Gene Expression and Radiation Response of Two Distinct Neuroblastoma Cell Lines

doi:10.21203/rs.3.rs-1720457/v1

Download PDF

Research Article

Gene Expression and Radiation Response of Two Distinct Neuroblastoma Cell Lines

https://doi.org/10.21203/rs.3.rs-1720457/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this older preprint version

Read the latest preprint version →

Background: External beam radiation is infrequently used for treatment of pediatric cancer such as neuroblastoma, typically reserved for palliative care in patients with aggressive metastatic disease who fail to respond to alternative treatments. Understanding effects of radiation on neuroblastoma cells could improve efficacy of this final means of therapy to decrease tumor burden and stabilize the disease.

Methods: In this study, clonogenic assays were used to establish radiation responses for SK-N-AS and SK-N-DZ cell lines; cells were then irradiated at doses that cause 90% cell killing based on clonogenic assay and their RNA isolated and subjected to microarray analysis. In addition, cells were transfected with pre-miRNA constructs that led to overexpression of microRNAs miR-34a and miR-1228 in combination with radiation exposure to determine possible miRNA regulation of radiation response.

Results: Statistically significant differences were detected for expression of several thousands of genes when the two cell lines were compared with each other. In contrast, radiation led only to minor gene expression differences of less than two-fold between irradiated and non-irradiated cultures in both cell lines. Overexpression of miR-34a and miR-1228 in either cell line did not change this outcome.

Conclusion: Neuroblastoma cell lines SK-N-AS and SK-N-DZ are phenotypically diverse and gene expression differences between them are extensive. However, the regulation of gene expression in both of these cell lines is in a stable equilibrium even when exposed to stressors such as radiation.

neuroblastoma

external beam radiation

radiotherapy

microRNAs

Neuroblastomas are pediatric solid tumors that develop from the peripheral sympathetic nervous system, originating from multipotent neural crest cells (1), and show significant diversity in disease severity and prognosis. Heterogeneity of neuroblastoma as a disease is echoed by heterogeneities between cell lines derived from individual neuroblastoma cancers (2, 3). This study is focused on cell lines SK-N-AS and SK-N-DZ, both isolated from bone marrow metastatic sites but showing many disparate responses to potential cancer treatments. Over the years, these two cell lines were sometimes used as a source for investigation of commonalities between different neuroblastomas, e.g. with regard to their proteomic profiles and secretion (4–6), cellular signaling (7) and response to therapy (8–12); and sometimes as models for differences between different neuroblastoma types. Implanted in mice, SK-N-AS cells generate larger tumors with larger diameter tumor vessels compared to SK-N-DZ cells, possibly attributed to the increased VEGF protein expression in the SK-N-AS cell line (13). In addition, only SK-N-AS cells cause osteolytic bone metastases in nude mice (14), which is ascribed to high COX-2 and prostaglandin E2 (PGE2) expression in this cell line. Different MYCN amplification status in these cells (15, 16), with SK-N-AS having only a single copy while SK-N-DZ carries amplified MYCN, led to their use in studies focused on MYC related treatments such as combination of uridine and dihydroorotate dehydrogenase (17). In a study focused on cell-line dependent response to the inhibitor of ganglioside sialidase, different responses to chemical agents were noted where de-differentiation and increased proliferation occurred in SK-N-DZ as a type of cholinergic/adrenergic cells, while no response was found in adrenergic type cell line SK-N-AS (18). Equal doses of deferoxamine had a cytolytic effect on SK-N-DZ cells but lead only to a growth reduction of SK-N-AS cells (19). On the other hand, IFN-gamma induced caspase-8 expression resulted in apoptosis of SK-N-AS cells, while in their counterpart SK-N-DZ cells activation of the same signaling cascade did not lead to TRAIL mediated apoptosis (20). Likewise, Toll-like receptor-3 (TLR3) expression is lower in SK-N-DZ cells and as a consequence, they survive treatment with poly(I:C) better than their TLR3-high counterpart SK-N-AS cells (21).

In this work, we compared radiation responses and gene expression of SK-N-AS and SK-N-DZ cells. Because ionizing radiation exposure has different effects on cells in different stages of cell cycle, it should be noted that these cell lines demonstrate different cell cycle arrest behaviors in response to, for example, doxorubicin treatment (22). Cell cycle arrest differences can be expected to lead to different responses to ionizing radiation, as previously demonstrated in preliminary work using a cesium-137 gamma irradiator on SK-N-AS and SK-N-DZ cells (23). In this study, we show that exposure to X-rays results in significantly greater survival of SK-N-AS cells compared to SK-N-DZ cell line. However, changes in gene expression in either cell line one hour after exposure to doses that cause 90% cell killing is surprisingly modest. In addition, we explored possible modulation of radiation response in the two cell lines associated with overexpression of hsa-miR-1228-3p (miR-1228) and hsa-miR-34a-5p (miR-34a) by transfection of synthetic pre-miRNAs. These two microRNAs have opposite patterns of baseline expression in untreated SK-N-AS and SK-N-DZ cells. Transfection of either of these two pre-miRNAs to SK-N-AS cells 24h before irradiation did not lead to significant modulation of survival in a clonogenic assay. Gene expression patterns in either cell line were not markedly different in response to microRNA overexpression with or without radiation exposure. Similarly, ELISA assays for nuclear expression of NF-kB proteins p65 and c-Rel and c-Myc protein showed that cell type differences lead to much more prominent gene expression differences than exposures to radiation, alone or in combination with microRNA treatments.

