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MicroRNAs define distinct human neuroblastoma cell phenotypes and regulate their differentiation and tumorigenicity
© Samaraweera et al.; licensee BioMed Central Ltd. 2014
Received: 19 November 2013
Accepted: 11 April 2014
Published: 2 May 2014
Neuroblastoma (NB) is the most common extracranial solid tumor in children. NB tumors and derived cell lines are phenotypically heterogeneous. Cell lines are classified by phenotype, each having distinct differentiation and tumorigenic properties. The neuroblastic phenotype is tumorigenic, has neuronal features and includes stem cells (I-cells) and neuronal cells (N-cells). The non-neuronal phenotype (S-cell) comprises cells that are non-tumorigenic with features of glial/smooth muscle precursor cells. This study identified miRNAs associated with each distinct cell phenotypes and investigated their role in regulating associated differentiation and tumorigenic properties.
A miRNA microarray was performed on the three cell phenotypes and expression verified by qRT-PCR. miRNAs specific for certain cell phenotypes were modulated using miRNA inhibitors or stable transfection. Neuronal differentiation was induced by RA; non-neuronal differentiation by BrdU. Changes in tumorigenicity were assayed by soft agar colony forming ability. N-myc binding to miR-375 promoter was assayed by chromatin-immunoprecipitation.
Unsupervised hierarchical clustering of miRNA microarray data segregated neuroblastic and non-neuronal cell lines and showed that specific miRNAs define each phenotype. qRT-PCR validation confirmed that increased levels of miR-21, miR-221 and miR-335 are associated with the non-neuronal phenotype, whereas increased levels of miR-124 and miR-375 are exclusive to neuroblastic cells. Downregulation of miR-335 in non-neuronal cells modulates expression levels of HAND1 and JAG1, known modulators of neuronal differentiation. Overexpression of miR-124 in stem cells induces terminal neuronal differentiation with reduced malignancy. Expression of miR-375 is exclusive for N-myc-expressing neuroblastic cells and is regulated by N-myc. Moreover, miR-375 downregulates expression of the neuronal-specific RNA binding protein HuD.
Thus, miRNAs define distinct NB cell phenotypes. Increased levels of miR-21, miR-221 and miR-335 characterize the non-neuronal, non-malignant phenotype and miR-335 maintains the non-neuronal features possibly by blocking neuronal differentiation. miR-124 induces terminal neuronal differentiation with reduction in malignancy. Data suggest N-myc inhibits neuronal differentiation of neuroblastic cells possibly by upregulating miR-375 which, in turn, suppresses HuD. As tumor differentiation state is highly predictive of patient survival, the involvement of these miRNAs with NB differentiation and tumorigenic state could be exploited in the development of novel therapeutic strategies for this enigmatic childhood cancer.
NB is the most common extracranial solid tumor in children. The outcome of patients has improved over the years and the estimated 5-year survival rate for non-high risk patients is 90%, whereas that for high-risk patients is 50% .
Amplification of the N-myc proto-oncogene and cellular heterogeneity are two key factors that influence patient survival. The three basic cell types in NB tumors and derived cell lines differ in their morphological, biochemical and tumorigenic properties — whereas N-type neuroblastic cells are mildly malignant and have neuronal characteristics, S-type cells are non-tumorigenic with features of non-neuronal (glial, melanocytic and smooth muscle) precursor cells. I-type cancer stem cells, which can differentiate into either N or S cells, express stem cell marker proteins and are highly tumorigenic [2–4]. Thus, the three basic cell phenotypes represent distinct differentiation states of NB with distinct tumorigenic properties. All three cell types are present in tumors . Clinically, cellular heterogeneity is predictive of patient outcome - patients with stroma-poor tumors comprising undifferentiated neuroblasts are frequently fatal whereas stroma-rich tumors or those with differentiated ganglion cells show a better prognosis . Therefore, one approach to controlling the malignant potential of this tumor involves exploiting its unique differentiation capacity.
MicroRNAs (miRNAs) are important regulators of gene expression and function and hence differentiation. A role for miRNAs in neuroblastoma has been extensively studied mainly focusing on their association with respect to N-myc amplification, chromosomal imbalances, prognosis and retinoic acid (RA)-induced differentiation as discussed in four reviews [6–9]. These studies have revealed that large scale chromosomal imbalances result in dysregulated miRNAs which have a functional role in neuroblastoma pathogenesis and tumorigenicity. MiRNAs associated with N-myc amplification such as miR-17-92 cluster members are shown to be associated with NB tumorigenicity. Also, miRNAs associated with RA-induced differentiation of NB has been extensively studied as RA is used clinically in treating NB patients. These studies, as reviewed by Stalling et al., indicate that miRNA and DNA methylation changes following RA-treatment play a critical role in NB differentiation . miRNAs modulated upon RA-treatment are shown to regulate key genes involved in differentiation, survival and tumorigenic properties of NB .
