MYCN is amplified during S phase, and c‑myb is involved in controlling MYCN expression and amplification in MYCN‑amplified neuroblastoma cell lines

Abstract

Neuroblastoma derived from primitive sympathetic neural precursors is a common type of solid tumor in infants. MYCN proto‑oncogene bHLH transcription factor (MYCN) amplification and 1p36 deletion are important factors associated with the poor prognosis of neuroblastoma. Expression levels of MYCN and c‑MYB proto‑oncogene transcription factor (c‑myb) decline during the differentiation of neuroblastoma cells; E2F transcription factor 1 (E2F1) activates the MYCN promoter. However, the underlying mechanism of MYCN overexpression and amplification requires further investigation. In the present study, potential c‑Myb target genes, and the effect of c‑myb RNA interference (RNAi) on MYCN expression and amplification were investigated in MYCN‑amplified neuroblastoma cell lines. The mRNA expression levels and MYCN gene copy number in five neuroblastoma cell lines were determined by quantitative polymerase chain reaction. In addition, variations in potential target gene expression and MYCN gene copy number between pre‑ and post‑c‑myb RNAi treatment groups in MYCN‑amplified Kelly, IMR32, SIMA and MHH‑NB‑11 cell lines, normalized to those of non‑MYCN‑amplified SH‑SY5Y, were examined. To determine the associations between gene expression levels and chromosomal aberrations, MYCN amplification and 1p36 alterations in interphases/metaphases were analyzed using fluorescence in situ hybridization. Statistical analyses revealed correlations between 1p36 alterations and the expression of c‑myb, MYB proto‑oncogene like 2 (B‑myb) and cyclin dependent kinase inhibitor 1A (p21). Additionally, the results of the present study also demonstrated that c‑myb may be associated with E2F1 and L3MBTL1 histone methyl‑lysine binding protein (L3MBTL1) expression, and that E2F1 may contribute to MYCN, B‑myb, p21 and chromatin licensing and DNA replication factor 1 (hCdt1) expression, but to the repression of geminin (GMNN). On c‑myb RNAi treatment, L3MBTL1 expression was silenced, while GMNN was upregulated, indicating G2/M arrest. In addition, MYCN gene copy number increased following treatment with c‑myb RNAi. Notably, the present study also reported a 43.545% sequence identity between upstream of MYCN and Drosophila melanogaster amplification control element 3, suggesting that expression and/or amplification mechanisms of developmentally‑regulated genes may be evolutionarily conserved. In conclusion, c‑myb may be associated with regulating MYCN expression and amplification. c‑myb, B‑myb and p21 may also serve a role against chromosome 1p aberrations. Together, it was concluded that MYCN gene is amplified during S phase, potentially via a replication‑based mechanism.

Keywords: neuroblastoma; MYCN proto‑oncogene bHLH transcription factor amplification; 1p36 deletion; c-MYB proto‑oncogene transcription factor; E2F transcription factor 1; L3MBTL1 histone methyl‑lysine binding protein; geminin DNA replication inhibitor; MYB proto‑oncogene like 2; p21/cyclin dependent kinase inhibitor 1A.

Figure 1.

Sequence comparison between MYCN upstream and ACE3, and transcription factor binding sites in MYCN upstream. (A) A 1,020 bp DNA sequence (top; bases 1–1,020) encompassing ACE3 from Drosophila melanogaster chorion gene cluster and a DNA sequence of ~65 kb (bottom) containing MYCN (bases 2,416,201 to 2,481,180) from human chromosome 2 genomic contig were compared using the LFASTAn program. (B) Using the TFSEARCH program, putative c-Myb and E2F1 binding sites upstream (−1,021 to −143) of human MYCN gene were detected. MYCN, MYCN proto-oncogene bHLH transcription factor; ACE3, amplification-control-element-on-3; c-Myb, transcriptional activator Myb; E2F1, E2F transcription factor 1.

Figure 2.

MYCN amplification and 1p36 alterations from interphase and metaphase in neuroblastoma cell lines. (A) Neuroblastoma cells were harvested for FISH experiments (magnification, ×40). (B) MYCN amplification status of interphase nuclei was determined by FISH. (C) Modal number, hsr and dmin from metaphase in neuroblastoma cell lines are demonstrated. (D) 1p36 alterations from metaphase and interphase in neuroblastoma cell lines are presented. Magnification for B-D, ×100. MYCN, MYCN proto-oncogene bHLH transcription factor; ACE3, amplification-control-element-on-3; FISH, fluorescence in situ hybridization; del, deletion; dmin, double minute; hsr, homogeneously staining region; imb, imbalance; SE, satellite enumeration.

Figure 3.