2.1 Cell Culture and Treatments

Human neuroblastoma cell lines SK-N-AS and SK-S-DZ (catalog numbers CRL-2137 and CRL-2149, respectively, ATCC, Manassas, VA) were maintained in high-glucose Dulbecco’s modified Eagle medium (Corning, Glendale, AZ) supplemented with 10% fetal bovine serum (Gibco^™, Waltham, MA). All cells were cultivated in presence of 1x antibiotic antimycotic solution (Corning, Glendale, AZ) and 1x Essential amino acids solution (Corning, Glendale, AZ), at 5% CO₂ at 37°C. For all transfection experiments, 6 x 10⁵ cells per well were seeded in 6 well plate one day prior transfection. Plates intended for SK-N-DZ cells wells coated with Geltrex (Invitrogen, Waltham, MA) before use.

Transfections were done with Ambion Pre-miR miRNA precursors (#AM17100; miR-34a assay #PM11030, miR1228 assay #PM13532; ThermoFisher Scientific, Waltham, MA), and respective negative controls (#AM17110, ThermoFisher) using Lipofectamine RNAiMax (Invitrogen, Waltham, MA). Total quantity of 700pmol of either pre-miR was resuspended into 2ml of medium with Lipofectamine mix following standard protocol. Overexpression of miRNA was confirmed by corresponding TaqMan Assay 2x mix (catalog #4427975, assay IDs #000425 for miR34a and #002919 for miR1228, Applied Biosystems, Waltham, MA).

For clonogenic assays, cells in T25 flasks were irradiated with 160 kVp X-rays at dose rate of 3.12Gy/min (RS200 Irradiator, RadSource, Buford, GA) with doses of 1, 2, 4, 6 or 7Gy. Cells were provided with fresh media immediately prior to irradiation and were trypsinized at 15 minutes post-irradiation to be seeded at different cell densities in 12, 24 and 48 well plates. Three to four weeks after plating, media was removed and cell colonies were fixed and stained over night with 0.001% crystal violet dissolved in 10% Neutral Buffered Formalin. The following day, plates were washed and dried prior to colony counting. This approach avoided extensive washes prior to fixation that could remove loosely attached SK-N-DZ cell colonies. Colonies with more than 50 cells were counted. Colonies that grew in six wells of a multi well plate (technical replicates) were counted for each separate experiment (biological replicate). Proportion of untreated and non-irradiated cells that successfully grew into colonies varied between experiments. This cell number was considered as 100% growth, and colony growth numbers for all treated cell samples were presented as percentages of this value.

For clonogenic assays with transfected cells, cells in six well plates were transfected with pre-miRNA molecules for 20 to 24h or non-transfected cells were irradiated with the same X-ray source with doses of 4 or 6 Gy. Again, cells were harvested by trypsinization and seeded at different cell densities; after several weeks of growth in the incubator, colonies were stained, washed, dried and counted as described above.

For RNA isolation and protein isolation, cells were irradiated twenty hours after transfection. Doses used for RNA and protein isolation from SK-N-AS cells were 4Gy and 6Gy, and for SK-N-DZ cells 2Gy and 4Gy. This choice of doses was made because of the roughly equivalent cell survival based on the clonogenic assay data. Cells were incubated 1h or 24h after irradiation prior to RNA isolation; cells used for preparation of nuclear extracts were harvested 24h after irradiation. In each case, experiments were done as biological triplicates for each transfection, irradiation and post-irradiation timepoint.

2.2 RNA Isolation for Quantitative Real-Time Polymerase Chain Reaction and Gene Expression Array

Total RNA and miRNA fractions were isolated with the mirVana^™ miRNA Isolation kit (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer's protocol. To confirm miRNA overexpression, cDNA was generated using specific primers for miR-34a, miR-1228, and endogenous control U6 snRNA (Thermo Fisher Scientific, Waltham, MA) and TaqMan MicroRNA Reverse Transcription kit (#4366496, Applied Biosystems, Waltham, MA). Overexpression of miRNA was confirmed using TaqMan assay for corresponding miRNA (Applied Biosystems, Waltham, MA).

cDNA for gene expression analysis was generated from 1 µg of DNase-treated total RNA using SuperScript® III First-Strand Synthesis SuperMix (Thermo Fisher Scientific, Waltham, MA). Next, RNA samples were diluted with DEPC treated water to final concentration of 50ng/ul and submitted to Northwestern University’s NUSeq Core Facility for gene expression analysis.

