
CALB2 Calbindin 2, 29 kDa (calretinin) NM_001740 6.503 4.3242
COL4A5 Collagen, type IV, α 5, transcript variant 2 NM_033380 6.438 2.1503
SLC6A15 Solute carrier family 6, member 15, transcript variant 1 NM_182767 6.234 0.9502
TSPAN8 Tetraspanin 8 NM_004616 5.955 1.4171
SLC7A14 mRNA for KIAA1613 protein AB046833 5.889 0.0000
THC2438492 Unknown THC2438492 5.588 0.0000
ENST00000222543 Similar to tissue factor pathway inhibitor 2 precursor (TFPI-2) ENST00000222543 5.577 3.2311
PSG7 Pregnancy specific β-1-glycoprotein 7 NM_002783 5.553 2.6056
DCBLD2 Discoidin, CUB and LCCL domain containing 2 NM_080927 5.547 0.7810
HTR1F 5-hydroxytryptamine (serotonin) receptor 1F NM_000866 5.539 1.6929
SLC7A14 Solute carrier family 7, member 14 NM_020949 5.424 1.2573
RNF128 Ring finger protein 128, transcript variant 1 NM_194463 5.387 4.2453
SHC3 SHC (Src homology 2 domain containing) transforming protein 3 NM_016848 5.329 2.0866
HLA-DR B HLA class II DR-β X12544 5.313 3.5143
SAMD3 Sterile α motif domain containing 3, transcript variant 2 NM_152552 5.254 0.0000
SEMA3E Semaphorin 3E NM_012431 5.227 2.0866
LOC284344 Similar to biliary glycoprotein 1 precursor AK097672 5.197 4.8517
PSG4 Pregnancy specific β-1-glycoprotein 4, transcript variant 2 NM_213633 5.164 1.2496
RGS4 Regulator of G-protein signaling 4 NM_005613 5.151 1.4010
AREG Amphiregulin (schwannoma-derived growth factor) NM_001657 5.043 4.0968
DNER Delta-notch-like EGF repeat-containing transmembrane NM_139072 4.943 2.4280
AK094786 cDNA FLJ37467 fis, clone BRAWH2011920 AK094786 4.787 1.2384
HGD Homogentisate 1,2-dioxygenase (homogentisate oxidase) NM_000187 4.784 1.8692
RGS18 Regulator of G-protein signaling 18 NM_130782 4.752 4.6632
SAMD3 Sterile α motif domain containing 3, transcript variant 1 NM_001017373 4.749 1.1850
SLC24A3 Solute carrier family 24, member 3 NM_020689 4.736 3.4520
F2RL1 Coagulation factor II (thrombin) receptor-like 1 NM_005242 4.683 2.6953
CST1 Cystatin SN NM_001898 4.625 1.6333
TMEM158 Transmembrane protein 158 NM_015444 4.618 3.4313
THC2455389 ORF2280 gene homolog THC2455389 4.608 3.1260
AK127194 cDNA FLJ45259 fis, clone BRHIP2020695 AK127194 4.588 3.1367
PSCDBP Pleckstrin, Sec7 and coiled-coil domains, binding protein NM_004288 4.539 2.4280
CLGN Calmegin NM_004362 4.533 3.7978
RP11-138L21.1 Similar to contactin associated protein (Caspr) AK054645 4.436 2.3612
BCAN Brevican BC005081 4.370 0.0000
NPTX1 Neuronal pentraxin I NM_002522 4.290 2.8954
HLA-DRB5 Major histocompatibility complex, class II, DR β5 NM_002125 4.284 3.3557
SULT4A1 Sulfotransferase family 4A, member 1 NM_014351 4.246 2.5183
THC2335868 ALU5_HUMAN (P39192) Alu subfamily SC THC2335868 4.231 0.0000
FATE1 Fetal and adult testis expressed 1 NM_033085 4.196 4.3477

List of downregulated genes in hMSCs from NB patients.