Expression of potential c-Myb target genes and MYCN gene copy number in neuroblastoma cell lines. (A) Heat map visualizing the mRNA expression of 10 genes and MYCN gene copy number (MYCN-DNA) from neuroblastoma cell lines compared with reference genes, HPRT1 and p53, respectively. The color key represents the column Z-score of gene expression levels and copy numbers. Dark pink indicates the lowest gene expression or copy number and dark green indicates the highest gene expression or copy number. (B) Expression levels of MYCN and B-myb as compared with reference HPRT1; (C) MYCN gene copy number as compared with reference p53 in neuroblastoma cell lines (see also Table III). MYCN, MYCN proto-oncogene bHLH transcription factor; B-myb, MYB proto-oncogene like 2; E2F1, E2F transcription factor 1; hCdt1, chromatin licensing and DNA replication factor 1; GMNN, geminin; p27, cyclin-dependent kinase inhibitor 1B; CDK2, cyclin dependent kinase 2; p21, cyclin-dependent kinase inhibitor 1A; L3MBTL1, L3MBTL1 histone methyl-lysine binding protein; c-Myb, transcriptional activator Myb; HPRT1, hypoxanthine phosphoribosyltransferase 1.

Figure 4.

Scatter plots for correlations of gene expressions and genomic alterations identified in neuroblastoma cell lines. The correlations of (A) MYCN copy number and 1p36-3/3 alteration, (B) c-myb and MYCN expression, (C) p21 expression and 1p36 imbalance, (D) 1p36-3/3 alteration and c-myb expression, (E) B-myb expression and 1p36 deletion, (F) 1p36 deletion and 1p36-3/3 alteration and (G) MYCN copy number and GMNN expression were presented. The scatter plots were produced using Excel. MYCN, MYCN proto-oncogene bHLH transcription factor; c-Myb, transcriptional activator Myb; B-myb, MYB proto-oncogene like 2; GMNN, geminin; p21, cyclin-dependent kinase inhibitor 1A.

Figure 5.

The effect of c-myb RNAi treatment on MYCN gene copy number. (A) Interference rate of c-myb mRNA was presented as compared with shRNA controls in MYCN-amplified neuroblastoma cell lines. The expression levels of c-myb mRNA were determined pre- and post-c-myb RNAi treatment of Kelly, SIMA, MHH-NB-11 and IMR32 cell lines, as normalized to the Cp values of target and reference genes of the SH-SY5Y control cell line. R-value was calculated using the mean Cp ± standard error of duplicate or triplicate experiments in case of discordant results. (B) MYCN/p53 DNA copy number ratios were determined by the delta-delta Cp method pre- and post-c-myb RNAi treatment as compared with shRNA control in MYCN-amplified neuroblastoma cell lines. Data are expressed as mean Cp ± standard error of the mean from ≥6 independent experiments. *P<0.05, Wilcoxon signed rank test (one-tailed). shRNA, short hairpin RNA; c-myb, transcriptional activator myb; RNAi, RNA interference; MYCN, MYCN proto-oncogene bHLH transcription factor.

Figure 6.

Alterations in potential c-Myb target gene expression and MYCN gene copy number following treatment with c-myb RNAi. MYCN gene copy number and expression levels of c-myb and potential target genes (B-myb, p21, hCdt1, E2F1, L3MBTL1, GMNN and MYCN) were calculated in MYCN-amplified neuroblastoma cell lines as normalized to the Cp values of target and reference genes of SH-SY5Y control cell line using the delta-delta Cp method (36). **Data are expressed as mean variation (%) ± standard error of the mean from Kelly, SIMA, MHH-NB-11 and IMR32 cell lines (n=4). *P<0.05; Wilcoxon signed rank test (one-tailed). shRNA, short hairpin RNA; RNAi, RNA interference; B-myb, MYB proto-oncogene like 2; p21, cyclin-dependent kinase inhibitor 1A; hCdt1, chromatin licensing and DNA replication factor 1; c-myb, transcriptional activator myb; E2F1, E2F transcription factor 1; L3MBTL1, L3MBTL1 histone methyl-lysine binding protein; GMNN, geminin; MYCN, MYCN proto-oncogene bHLH transcription factor.

Figure 7.

Involvement of c-myb and E2F1 in the expression and amplification of MYCN. (A) Model mechanism indicates the possible contribution of c-myb and E2F1 genes in the apoptotic elimination of MYCN-amplified neuroblastoma (MNA NB) cells via the upregulation of MYCN expression. MYCN overexpression indirectly activates the E2F1-induced apoptosis signaling pathway, potentially by inhibiting miR-20a and miR-92a, which prevent upregulation of E2F genes. (B) Flow schema showing the alterations in MYCN gene copy number and expression of c-myb, E2F1 and potential target genes following treatment with c-myb RNAi in MYCN-amplified neuroblastoma cell lines. *Spearman's correlation coefficient, two-tailed (r_s=1.0, P<0.01). miR, microRNA; RNAi, RNA interference. c-myb, transcriptional activator myb; MYCN, MYCN proto-oncogene bHLH transcription factor; E2F1, E2F transcription factor 1; p-73, tumor protein p73; p21, cyclin-dependent kinase inhibitor 1A; L3MBTL1, L3MBTL1 histone methyl-lysine binding protein; B-myb, MYB proto-oncogene like 2; hCdt1, chromatin licensing and DNA replication factor 1; Rb, RB transcriptional corepressor; GMNN, geminin.