The Human Clariom™ D GeneChip (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA), which allows to probe the expression of over 540,000 transcripts was used for the microarray study. The GeneChip WT PLUS Reagent Kit was used for RNA sample preparation for array hybridization. For each sample, 50 ng total RNA was used for cRNA target preparation, array hybridization, washing, staining and image scanning. The washing and staining steps were performed on a GeneChip Fluidics Station 450 and the scanning of hybridized arrays was conducted on a GeneChip Scanner 3000 7G. After generation, the array data was first examined for quality using QC criteria set by Affymetrix. All hybridizations were done in triplicate for biological replicates for each transfection, irradiation and post-irradiation timepoint. Sample and hybridization quality controls met the criteria for this array type.

Probeset data from the raw CEL files were summarized using the “affy” package (v. 1.68) (24) of Bioconductor (v. 3.12) (25) in R (v. 4.0.4) (26). Genes were annotated with a custom CDF (v. 25) from the Molecular and Behavioral Neuroscience Institute (Brainarray) at the University of Michigan (27).

Signals were then standardized across arrays using the Supervised Normalization of Microarrays (SNM) method (v. 1.38) (28) in R/Bioconductor. The SNM model was fit with three biological variables: cell line, radiation dose level, and miRNA treatment. Variance due to batch processing was removed by modeling the microarray scan date as the adjustment variable and setting the “Rm = True” option.

Differential expression analysis was performed using the R package Limma (v. 3.46) (29). Normalized intensities were fit to a linear model with coefficients for each of the factor combinations of cell line, radiation dose and miRNA treatment. All pairwise comparisons of interest were extracted from this model as contrasts using empirical Bayes smoothing (30). The false discovery rate (FDR) was controlled using the Benjamini and Hochberg correction. Probes with FDR < 0.05 and fold-change greater than 1.5 (equal to abs(log2FC) > 0.58) were judged to be differentially expressed.

For cellular pathways analyses, the gene expression lists were ordered based on differential gene expression in SK-N-AS vs. SK-N-DZ cells and submitted to g-profiler (https://biit.cs.ut.ee/gprofiler_beta/gost) (31). Query conditions were: ordered gene list: TRUE; sources of pathways GO:MF, GO:CC, GO:BP, KEGG, REAC, TF, MIRNA, HPA, CORUM, HP, WP; significance threshold method g_SCS, threshold 0.05.

2.3 Quantitative real-time polymerase chain reaction (qPCR)

Quantitative real-time polymerase chain reaction (qPCR) was performed with the Fast SYBR Green Master Mix (Applied Biosystems, Waltham, MA) by using the Applied Biosystems thermal cycler Model 7300. Expression was normalized with GAPDH as before (32), and relative quantification was calculated using the ΔΔCt method.

Results are represented as fold increase in the test samples compared with the sample transfected with control pre-miRNA. For qPCR, all samples were analyzed in three technical and biological replicates. The sequences of oligonucleotides used as qPCR primers are listed in Supplemental Table S1.

2.4 Isolation of nuclear extracts and ELISA assays for c-Myc and NF-kB

Content of c-Myc and NF-kB in nuclear extracts was detected using TransAM™ (Active Motif, Carlsbad, CA) ELISA based kits. Nuclear extracts were isolated from non-transfected and transfected, irradiated and non-irradiated SK-N-AS and SK-N-DZ cells using Nuclear Extract Kit (Active motif, Carlsbad, CA). Transfections were performed as described previously, cells were irradiated 24h after transfection and extracts isolated after a 24h post-irradiation incubation. Ten to seventeen micrograms per well were used for nuclear protein extract ELISAs for detection of NF-kB proteins p65, c-Rel and c-Myc. Detection was performed according manufacturer instructions using ClarioStar plate reader (BMG Labtech, Cary, NC 27513). The experiments were done in triplicates, with cell nuclear extracts isolated for three independently performed transfection-irradiation experiments.

3.1 Clonogenic assays show marked differences in radiation response between neuroblastoma cell lines SK-N-AS and SK-N-DZ

Neuroblastic SK-N-DZ cells (3, 33) have low adhesion, which hampers manipulation required for irradiation of already plated cell culture dishes with single cells. In previous ionizing radiation experiments using Cs-137 gamma rays, we have used MTS assays in order to circumvent this problem (23). In this work, we utilized a different approach by irradiating relatively confluent cell culture dishes with X-rays, and seeding clonogenic assays after a 15 minutes post-irradiation incubation. The same approach was used for SK-N-AS cell line, which is more substrate-adherent. Single cells generated by trypsinization of irradiated or sham irradiated 70% confluent cells were permitted to attach to the plate and grow into colonies over a period of 4–6 weeks. Figure 1 shows the clonogenic assay data for SK-N-AS and SK-N-DZ cell lines.

