Neuroblastoma (NB) is the most common extra-cranial solid pediatric tumor. The prognosis of patients with NB has been improved during the last decades. However, treatment results for patients with advanced tumor stages are still unsatisfying. NB cells are characterized by a high tendency for spontaneous or induced differentiation. During differentiation, down-regulation of the basic helix-loop-helix transcription factor achaete-scute complex homolog 1 (ASCL1) has been observed but the consequences of ASCL1 down-regulation have not been elucidated. We used RNA interference to knock-down ASCL1 in NB cells. DNA microarray analysis was used for the identification of ASCL1-regulated genes. Furthermore, conventional and quantitative reverse transcription-polymerase chain reaction (RT-PCR) was used for validation of ASCL1-regulated genes. Down-regulation of ASCL1 influenced the expression of several genes. After down-regulation of ASCL1, we observed very high expression of insulin-like growth factor 2 (IGF2), a factor that is known to be induced during differentiation of NB cells. RT-PCR indicated up-regulation of multiple IGF2 transcript variants after ASCL1 knock-down. Our data suggest that the ASCL1-pathway is responsible for the up-regulation of IGF2 during NB differentiation.
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Achaete-scute complex homolog 1 (ASCL1) is a member of the basic helix-loop-helix (bHLH) family of transcription factors and is required for proper development of the sympathetic nervous system . ASCL1 is expressed at early stages of neurogenesis and is transiently expressed in migrating sympatho-adrenal precursor cells. ASCL1 plays a key role in neuronal fate determination and cell type specification and is essential for the formation of multiple central nervous system and peripheral nervous system neural lineages . ASCL1 is expressed in a majority of primary neuroblastoma cells. It was observed that when neuroblastoma cells are induced to differentiate by inducing agents like retinoic acid, the expression of ASCL1 is rapidly down-regulated . One important regulator of ASCL1 expression is the bHLH protein hairy and enhancer of spilt 1 (HES1). HES1 belongs to the highly conserved family of hairy-related bHLH proteins. HES1 is a positive regulator of the NOTCH signaling pathway. A very rapid up-regulation of HES1 was found during neuroblastoma differentiation that was followed by ASCL1 down-regulation.
The NOTCH signaling pathway is a highly conserved cell-signaling pathway. Since the first description in 1917 , it was noticed that Notch signaling plays a key role during several differentiation processes including neuronal development . Treatment of tumor cells with biological response modifiers often leads to alterations in the NOTCH pathway . NOTCH is a transmembrane protein with a large extracellular domain, a single transmembrane pass and a small intracellular domain. After ligand binding, proteolytic processing of NOTCH liberates the intracellular domain followed by translocation of this domain to the nucleus. The NOTCH signaling pathway is not only involved in neuronal development but also in the biology of lymphoid cells including leukemia cells [8-10].
Given the importance of the NOTCH-ASCL1 pathway for neuroblastoma, ASCL1 regulated genes might be interesting targets for the development of future treatment strategies. Therefore, we analyzed the gene expression profile of NB cells after RNA interference mediated knock-down of ASCL1.
Materials and methods
The neuroblastoma cell line SH-SY5Y, a sub-clone of the parental SK-N-SH cell line [11, 12] was purchased from the Deutsche Sammlung für Zellkulturen und Mikroorganismen (Braunschweig, Germany).
Transfection of SH-SY5Y cells with small interfering RNAs (siRNAs) was performed by electro-transfection using the Amaxa system (Amaxa GmbH, Cologne, Germany). siRNAs were synthesized by Qiagen (Hilden, Germany). ASCL1 knock-down was assessed by real time quantitative polymerase chain reaction (see below). The following siRNAs were used: ASCL1_siA: 5'-CTC CAA CGA CTT GAA CTC CAT-3'; ASCL1_siB 5'-CCG CGT CAA GTT GGT CAA CCT-3'; Control_si: 5'-AAT TCT CCG AAC GTG TCA CGT-3'. Cells treated with electroporation medium without siRNAs were used as control.
Gene expression analysis
DNA microarray analysis was performed as described  by using Affymetrix HG_U133Plus2.0 microarrays. The following conditions have been used for identification of differentially expressed genes: (i) a fold change of greater than 3 between the ASCL1 knock-down sample and the control siRNA sample, (ii) a signal intensity of greater than 100 in the sample with higher expression, (iii) a fold change of lower than 2 between the medium sample and the control siRNA sample. Data visualization was performed with the genesis software package .