Gene symbol Gene description GenBank accession no. Fold-change Q-value
IFI27 Interferon, α-inducible protein 27 NM_005532 0.0487 4.052
CRIP1 Cysteine-rich protein 1 (intestinal) NM_001311 0.0994 4.324
CCL8 Chemokine (C-C motif) ligand 8 NM_005623 0.1021 3.438
LSP1 Lymphocyte-specific protein 1, transcript variant 3 NM_001013254 0.1188 1.462
CDCA7 Cell division cycle associated 7, transcript variant 1 NM_031942 0.1509 0.000
ENST00000372045 cDNA clone CS0DI016YJ18 (CR623913) ENST00000372045 0.1757 2.385
IL21R Interleukin 21 receptor, transcript variant 2 NM_181078 0.1975 4.052
C2 Complement component 2 NM_000063 0.2005 4.448
MT1JP MTB AF348994 0.2014 4.517
IFITM1 Interferon induced transmembrane protein 1 (9–27) NM_003641 0.2049 3.995
ENST00000313624 cDNA clone DKFZp667P0410 (AL831953) ENST00000313624 0.2118 3.438
FAM70A Family with sequence similarity 70, member A NM_017938 0.2231 2.385
ISG20 Interferon stimulated exonuclease gene 20 kDa NM_002201 0.2419 2.385
DKFZP761M1511 cDNA FLJ39342 fis, clone OCBBF2018873 AK096661 0.2459 3.995
MBOAT1 cDNA FLJ16207 fis, clone CTONG2019822 AK131269 0.2475 0.000
JPH2 Junctophilin 2, transcript variant 1 NM_020433 0.2524 1.448
C1R Complement component 1, r subcomponent NM_001733 0.2639 4.724
ENPP2 Ectonucleotide pyrophosphatase/phosphodiesterase 2 (autotaxin), transcript variant 1 NM_006209 0.2789 1.462
TMEM119 Transmembrane protein 119 NM_181724 0.2826 2.385
E2F2 E2F transcription factor 2 NM_004091 0.2863 2.385
OLFML2B Olfactomedin-like 2B NM_015441 0.2879 2.854
EXO1 Exonuclease 1, transcript variant 3 NM_003686 0.2881 2.487
CCNE2 Cyclin E2, transcript variant 1 NM_057749 0.2951 1.448
C12orf46 Chromosome 12 open reading frame 46 NM_152321 0.3028 2.385
ECGF1 Endothelial cell growth factor 1 (platelet-derived) NM_001953 0.3067 1.462
SLC2A12 Solute carrier family 2 (facilitated glucose transporter), member 12 NM_145176 0.3321 4.724
NAV2 Steerin3 protein, alternative exon 1b AJ488202 0.3387 3.145
A_24_P927205 Unknown A_24_P927205 0.3480 4.052
IL7 Interleukin 7 NM_000880 0.3811 4.463
ENST00000270031 Unknown ENST00000270031 0.3965 4.448
GBP1 Guanylate binding protein 1, interferon-inducible, 67 kDa NM_002053 0.4015 2.385
RAB42 RAS oncogene family NM_152304 0.4214 4.517
HELLS Helicase, lymphoid-specific NM_018063 0.4240 4.448
BARD1 BRCA1 associated RING domain 1 NM_000465 0.4244 3.699
RAD51AP1 RAD51 associated protein 1 NM_006479 0.4256 2.392
FLJ39660 cDNA clone DKFZp434P055 AL834537 0.4292 2.576
HIRA Histone cell cycle regulation defective homolog A NM_003325 0.4327 4.448
THC2376015 Unknown THC2376015 0.4385 4.168
POLE2 Polymerase (DNA directed), ɛ 2 (p59 subunit) NM_002692 0.4623 2.854
GBP2 Guanylate binding protein 2, interferon-inducible NM_004120 0.4708 4.183
ATAD2 ATPase family, AAA domain containing 2 NM_014109 0.4838 4.069

Main GO terms of the different GO categories enriched in upregulated genes in hMSCs from NB patients.