3.2 Neuroblastoma cell lines SK-N-AS and SK-N-DZ show opposite pattern of expression for microRNAs miR-1228 and miR-34a

A preliminary gene expression evaluation of cell lines SK-N-AS and SK-N-DZ (not published) showed numerous messenger RNA expression differences as well as opposite expression patterns for microRNAs miR-1228 and miR-34a. We repeated evaluation of expression for the two miRs in the two cell lines by qRT-PCR (Supplemental Fig. 1) and found a robust and reproducible expression of miR-1228 in SK-N-AS cells while expression of miR-34a was several-fold lower in the same cell line. MicroRNA miR-1228 is one of the handful of miRs known to be increased in response to ionizing radiation in human embryonic stem cell line H1 (34). MiR-1228 is also a so-called mirtron – a micro RNA released from an intron of low density lipoprotein receptor related protein 1 precursor (LDP1) (35).

Conversely, in SK-N-DZ cells (Supplemental Fig. 1) relative quantification obtained by qPCR showed a several fold higher expression of miR-34a compared to miR-1228. In previous neuroblastoma studies, expression of miR-34a was found to be associated with a good prognosis (36, 37). In neuroblastoma cell lines in vitro, overexpression of miR-34a was found to be cytotoxic (38, 39).

3.3 Clonogenic assays show no cell survival differences between native and microRNA transfected neuroblastoma cells SK-N-AS

SK-N-AS cells were transfected with pre-miRNAs for mature miR-1228 and miR-34a as described in the methods section. Successful transfection was monitored by fluorescence readings. Twenty hours after transfection cells were irradiated and 15 minutes later trypsinized and seeded to grow into colonies. Four biological replicates and three technical replicates of each experiment were done; no effects of miRs were noted. Across different experimental conditions, percentage of cells growing into colonies after 4Gy exposure varied between 0.05 and 4.03% for non-transfected cells, 0.02 and 3.8% for control miR transfected cells; 0.15 to 3.56% for cells transfected with miR-1228, and 0.14 to 4.28% for cells transfected with miR-34a. Similarly, percentage of cells growing into colonies after 6Gy exposure was 0.11 to 1.81% for non-transfected cells, 0.16 to 1.63% for control miR transfected cells; 0.13 to 1.73% for cells transfected with miR-1228; and 0.18 to 1.94% for cells transfected with miR34a. It should be noted that these numbers are lower than what was shown for SK-N-AS cell line data obtained in previous clonogenic experiments series. This may be the result of the fact that the cells in this experiment have undergone two trypsinization steps within a period of 48h and their capacity to re-attach to the plate may have been diminished. Regardless, the data are fairly consistent for each radiation dose irrespective of the pre-miR treatment, suggesting that the overexpression of miR-1228 or miR-34a has no significant effect on viability of SK-N-AS cells in combination with 4 or 6 Gy radiation exposure.

3.4 Clariom™ D Assay Gene Expression Study of Neuroblastoma Cell Lines SK-N-AS and SK-N-DZ

Gene expression differences between different neuroblastoma cell lines are well documented (2, 40), however, gene expression comparisons between cell lines SK-N-AS and SK-N-DZ were not done until this work. In this study, cells were irradiated with X-ray doses that cause comparatively similar degree of cell death: 6 Gy for cell line SK-N-AS and 4 Gy for SK-N-DZ. For each cell line, we have also irradiated the cells transfected to overexpress microRNAs miR-1228 and miR-34a for 24h prior to X-ray radiation exposure. Three biological replicates for each treatment were done and the cells were harvested for RNA isolation for microarray work 1h after radiation exposure. Gene expression was evaluated with Clariom D gene expression array; the data have been deposited into GEO database (GSE197124). Gene expression normalization and differential expression evaluation was carried out as described in the Methods section. Interestingly, despite the fact that the dose of radiation used with each cell line was cytotoxic and resulting in no more than 10% of the cells growing into colonies, at the one hour post-irradiation timepoint gene expression differences between irradiated and non-irradiated cells were almost non-existent. For example, non-transfected or control miR transfected SK-N-AS cells did not show any differentially expressed genes with change above 1.5-fold after radiation with 6Gy. In SK-N-AS cells transfected to overexpress miR-1228, only instances of gene expression above 1.5-fold were found: the C-C motif chemokine ligand 2 (CCL2, ENSEMBL ID ENSG00000108691) and the U5B small nuclear RNA 1 (RNU5B-1, ENSEMBL ID ENSG00000200156). The single overexpressed gene in response to radiation in miR-34a-transfected SK-N-AS cells was NF-kB inhibitor alpha (NFKBIA, ENSEMBL ID ENSG00000100906). Irradiation of SK-N-DZ cells with 4Gy dose of X-rays did not cause any gene expression differences that crossed the threshold of 1.5-fold change in gene expression and were statistically significant.

Conversely, statistically significant (p < 0.01) gene expression differences between the two cell lines were marked at the 1.5 threshold (Fig. 2) with more than 10% or 4000 differentially expressed genes out of the total 38833 genes. Exposure to radiation did not disturb this ratio.