The present study is mainly focused on investigating the association of miRNAs with respect to the different cell phenotypes derived from NB and their role in regulating their intrinsic differentiation and tumorigenic properties with use of large panel of NB cell lines.
Cell culture and differentiation
The thirteen different human NB cell lines or clones, established from 8 patients’ tumors or bone marrow aspirates, used for these studies have been published previously (4). Seven cell lines or clones were isolated at Memorial Sloan-Kettering Cancer Center or Fordham University [SH-SY5Y, SH-EP1, BE(1)n, BE(2)-M17V, BE(2)-C, SK-N-LD, and SK-N-HM], three [SMS-KCN, SMS-LHN, and CB-JMN] were obtained from Dr. C. Patrick Reynolds (Texas Tech University Health Sciences Center) and SMS-KCN subsequently cloned [KCN-83n and KCNs], and one cell line, LA-N-1, was obtained from Dr. Robert C. Seeger (Children’s Hospital of Los Angeles) and cloned [LA1-55n and LA1-5s]. All cell lines were maintained in a 1:1 mixture of Eagle’s Minimum Essential Medium with non-essential amino acids and Ham’s Nutrient Mixture F12 (Invitrogen Corporation, Carlsbad, CA), supplemented with 10% fetal bovine serum (Hyclone, Logan, UT) without antibiotics.
miRNAs were isolated using the miRVana miRNA isolation kit from Ambion (Austin, TX). Processing and initial microarray analysis of miRNA expression levels was done by LC Sciences (Houston, TX). Levels of 313 different miRNAs were assayed by these arrays. Three groups of miRNAs were deleted prior to analysis: i) miRNAs whose expression was barely detectable in all samples (i.e., with a mean fluorescence ≤ 100); ii) those with statistically non-significant differences (p ≥ 0.05) between (N and I) and Mix; and iii) data from hybridizations to the complementary strand of the miRNAs (S-hsa-miRNAs). The expression levels of miRNAs in different groups were analyzed by Student’s t-test.
Unsupervised clustering based on miRNA expression profiles was generated using MultiExperiment Viewer (MeV) version 4 (http://www.tm4.org/mev.html) using a complete linkage-clustering algorithm with a Spearman rank correlation metric.
Semi-quantitative RT-PCR was performed using the mirVana RT-PCR miRNA Detection Kit (Ambion, Austin, TX).
cDNAs for miRNAs were synthesized using the TaqMan® MicroRNA Reverse Transcription kit and miRNA-specific primers and quantified using TaqMan assays (Applied Biosystems, Foster City, CA) by comparative ΔΔCt method. Expression levels of miRNAs were normalized to U6 and expressed as a fold change compared to the levels of a standard sample of SH-SY5Y or LA1-5s.
Generation of miR-124-overexpressing BE (2)-C clones
miR-124-overexpressing lentivirus was purchased from SBI Biosciences (Mountain View, CA). BE(2)-C cells were infected at a multiplicity of infection of 10 according to manufacturer’s instructions and cloned using cloning cylinders.
Cells growing in multiwell plates were incubated in triplicate with 250 nM (1.2 μCi) 3H-norephinephrine (PerkinElmer, Waltham, MA) for 45 min, washed 2 times, and lysed ; radioactivity was measured by liquid scintillation spectrometry and normalized to cell number.
Colony forming efficiency
Colony-forming efficiencies (CFE) in soft agar were measured as described previously  and determined in quadruplicate in three independent experiments.
Chromatin immunoprecipitation (ChIP) assays
ChIP assays used the EZ ChIP™ Chromatin Immunoprecipitation Kit (Upstate Biological, Lake Placid, NY). Chromatin isolated from BE(2)-C cells was incubated with anti-N-myc rabbit polyclonal antibody (sc-791) (Santa Cruz Biotechnology, Santa Cruz, CA); mouse monoclonal anti-RNA polymerase II antibody (clone CTD4H8) (Upstate Biological, Lake Placid, NY); or rabbit anti-goat IgG (Chemicon International, Temecula, CA). The primer sets used for amplification are available upon request.
Stable and transient transfections
Stable SH-SY5Y sense-N-myc and LA1-55n antisense N-myc transfectants have been described . miRNA inhibitors for miR-375, miR-335, and control oligos (100 nM) (Ambion, Austin, TX) were transiently transfected into BE(2)-C or SH-EP1 cells for 48 hrs using Lipofectamine 2000 (Invitrogen Corp., Carlsbad, CA).
Western blot analysis
Western blot analysis of proteins was performed as previously described . Primary antibodies used were rabbit anti-N-myc [(C-19) (SC 791)] (Santa Cruz Biotechnology Inc., Santa Cruz, CA), human antisera against Hu proteins (a kind gift of Dr. Myrna Rosenfeld, University of Pennsylvania Medical School) and, as controls, mouse anti-actin [(AC-74) (076 K4762)] (Invitrogen Corp., Carlsbad, CA) and mouse anti-Hsp72/73 [(W27) (HSP01)] (EMD Chemicals Inc., Gibbstown, NJ).