GO term Count P-value Genes
Biological process
  GO:0007166. Cell surface receptor linked signal transduction 33 0.09 GPR128, CDK5R1, TACR3, EDN1, F2RL1, PTPN22, OXTR, OPN1SW, LGR6, OR4F4, DNER, TGM2, HHIP, SHC3, HTR1F, PTPRC, RET, VAV3, GPR135, ITGA2, RGS18, GPR132, OR10J5, CCND1, GPR34, OR12D3, OR5AK2, HIPK2, GPR56, CRH, STC1, IGFBP1, AREG
  GO:0050877. Neurological system process 27 0.01 RTP3, SYT1, UTS2, SNCA, SLC6A4, OXTR, OPN1SW, OR4F4, ESPN, DFNB31, NPTX1, DMD, SHC3, HAP1, HTR1F, DTNA, PRKCA, COCH, ITGA2, OR10J5, PRKCG, OR12D3, OR5AK2, CRH, USH1C, CLN8, TCF15
  GO:0007242. Intracellular signaling cascade 25 0.05 DCBLD2, EDN1, OXTR, TLR7, TGM2, SHC3, KNDC1, HTR1F, PRKCA, PTPRC, RET, VAV3, PRKCG, RGNEF, CCND1, UACA, CNIH3, RGS4, C7ORF16, HIPK2, ASB1, TREM1, ABL1, GRB14, DUSP6
  GO:0042981. Regulation of apoptosis 21 0.01 PRKCA, PTPRC, IER3, CDK5R1, VAV3, SNCA, SOX9, GDNF, ACVR1C, CDKN2A, UACA, TNFRSF10D, HIPK2, CRH, TGM2, BIK, CTSB, ABL1, CLN8, PHLDA1, ANGPTL4
  GO:0007155. Cell adhesion 17 0.03 DCBLD2, PTPRC, OLFM4, CDK5R1, RET, PCDH10, BCAN, ITGA2, MGP, CDH2, PCDH7, SOX9, BTBD9, GPR56, CNTNAP3, ABL1, CDH10
  GO:0010605. Negative regulation of macromolecule metabolic process 17 0.04 PRKCA, PTPRC, SNCA, EDN1, PRKCG, SOX9, NR0B1, PKIA, PROX1, CDKN2A, HIPK2, RNF128, HES2, ASB1, PRDM1, CLN8, C1D
  GO:0007610. Behavior 13 0.02 PRKCA, SNCA, OXTR, PRKCG, GDNF, ESPN, C7ORF16, HIPK2, CRH, SERPIND1, SHC3, CLN8, TCF15
  GO:0006928. Cell motion 12 0.06 PRKCA, KLF7, CDK5R1, RET, VAV3, ANK3, DNER, PRSS3, ITGA2, CDH2, GDNF, TNP2
  GO:0060341. Regulation of cellular localization 11 0.01 PRKCA, SYT1, CDKN2A, UACA, SNCA, EDN1, CRH, OXTR, PKIA, GDNF, ACVR1C
  GO:0048584. Positive regulation of response to stimulus 10 0.01 PRKCA, PTPRC, HIPK2, F2RL1, CRH, TGM2, PTPN22, ITGA2, PRKCG, TLR7
Cellular component
  GO:0031224. Intrinsic to membrane 98 0.04 GPR128, SYT1, SLC9A7, KLRC2, SLC6A4, F2RL1, TSPAN8, TLR7, PAPPA, CREB3L3, HTR1F, TMEFF2, LRRC3, RET, GPR135, PLXNB3, TMEM132B, GPR132, CYP2E1, PTPRO, HLA-DQA1, SLITRK1, GPR56, NEU3, RTP3, DCBLD2, OPN1SW, RIC3, HLA-DRB5, MFAP3L, CACNA2D1, MLC1, PCDH10, ITGA2, SLC6A15, NCKAP1L, SLAMF8, REEP2, KCTD4, P2RX5, GPR34, OR12D3, CLGN, OR5AK2, CNIH3, AREG, CD200, SLC45A3, KCNJ16, SLC16A14, IER3, SLC5A4, MSR1, TACR3, SLC20A1, DPP10, BCAN, CDCP1, LGR6, ACVR1C, ST6GALNAC5, SLC24A3, CNTNAP3, HHIP, CEACAM3, PPAP2C, MMP16, OR10J5, PCDH7, SLC7A14, PSG8, PSG7, TNFRSF10D, PSG4, TREM1, CLN8, GPAM, KCNH5, TMCO2, FATE1, MFSD4, TMEM158, OXTR, CDH2, C12ORF53, OR4F4, LOC151162, DNER, TMEM35, RNF128, HS6ST3, PTPRC, KIAA1244, TMPRSS9, TMEM51, GDPD1, CYBB, SLC17A3, BIK, CDH10
  GO:0005576. Extracellular region 54 6×10−5 UTS2, MSR1, EDN1, BCAN, CDCP1, GDNF, PSG11, SERPINA9, PAPPA, SEMA3E, SERPINE1, CNTNAP3, HHIP, ANGPT2, TFPI2, PRSS35, COCH, TMEFF2, SCUBE3, CA11, CRISP1, CST2, MGP, PSG3, CST1, MMP16, PSG1, C17ORF69, MMP12, PSG9, PSG8, PSG7, PSG4, TFPI, STC1, CTSB, TREM1, GPHA2, CA2, OLFM4, DEFB126, MIA2, DEFB114, PRSS2, PRSS3, ANGPTL4, COL4A5, TSLP, IGSF21, UACA, MCFD2, CRH, AREG, IGFBP1, SERPIND1, TLL2
  GO:0042995. Cell projection 18 0.04 SYT1, CDK5R1, FBXO2, SNCA, OXTR, ITGA2, PRKCG, CDH2, OPN1SW, ESPN, DFNB31, PPP1R9A, GPR34, ANK3, DNER, DRP2, USH1C, CA2
Molecular function
  GO:0005509. Calcium ion binding 32 6×10−7 SYT1, CDK5R1, SNCA, CDH2, CALB2, NPTX1, SLC24A3, PRSS2, DNER, DMD, PRSS3, TGM2, DTNA, PRKCA, CACNA2D1, RET, SCUBE3, PCDH10, MGP, ITGA2, PRKCG, MMP16, PCDH7, PADI1, MMP12, CLGN, ANXA10, EFHB, DRP2, MCFD2, TLL2, CDH10
  GO:0004857. Enzyme inhibitor activity 14 5×10−5 SNCA, CST2, CST1, PKIA, SERPINA9, CDKN2A, PPP1R1C, C7ORF16, SERPINE1, TFPI, SERPINB4, SERPIND1, TFPI2, ANGPTL4