The complete gene expression datasets are available in GEO (GSE197124); however, three tables of genes of interest are provided in supplemental data (Tables S2-4). A Venn diagram was generated for non-transfected cells and comparisons between non-irradiated SK-N-AS and SK-N-DZ cells. The gene list in Table S2 contains genes that are expressed differently between the two cell lines only when they are not irradiated, Table S3 contains genes that are differentially expressed between the two cell lines regardless of irradiation, while Table S4 contains genes that are differentially expressed between the two cell lines only when they were exposed to X-rays. Genes known to be overexpressed in SK-N-DZ cell line such as MYCN and miR-34a appeared in these lists as downregulated in SK-N-AS cells in comparison with SK-N-DZ cells. Unexpectedly, miR-1228 did not appear in any of the three lists as one of the upregulated genes, potentially a result of the type of array used for this study.

Next, the gene expression lists were submitted to g-profiler as ordered lists based on intensity of gene expression and the list of pathways that are activated by these gene expression patterns are provided in Tables S5-7. In SK-N-AS cells, in comparison with SK-N-DZ cells, and regardless of radiation (Table S5) the most prominent differentially expressed pathways are associated with cell adhesion (specifically cell adhesion molecule binding, cadherin binding, collagen binding and extracellular matrix binding) as could be expected based on cell phenotype differences. Interestingly, a number of transcription factors including many from interferon regulatory transcription factor (IRF) family are also found to be associated with differential gene expression in SK-N-AS cells. In the same cell type, genes differentially expressed after irradiation (Table S6) show evidence of activation of the NF-kB pathway. For example, six different references to NF-kB pathway protein complexes are noted in CORUM database of mammalian protein complexes (41). Conversely, in SK-N-DZ cells very few pathways were found associated with ordered list of differentially expressed genes (Table S7).

3.5 Selected gene and protein expression differences

A subset of genes were selected from the microarray gene expression analysis to confirm findings. Their expression at 1 or 24 h after irradiation was evaluated in untreated cells or cells transfected with control miR, or pre-miRNAs that lead to overexpression of miR-34a and miR-1228 (Supplemental Figures S2, S3 and S4).

A small selection of proteins was also examined by subsequent ELISA analyses in both cell lines. In SK-N-AS cells, transfection with pre-miRNA for miR-34a led to about 1.5-fold increase in expression of inhibitor of NF-kB alpha. Nuclear concentration of some of the NF-kB proteins may thereby be impacted, and we chose p65 and c-Rel for further investigation. While p65 is one of the NF-kB proteins dependent on IKB expression, c-Rel is not and we anticipated that the differences in p65 vs. c-Rel expression pattern will confirm the role of IKB in modulation of radiation response in SK-N-AS cells. However, levels of expression of both proteins were low and no significant trend in protein quantities could be detected (Figures S5 and S6).

Finally, we also conducted ELISA evaluation of c-Myc protein in the two cell lines (Figure S7). As expected, SK-N-AS cell line that is not overexpressing MYCN had higher expression of c-Myc protein. In SK-N-DZ cell line, which has MYCN gene amplification, expression of c-Myc was negligible.

Differences between the N-type and S-type neuroblastoma cell lines such as SK-N-DZ and SK-N-AS, respectively, are very pronounced and their responses to radiation stress are equally heterogeneous. Studies using Chip-Seq to investigate transcription factors regulating gene expression in different neuroblastoma cell lines, including the two used here, identified 37 transcription factors with distinctive pattern of presence in the cell lines SK-N-AS (considered “intermediate type”) and cell line SK-N-DZ (considered a “type I” cell type).

In this work, we exposed the two cell lines to doses of radiation which caused 90% cell killing for each cell line. In most other cancer cell lines, analogous stress exposures lead to marked gene expression differences. Here, we found only subtle differences in gene expression between non-irradiated and irradiated cells regardless of miR-1228 or miR-34a overexpression. At the same time, gene expression differences between the two cell lines included many hundreds of genes (Fig. 2).

Low fold-changes in gene expression array studies of neuroblastoma cell lines exposed to different growth and treatment conditions have been observed in the past as well. For example, SK-N-DZ cells grown as neurospheres show altered gene expression for some 11% of the registered genes only if fold changes criterium is set to “anything above 1” (40). In this work, not a single gene was found to be modulated above 1.5-fold in SK-N-DZ cells in response to radiation. In SK-N-AS cell line, non-transfected or control transfected cell lines showed no changes in gene expression 1h after exposure to 6Gy. Only changes after irradiation found in SK-N-AS cells were noted in cells transfected with miR-34a, which showed an increase in inhibitor of NF-kB alpha (NFKBIA), or miR-1228, which showed higher expression of U5B small nuclear RNA 1 (RNU5B-1) and the C-C motif chemokine ligand 2 (CCL2). Transfections of SK-N-AS cells with either pre-miRNA did not lead to any significant change of cell survival. At the same time, gene expression differences between the two cell lines included several thousands of genes (Fig. 2), about 1% of all the genes expressed in each cell type. Taken together, these findings suggests that gene expression regulation in neuroblastoma cell lines depends on stably engaged gene regulation patterns as suggested by genomic DNA-enhancer studies (42). It is probable that the gene expression patterns such as these found in two different types of neuroblastoma cell lines are not easily altered in response to stress.