Results and discussion
miRNAs define distinct NB cell phenotypes
To specifically identify miRNAs associated with the non-neuronal vs neuroblastic phenotype, miRNA levels were compared among cell lines representing these two phenotypes. The expression levels of twenty miRNAs that were highly significantly different between the two groups were ranked according to fold change (Additional file 1: Table S1). The majority of these miRNAs are highly expressed in the S-type cell containing mix and five out of top seven candidates that showed the highest fold change were selected for further analysis (miR-21, miR-31, miR-222, miR-221 and miR-335).
A second grouping compared expression levels between the two neuroblastic phenotypes (N vs I) to identify miRNAs that reflect the differences in neuronal maturation and/or malignant potential (Additional file 2: Table S2) as N-cells show more neuronal features and are less malignant than I-cells . We also took into account published studies of miRNAs associated with neuronal differentiation in neuroblastoma. Three additional miRNAs, all showing higher expression in N compared to I cells— miR-124, miR-375 and miR-10b ― were selected for further analysis.
Expression levels of two miRNAs, miR-124 and miR-375, were higher in the neuroblastic phenotype (Figure 2D and E). The six N-type cell lines have the highest levels of miR-124 expression [12.5-fold higher compared to I-type lines] suggesting its association with neuronal differentiation; S-cells have barely detectable levels of this miRNA. The second miRNA associated with a neuroblastic lineage, miR-375, is expressed at similar levels in both N- and I-cells while being barely detectable in S-type cells (Figure 2E).
Drug-induced irreversible differentiation of I-type NB cancer stem cell confirms the association miRNAs with cell phenotype
RA-induced differentiation increases miR-124 expression 2.0-fold (P < 0.01), whereas BrdU treatment causes a 5.0-fold reduction (P < 0.01) (Figure 3B). Similarly, miR-375 levels in I cells treated with BrdU decrease ~ 50-fold (P < 0.01) (Figure 3B). Thus, expression of these miRNAs characterizes the non-neuronal, non-tumorigenic NB cell phenotype.
Functional role for miR-335 in S-cell phenotype
MiR-124 induces neuronal differentiation of I-type NB stem cells with concomitant reduction in malignant potential
13-cis retinoic acid treatment increases the survival of patients with high-risk NB . Thus, we hypothesized that neuronal differentiation following miR-124 overexpression might also decrease cell tumorigenicity. Colony-forming efficiency (CFE) in soft agar revealed that, whereas control cells have a CFE of 29.5%, miR-124-infected BE(2)-C cells have a CFE of 5.2% (Figure 5F), a significant 5.7-fold reduction in malignant potential (P < 0.001).
Several other researchers have shown that miR-124 expression is related to neuronal differentiation [21, 22]. Consistent with our findings, Le et al. showed that over expression of miR-124 in SH-SY5Y cells induces neurite outgrowth . Clinically, neuronal differentiation in NB tumors is associated with reduced malignancy and tumor regression . Therefore, miR-124 has the potential for use as a therapeutic miRNA in NB.
N-myc regulates expression of miR-375
ChIP experiments confirmed that N-myc binds to one of two E-box sequences in the promoter region of the miR-375 gene (Figure 6C). N-myc binding specificity was confirmed with GAPD as a positive control [which contains a non-canonical E-box to which N-myc binds]  and HOOK1 as a negative control (which lacks E-boxes). This experiment also shows that RNA Polymerase II is associated with the promoter region of miR-375 in BE(2)-C cells (Figure 6C).
HuD is regulated by miR-375
Our study shows that expression of specific miRNAs defines different NB cell phenotypes and are responsible for their associated tumorigenic and differentiation properties. The expression of three miRNAs, miR-21, miR-221 and miR-335, are exclusive to non-tumorigenic NB cell phenotype. Evidence suggests miR-335 maintains the non-neuronal features possibly by blocking neuronal differentiation. MiR-124 expression is exclusive to neuroblastic cells and overexpression of this miRNA in NB stem cells induces terminal differentiation with concomitant reduction in their malignant potential, suggesting a therapeutic potential for this miRNA in treating NB. The expression of miR-375 is associated with tumorigenic neuroblastic cell phenotype and we report that its expression is regulated by N-myc. MiR-375 downregulates HuD, a gene involved in neuronal differentiation. The differentiation state of the tumor is highly predictive of survival of NB patients. Thus, the involvement and association of these miRNAs in differentiation of NB could be used as prognostic markers and also in development of novel therapeutic strategies for this enigmatic childhood cancer.
This research and all authors (LS, BG, RH, BS and RR) of the study were supported by NIH grant CA077593.
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