Regulation of MYCN expression in human neuroblastoma cells

Abstract

Background: Amplification of the MYCN gene in neuroblastoma (NB) is associated with a poor prognosis. However, MYCN-amplification does not automatically result in higher expression of MYCN in children with NB. We hypothesized that the discrepancy between MYCN gene expression and prognosis in these children might be explained by the expression of either MYCN-opposite strand (MYCNOS) or the shortened MYCN-isoform (DeltaMYCN) that was recently identified in fetal tissues. Both MYCNOS and DeltaMYCN are potential inhibitors of MYCN either at the mRNA or at the protein level.

Methods: Expression of MYCN, MYCNOS and DeltaMYCN was measured in human NB tissues of different stages. Transcript levels were quantified using a real-time reverse transcriptase polymerase chain reaction assay (QPCR). In addition, relative expression of these three transcripts was compared to the number of MYCN copies, which was determined by genomic real-time PCR (gQPCR).

Results: Both DeltaMYCN and MYCNOS are expressed in all NBs examined. In NBs with MYCN-amplification, these transcripts are significantly higher expressed. The ratio of MYCN:DeltaMYCN expression was identical in all tested NBs. This indicates that DeltaMYCN and MYCN are co-regulated, which suggests that DeltaMYCN is not a regulator of MYCN in NB. However, the ratio of MYCNOS:MYCN expression is directly correlated with NB disease stage (p = 0.007). In the more advanced NB stages and NBs with MYCN-amplification, relatively more MYCNOS is present as compared to MYCN. Expression of the antisense gene MYCNOS might be relevant to the progression of NB, potentially by directly inhibiting MYCN transcription by transcriptional interference at the DNA level.

Conclusion: The MYCNOS:MYCN-ratio in NBs is significantly correlated with both MYCN-amplification and NB-stage. Our data indicate that in NB, MYCN expression levels might be influenced by MYCNOS but not by DeltaMYCN.

Figure 1

Schematic overview of (A) genomic organization, (B) transcripts, and (C) protein isoforms of MYCN and MYCNOS. The localization of primer sites (small arrows) and the C-19 antibody epitope are indicated. (D) RT-PCR with primers on exon 1 and 3 on NB cDNA of patient 13 and NB cell line IMR-32 give products of 1007 bp (MYCN) and 100 bp (ΔMYCN). The identity of both products was verified by sequence analysis.

Figure 2

Detection of MYCN and ΔMYCN in IMR-32 cells. (A) Western blot, visualizing two proteins in the IMR-32 (NB cell line with MYCN-amplification) lysate with the C-19 antibody that recognizes the C-terminal epitope of both MYCN and ΔMYCN. In the BLM (melanoma cell line without MYCN-amplification) lysate, these proteins were not present. (B) Western blot of IMR-32 whole lysate (1), whole lysate minus precipitate (2) and the precipitate (3) using the C-19 antibody. Arrows indicate the positions of MYCN (65 kDa) and ΔMYCN (45 kDa). The additional band in lane 3 is caused by deposition of Ig-heavy chains (50 kDa). Exposure times are indicated below the blots.

Figure 3

MYCN, ΔMYCN, and MYCNOS mRNA expression levels in NBs. (A) Expression levels of MYCN, ΔMYCN and MYCNOS in MYCN-amplified (closed-triangles) compared to non-amplified (open triangles) tumours. (B) Relative expression-levels of MYCN (closed triangles), ΔMYCN (closed circles) and MYCNOS (open circles) correlated to MYCN-amplification in NBs. (C) Difference of the MYCN:MYCNOS-ratio between NBs with MYCN-amplification (closed circles) and NBs without MYCN-amplification (open circles). (D) Correlation of the number of MYCN copies with MYCN:MYCNOS mRNA ratios (closed circles and non-interrupted line) and with MYCN:MYCN mRNA ratios (open triangles and dotted line). (E) Correlation between NB stage and MYCN:MYCNOS-ratio. (F) Correlation between NB stage and MYCN: ΔMYCN-ratio. NB IV-s is a special type of NB characterized by metastatic disease with spontaneous regression and good survival [31].

Figure 4

Overexpression of MYCNOS in the NB cell line IMR-32. (A) Transfection-efficiency was measured by GFP expression as analysed by flow cytometry, 74% of the NB cells expressed GFP 72 hours after transfection. (B) In the MYCNOS transfected IMR-32 cells, MYCNOS was 50× upregulated compared to not transfected and mock-transfected IMR-32 cells. Endogenous MYCN expression was not significantly affected. (C) Western blot showing that MYCN protein expression in MYCNOS transfected IMR-32 cells was unaffected. Lane 1 is loaded with lysate from untransfected cells, lane 2 with lysate from cells transfected with the empty vector, and lane 3 with lysate from MYCNOS transfected cells.