In conclusion, neuroblastoma cells demonstrate subtle gene expression differences at early timepoints after exposure to external radiation, indicating stable gene regulation patterns regardless of tumor cell subtype. While this makes conventional therapy difficult to effectively utilize, it also provides justification for continued attempts to develop molecular targeting of neuroblastoma. Further studies are needed to determine how to potentially leverage such information in a clinical setting.

CCL2: C-C motif chemokine ligand 2

cDNA: complementary DNA

c-Rel: REL proto-oncogene, NF-kB subunit

cRNA: complementary RNA

c-Myc: MYC proto-oncogene, bHLH transcription factor

CORUM: Comprehensive resource of mammalian protein complexes

COX-2: cyclooxygenase-2, or prostaglandin-endoperoxide synthase 2

Cs-137: Cesium-137

ΔΔCt: delta-delta-Ct (cycle threshold)

DNA: deoxyribonucleic acid

DEPC: diethyl pyrocarbonate

ELISA: enzyme-linked immunoassay

FDR: False Discovery Rate

GAPDH: glyceraldehyde-3-phosphate dehydrogenase

GEO: Gene Expression Omnibus database

Gy: Gray (radiation SI unit)

IFN-gamma: interferon gamma

IRF: interferon regulatory transcription factor

kVp: kilovoltage peak

LDP1: low density lipoprotein receptor related protein 1

miR/miRNA: microRNA

miR-34a: hsa-miR-34a-5p

miR-1228: hsa-miR-1228-3p

MTS assay: 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay

MYC: MYC proto-oncogene, bHLH transcription factor

MYCN: n-MYC; MYCN proto-oncogene, bHLH transcription factor

NF-kB: Nuclear Factor kappa B

NFKBIA: NF-kB inhibitor alpha

Pre-miRNA: precursor microRNA

RNU5B-1: U5B small nuclear RNA 1

TLR-3: Toll-like receptor-3

TRAIL: TNF-related apoptosis-inducing ligand

P65: transcription factor p65, RELA

PGE2: prostaglandin E2

qPCR: quantitative real-time Polymerase Chain Reaction

RNA: ribonucleic acid

snRNA: small nuclear RNA

VEGF: vascular endothelial growth factor

Ethics approval and consent to participate: Not applicable

Consent for publication: Not applicable

Availability of data and materials: The microarray dataset generated in this study is openly available in NCBI’s Gene Expression Omnibus (Edgar, et. al. 2002) and is accessible through GEO Series accession number GSE197124. All other data generated or analyzed during this study are included in this article and its supplementary information files (Supplementary Tables 1-7; Supplementary Figures 1-7).

Competing interests: The authors declare that they have no competing interests

Funding: This research was funded by NIH, NCI grant R01CA221150, and NIH SBIR grant 75N91019C00043. In addition, this work was supported by the Northwestern University NUSeq Core Facility.

Authors’ Contributions: CR participated in project conceptualization, investigation, and data analysis; writing of the original draft, editing, and submission. JP contributed to project conceptualization, investigation, and manuscript writing and editing. AA aided in experiments and conducted formal data analysis. YL assisted in experiments and data collection. TP helped develop project concept and investigation, and was a major contributor to manuscript writing and editing. RP established methodology and assisted in funding acquisition. GW aided in project conceptualization and overall supervision, acquisition of funding, and writing and submission of manuscript.

Acknowledgements: The authors declare no conflict of interest.