Main GO terms of the different GO categories enriched in downregulated genes in hMSCs from NB patients.

GO term Count P-value Genes
Biological process
GO:0006955. Immune response 9 0.00003 EXO1, IL7, ENPP2, CCL8, RSAD2, C1R, C2, GBP2, GBP1
GO:0006259. DNA metabolic process 7 0.003 EXO1, CCNE2, RAD51AP1, POLE2, HELLS, ISG20, BARD1
GO:0006952. Defense response 5 0.02 LSP1, CCL8, RSAD2, C1R, C2
GO:0007049. Cell cycle 5 0.06 EXO1, CCNE2, E2F2, HELLS, BARD1
GO:0006959. Humoral immune response 4 0.0004 EXO1, IL7, C1R, C2
GO:0002252. Immune effector process 4 0.002 EXO1, RSAD2, C1R, C2
GO:0046649. Lymphocyte activation 4 0.01 EXO1, IL7, IL21R, HELLS
GO:0006281. DNA repair 4 0.01 EXO1, RAD51AP1, POLE2, BARD1

List of neuronal-related genes upregulated in hMSCs from NB patients.

Gene symbol Gene description GenBank accession no. Fold change Q-value
ITGA2 Integrin, α 2 (CD49B, α 2 subunit of VLA-2 receptor) NM_002203 11.435 0.0000
HTR1F 5-hydroxytryptamine (serotonin) receptor 1F NM_000866 5.539 1.6929
SHC3 SHC (Src homology 2 domain containing) transforming protein 3 NM_016848 5.329 2.0866
NPTX1 Neuronal pentraxin I NM_002522 4.290 2.8954
DFNB31 Autosomal recessive deafness type 31 protein 2 AK056190 3.487 0.0000
SYT1 Synaptotagmin I NM_005639 3.452 4.6886
RTP3 Receptor transporter protein 3 NM_031440 3.282 2.3923
OXTR Oxytocin receptor NM_000916 3.274 3.5862
SNCA Synuclein, α (non A4 component of amyloid precursor) NM_007308 2.980 2.8954
ESPN Espin NM_031475 2.518 1.5910
CRH Corticotropin releasing hormone NM_000756 2.496 3.3557
CLN8 Ceroid-lipofuscinosis, neuronal 8 (epilepsy, progressive with mental retardation) NM_018941 2.479 1.0034
COCH Coagulation factor C homolog, cochlin NM_004086 2.420 3.6986
DMD Dystrophin (muscular dystrophy, Duchenne and Becker types) NM_004010 2.408 4.0521
DTNA Dystrobrevin, α, transcript variant 1 NM_001390 2.330 2.4868
HAP1 Huntingtin-associated protein 1 (neuroan 1), transcript variant 1 NM_003949 2.311 2.1529
OR12D3 Olfactory receptor, family 12, subfamily D, member 3 NM_030959 2.301 1.0207
OR5AK2 Olfactory receptor, family 5, subfamily AK, member 2 NM_001005323 2.293 0.7810
TCF15 Transcription factor 15 (basic helix-loop-helix) NM_004609 2.151 2.5183
PRKCG Protein kinase C, γ NM_002739 2.138 3.4520
PRKCA Protein kinase C, α NM_002737 2.132 1.3963
OR4F4 Olfactory receptor, family 4, subfamily F, member 4 NM_001004195 2.104 1.6012
OPN1SW Opsin 1 (cone pigments), short-wave-sensitive (color blindness, tritan) NM_001708 2.069 1.1850
UTS2 Urotensin 2, transcript variant 1 NM_021995 2.067 1.3372
OR10J5 Olfactory receptor, family 10, subfamily J, member 5 NM_001004469 2.052 1.0034
USH1C cDNA: FLJ21290 fis, clone COL01954 AK024943 2.011 1.0034
SLC6A4 Solute carrier family 6 (neurotransmitter transporter, serotonin), member 4 NM_001045 2.001 4.5173