Neuroblastoma, the most common extracranial tumor of childhood, is precursor of sympathetic ganglia derived from malignant tumors originating from neural crest (1). It accounts for approximately 10% of all pediatric cancers (2), and patients less than 1 year old or with lower stage usually have better outcome than older patients or those with advanced stage diseases (3).
Figure 1-Primers used in RT-PCR
Figure 2-Expression of NELL2 and Fzd6 in differentiated BE(2)-C neuroblastoma cells. (A) RT-PCR examining NELL2 was performed on RNA isolated from untreated, RA treated and BUdR treated BE(2)-C cells. Primers are located in exon5 and exon9. (B) RT-PCR using Fzd6 primer set 1 was performed on RNA isolated from untreated, RA treated and BUdR treated BE(2)-C cells. Primers are located in exon4 and exon5. (C) RT-PCR of Fzd6 using primer set 2. Primers are located in exon4 and exon6. All the amplified products were fractionated on a 1.2% agarose gel. RT-PCR amplification of GAPDH mRNA was used to monitor the amount of RNA present in the samples.
Figure 3-Alignments of PCR products with reported sequences in NCBI.(A) Alignment of PCR product using NELL2 primers with a previously reported sequence (NM_006159). (B) Alignment of PCR product using Fzd6 primer set1 with a previously reported sequence (NM_003506). (C) Alignment of large PCR product using Fzd6 primer set 2 with a previously reported sequence (NM_003506). (D) RT-PCR, using Fzd6 primer set 2 identifies a splice variant of FZD6 mRNA. Alignment of small PCR product using Fzd6 primer set 2 with a previously reported sequence (NM_003506).
Figure 4-Excision of exon5 in the splice variant. (A) Splicing of the Fzd6 transcript. The large Fzd6 isoform is due to removal of introns 4 and 5 and in retention of exon5. The small Fzd6 isoform comes from the removal of introns 4 and 5 and exon 5. The primers are indicated as arrows. (B) Alignment of large and small Fzd6 cDNA and amino acid sequences. An alignment of a portion of exons 4-6 of large Fzd6 cDNA with that of small Fzd6 cDNA, demonstrating that the exclusion of exon5 results in a frameshift. The seventh transmembrane domain of Fzd6 protein is indicated in the large allele in red color. (C) A cartoon showing the effect of alternative splicing on protein level. The seventh transmembrane domain and the C-terminal are removed.
The detected expression of NELL2 in BE(2)-C neuroblastoma cell line and its differentiated cells confirms the previous report that NELL2 is highly expressed in neuroblastoma cells (18). Additionally, it has been proposed that the spatial and temporal expression of NELL2 is highly regulated, with its predominant expression in neuronal cell lineage (17, 18). Not surprisingly, BUdR treated cells showed the lowest level of NELL2 expression in that BUdR could induce I-type cells to differentiate into Schwann cells. The expression level of NELL2 in I-type BE(2)-C cells and RA treated cells was about the same, suggesting its role in stem cell renewal and differentiation and neuroblastoma cells signaling.
There appears to be no expression difference of large Fzd6 isoform using two primer sets in three cell samples. The present study indicates that Fzd6 might not be a critical player in neuroblastoma cell differentiation and progression of neuroblastoma. However, the possibility that the deregulation of Fzd6 may contribute to the tumorigenicity cannot be excluded. Moreover, the Fzd6 protein expression studies would facilitate the interpretation of Fzd6 in neuroblastoma.
The differential expression of the two Fzd6 isoforms in all three samples were also examined, and no significant difference was found (date not shown). The function of such a splice variant is unknown. Future studies on other tissue samples and research using such methods as Western blot, immunohistochemistry and functional assay would be necessary to determine the role of the splice variant of Fzd6.
I would like to thank Bo Liu and Leleesha Samaraweera for their continuous help through this project. Sincere thanks to Dr. Berish Rubin for his guidance and providing the opportunity to do the present project. I am grateful to Barbara Spengler and Dr. Robert Ross for the cells. A special thanks to Jinsong Qiu and Dan Han for valuable discussions and expertise.
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