Johnsen JI, Dyberg C, Wickström M. Neuroblastoma-A Neural Crest Derived Embryonal Malignancy. Frontiers in molecular neuroscience. 2019;12:9.
Gautier M, Thirant C, Delattre O, Janoueix-Lerosey I. Plasticity in Neuroblastoma Cell Identity Defines a Noradrenergic-to-Mesenchymal Transition (NMT). Cancers. 2021;13(12).
Ciccarone V, Spengler BA, Meyers MB, Biedler JL, Ross RA. Phenotypic diversification in human neuroblastoma cells: expression of distinct neural crest lineages. Cancer research. 1989;49(1):219–25.
Escobar MA, Hoelz DJ, Sandoval JA, Hickey RJ, Grosfeld JL, Malkas LH. Profiling of nuclear extract proteins from human neuroblastoma cell lines: the search for fingerprints. Journal of pediatric surgery. 2005;40(2):349–58.
Sandoval JA, Hoelz DJ, Woodruff HA, Powell RL, Jay CL, Grosfeld JL, et al. Novel peptides secreted from human neuroblastoma: useful clinical tools? Journal of pediatric surgery. 2006;41(1):245–51.
Yang TW, Sahu D, Chang YW, Hsu CL, Hsieh CH, Huang HC, et al. RNA-Binding Proteomics Reveals MATR3 Interacting with lncRNA SNHG1 To Enhance Neuroblastoma Progression. Journal of proteome research. 2019;18(1):406–16.
Zins K, Schäfer R, Paulus P, Dobler S, Fakhari N, Sioud M, et al. Frizzled2 signaling regulates growth of high-risk neuroblastomas by interfering with β-catenin-dependent and β-catenin-independent signaling pathways. Oncotarget. 2016;7(29):46187–202.
Di Francesco AM, Meco D, Torella AR, Barone G, D'Incalci M, Pisano C, et al. The novel atypical retinoid ST1926 is active in ATRA resistant neuroblastoma cells acting by a different mechanism. Biochemical pharmacology. 2007;73(5):643–55.
Chuang JH, Chou MH, Tai MH, Lin TK, Liou CW, Chen T, et al. 2-Deoxyglucose treatment complements the cisplatin- or BH3-only mimetic-induced suppression of neuroblastoma cell growth. The international journal of biochemistry & cell biology. 2013;45(5):944–51.
Hanmod SS, Wang G, Edwards H, Buck SA, Ge Y, Taub JW, et al. Targeting histone deacetylases (HDACs) and Wee1 for treating high-risk neuroblastoma. Pediatric blood & cancer. 2015;62(1):52–9.
Huang CC, Wang SY, Lin LL, Wang PW, Chen TY, Hsu WM, et al. Glycolytic inhibitor 2-deoxyglucose simultaneously targets cancer and endothelial cells to suppress neuroblastoma growth in mice. Disease models & mechanisms. 2015;8(10):1247–54.
Dower CM, Bhat N, Gebru MT, Chen L, Wills CA, Miller BA, et al. Targeted Inhibition of ULK1 Promotes Apoptosis and Suppresses Tumor Growth and Metastasis in Neuroblastoma. Molecular cancer therapeutics. 2018;17(11):2365–76.
Marcus K, Johnson M, Adam RM, O'Reilly MS, Donovan M, Atala A, et al. Tumor cell-associated neuropilin-1 and vascular endothelial growth factor expression as determinants of tumor growth in neuroblastoma. Neuropathology: official journal of the Japanese Society of Neuropathology. 2005;25(3):178–87.
Tsutsumimoto T, Williams P, Yoneda T. The SK-N-AS human neuroblastoma cell line develops osteolytic bone metastases with increased angiogenesis and COX-2 expression. Journal of bone oncology. 2014;3(3–4):67–76.
Ochiai H, Takenobu H, Nakagawa A, Yamaguchi Y, Kimura M, Ohira M, et al. Bmi1 is a MYCN target gene that regulates tumorigenesis through repression of KIF1Bbeta and TSLC1 in neuroblastoma. Oncogene. 2010;29(18):2681–90.
Thiele CJ. Neuroblastoma. In: Masters JRW, Palsson B, editors. Human Cell Culture: Cancer Cell Lines Part 1. Dordrecht: Springer Netherlands; 1999. p. 21–53.
Yu Y, Ding J, Zhu S, Alptekin A, Dong Z, Yan C, et al. Therapeutic targeting of both dihydroorotate dehydrogenase and nucleoside transport in MYCN-amplified neuroblastoma. Cell death & disease. 2021;12(9):821.
von Reitzenstein C, Kopitz J, Schuhmann V, Cantz M. Differential functional relevance of a plasma membrane ganglioside sialidase in cholinergic and adrenergic neuroblastoma cell lines. European journal of biochemistry. 2001;268(2):326–33.
Helson C, Helson L. Deferoxamine and human neuroblastoma and primitive neuroectodermal tumor cell lines. Anticancer research. 1992;12(2):481–3.
Johnsen JI, Pettersen I, Ponthan F, Sveinbjørnsson B, Flaegstad T, Kogner P. Synergistic induction of apoptosis in neuroblastoma cells using a combination of cytostatic drugs with interferon-gamma and TRAIL. International journal of oncology. 2004;25(6):1849–57.
Chuang JH, Chuang HC, Huang CC, Wu CL, Du YY, Kung ML, et al. Differential toll-like receptor 3 (TLR3) expression and apoptotic response to TLR3 agonist in human neuroblastoma cells. Journal of biomedical science. 2011;18(1):65.
Ödborn Jönsson L, Sahi M, Lopez-Lorenzo X, Keller FL, Kostopoulou ON, Herold N, et al. Heterogeneities in Cell Cycle Checkpoint Activation Following Doxorubicin Treatment Reveal Targetable Vulnerabilities in TP53 Mutated Ultra High-Risk Neuroblastoma Cell Lines. International journal of molecular sciences. 2021;22(7).
Liu W, Mirzoeva S, Yuan Y, Deng J, Chen S, Lai B, et al. Development of Fe(3)O(4) core-TiO(2) shell nanocomposites and nanoconjugates as a foundation for neuroblastoma radiosensitization. Cancer nanotechnology. 2021;12(1):12.
Gautier L, Cope L, Bolstad BM, Irizarry RA. affy–analysis of Affymetrix GeneChip data at the probe level. Bioinformatics (Oxford, England). 2004;20(3):307–15.
Bioconductor. [Available from: https://www.bioconductor.org/.
Team RC. R: A language and environment for statistical computing. R Foundation for Statistical Computing Vienna, Austria [Available from: https://www.R-project.org/.
Dai M, Wang P, Boyd AD, Kostov G, Athey B, Jones EG, et al. Evolving gene/transcript definitions significantly alter the interpretation of GeneChip data. Nucleic acids research. 2005;33(20):e175.
Mecham BH, Nelson PS, Storey JD. Supervised normalization of microarrays. Bioinformatics (Oxford, England). 2010;26(10):1308–15.
Ritchie ME, Phipson B, Wu D, Hu Y, Law CW, Shi W, et al. limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic acids research. 2015;43(7):e47.
Phipson B, Lee S, Majewski IJ, Alexander WS, Smyth GK. ROBUST HYPERPARAMETER ESTIMATION PROTECTS AGAINST HYPERVARIABLE GENES AND IMPROVES POWER TO DETECT DIFFERENTIAL EXPRESSION. The annals of applied statistics. 2016;10(2):946–63.
Raudvere U, Kolberg L, Kuzmin I, Arak T, Adler P, Peterson H, et al. g:Profiler: a web server for functional enrichment analysis and conversions of gene lists (2019 update). Nucleic acids research. 2019;47(W1):W191-w8.
Stanisavljevic D, Petrovic I, Vukovic V, Schwirtlich M, Gredic M, Stevanovic M, et al. SOX14 activates the p53 signaling pathway and induces apoptosis in a cervical carcinoma cell line. PloS one. 2017;12(9):e0184686.
Esumi N, Imashuku S, Tsunamoto K, Todo S, Misawa S, Goto T, et al. Procoagulant activity of human neuroblastoma cell lines, in relation to cell growth, differentiation and cytogenetic abnormalities. Japanese journal of cancer research: Gann. 1989;80(5):438–43.
Sokolov MV, Panyutin IV, Neumann RD. Unraveling the global microRNAome responses to ionizing radiation in human embryonic stem cells. PloS one. 2012;7(2):e31028.
Berezikov E, Chung WJ, Willis J, Cuppen E, Lai EC. Mammalian mirtron genes. Molecular cell. 2007;28(2):328–36.
Welch C, Chen Y, Stallings RL. MicroRNA-34a functions as a potential tumor suppressor by inducing apoptosis in neuroblastoma cells. Oncogene. 2007;26(34):5017–22.
Tivnan A, Tracey L, Buckley PG, Alcock LC, Davidoff AM, Stallings RL. MicroRNA-34a is a potent tumor suppressor molecule in vivo in neuroblastoma. BMC cancer. 2011;11:33.
Cheng X, Xu Q, Zhang Y, Shen M, Zhang S, Mao F, et al. miR-34a inhibits progression of neuroblastoma by targeting autophagy-related gene 5. European journal of pharmacology. 2019;850:53–63.
De Antonellis P, Carotenuto M, Vandenbussche J, De Vita G, Ferrucci V, Medaglia C, et al. Early targets of miR-34a in neuroblastoma. Molecular & cellular proteomics: MCP. 2014;13(8):2114–31.
Ordóñez R, Gallo-Oller G, Martínez-Soto S, Legarra S, Pata-Merci N, Guegan J, et al. Genome-wide microarray expression and genomic alterations by array-CGH analysis in neuroblastoma stem-like cells. PloS one. 2014;9(11):e113105.
Giurgiu M, Reinhard J, Brauner B, Dunger-Kaltenbach I, Fobo G, Frishman G, et al. CORUM: the comprehensive resource of mammalian protein complexes-2019. Nucleic acids research. 2019;47(D1):D559-d63.
Boeva V, Louis-Brennetot C, Peltier A, Durand S, Pierre-Eugène C, Raynal V, et al. Heterogeneity of neuroblastoma cell identity defined by transcriptional circuitries. Nature genetics. 2017;49(9):1408–13.

No competing interests reported.

Download PDF

Version 1

posted

You are reading this older preprint version

Read the latest preprint version →

Gene Expression and Radiation Response of Two Distinct Neuroblastoma Cell Lines

Status:

Version 1

Abstract

Figures

1. Background

2. Methods

2.1 Cell Culture and Treatments

2.2 RNA Isolation for Quantitative Real-Time Polymerase Chain Reaction and Gene Expression Array

2.3 Quantitative real-time polymerase chain reaction (qPCR)

2.4 Isolation of nuclear extracts and ELISA assays for c-Myc and NF-kB

3. Results

3.1 Clonogenic assays show marked differences in radiation response between neuroblastoma cell lines SK-N-AS and SK-N-DZ

3.3 Clonogenic assays show no cell survival differences between native and microRNA transfected neuroblastoma cells SK-N-AS

3.4 Clariom™ D Assay Gene Expression Study of Neuroblastoma Cell Lines SK-N-AS and SK-N-DZ

3.5 Selected gene and protein expression differences

4. Discussion

5. Conclusions

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1