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Conventional and quantitative PCR was performed essentially as described [15, 16]. The following primers have been used: beta-actin (ACTB): 5'-GGC ATC GTG ATG GAC TCC G-3', 5'-GCT GGA AGG TGG ACA GCG A-3'; ASCL1: 5'-TCG CAC AAC CTG CAT CTT TA-3', 5'-CTT TTG CAC ACA AGC TGC AT-3'; insulin-like growth factor 2 (IGF2): 5'-TTC AGT TGG CAT TTG AGC AG-3' (IGF2_a), 5'-GAA ACT GCC TGG ACG ATG AT-3' (IGF2_b), 5'-CTC CCG GAC ACT GAG GAC T-3' (IGF2_c), 5'-GTG CGT TGG ACT TGC ATA GA-3' (IGF2_d), 5'-CTC TCC GTG CTG TTC TCT CC-3' (IGF2_e), 5'-GCT GAC CTC ATT TCC CGA TA-3' (IGF2_f), 5'-CGG GCC AGA TGT TGT ACT TT-3' (IGF2_g), 5'-GGT GCT TCT CAC CTT CTT GG-3' (IGF2_h), 5'-GGG GTA TCT GGG GAA GTT GT-3' (IGF2_i); H19, imprinted maternally expressed transcript (H19): 5'-CAA CCA CTG CAC TAC CTG GA-3', 5'-GCT CAC ACT CAC GCA CAC TC-3'. PCR conditions were: 94°C, 45 sec; 60°C, 45 sec; 72°C, 60 sec (35 cycles). Primer combinations used for identification of different IGF2 transcript variants are shown in Fig. 1. Quantitative RT-PCR was performed by using the QuantiTect SYBR RT-PCR Kit (Qiagen) using the following conditions: 94°C, 45 sec; 62°C, 45 sec; 72°C, 60 sec. Target genes were amplified with 40 cycles using a Rotor Gene RG-3000 (Corbett Research, Cambridgeshire, UK) and Rotor Gene 6 software. Expression values were calculated by standard DDct calculation .
Sequencing of RT-PCR products and in silico analysis of heteroduplexes
After reverse transcription of RNA, PCR (35 cycles) was performed with primers IGF2_c and IGF2_i. After agarose gel electrophoresis, PCR products were purified using a Mini Elute Gel Extraction Kit (Quiagen). PCR products were sequenced by using an Abi Prism 3100 DNA Sequencing System (Applied Biosystems, Warrington, U.K.) using the BigDye Terminator v1.1 Cycle Sequencing Kit (Applied Biosystems) in combination with primers IGF2_c or IGF2_i. Correctness of automatically determined sequences was checked by visual examination of the corresponding chromatograms.
Prediction of structures and free energies of DNA homo- and heteroduplexes was performed with RNAstructure version 5.03 .
Results and discussion
For analysis of ASCL1 dependent gene expression in NB cells we used SH-SY5Y cells. SH-SY5Y cells as well as the parental SK-N-SH cell line are widly used models for neuronal cells [19-21]. After transfection of ASCL1-specific siRNA we observed regulation of several genes (Fig. 2). Among the probe sets corresponding to down-regulated genes after transfection with ASCL1-specific siRNA we found probe sets corresponding to ASCL1, indicating that siRNA-mediated knock-down in our cells was successful (Fig. 2). Quantitative RT-PCR proofed down-regulation of ASCL1 to approximately 10% (data not shown).
Differentially expressed genes included known genes from the NOTCH pathway, e.g. jagged 2 (JAG2) as well as unrelated genes, e.g. the ribosomal protein S11 (RPS11) or the histone H3FA (Fig. 2). According to our microarray data we found that insulin-like growth factor 2 (IGF2) was the gene with strongest up-regulation after knock-down of ASCL1 (Fig. 2). Different probe sets corresponding to IGF2 showed a highly similar signal intensity pattern (Fig. 3). Up-regulation of IGF2 after down-regulation of ASCL1 was validated by conventional and quantitative RT-PCR (Fig. 3).
Probe sets on the microarrays corresponding to IGF2 recognize all known transcript variants from IGF2. Similarly, the primers used for validation of microarray data (primers IGF2_h and IGF_i) can not be used for discrimination between different transcript variants or isoforms of IGF2 (see Fig. 1). Therefore, we designed additional primer combinations with specificity for individual transcript variants (see Fig. 1). Quantitative (Fig. 4) as well as conventional (Fig. 5) RT-PCR indicated up-regulation of all three major transcript variants of IGF2 with a dominance of transcript variant 1. Only very weak signals were obtained for transcript variant 2. Interestingly, when we performed conventional RT-PCR using primers (IGF2_c and IGF2_i) with specificity for transcript variant 3 (encoding the longer isoform 2 of IGF2), we observed multiple bands (Fig. 5). Sequencing of these PCR products revealed that the lower band corresponds to a splice variant that lacks an internal exon including the start codon for isoform 2 of IGF2 (see Fig. 1). The open reading frame (orf) of this splice variant is identical to the orf of transcript variants 1 and 2, encoding isoform 1 of IGF2. The corresponding sequences have been submitted to GenBank (accession numbers HM481219 and HM481220). Sequencing of an additional band from the agarose gel revealed that this product was derived by heteroduplex formation between the two splice variants (Fig. 6). This heteroduplex formation might be favored because the heteroduplex has a lower calculated free energy than the homoduplex formed by hybridization of the sense and antisense strands of the shorter splice variant (Fig. 6).
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During the last decades, prognosis of patients with neuroblastoma has been significantly improved. Differentiation therapy by using retinoids is an important component of the currently used therapy for neuroblastoma . Retinoids like all-trans retinoic acid (ATRA, tretinoine, 3,7-dimethyl-9-(2,6,6-trimethyl-1-cyclohexenyl)-nona-2,4,6,8-tetraenic acid), 13-cis retinoic acid (13-cis-RA, isotretinoine, 3,7-dimethyl-9-(2,6,6-trimethyl-1-cyclohexenyl)nona-2,4,6,8-tetraenic acid) or fenretinid (4-HPR, (2E,4E,6E,8E)-N-(4-hydroxyphenyl)-3,7-dimethyl-9-(2,6,6-trimethyl-1-cyclohexenyl)nona-2,4,6,8-tetraenamide) are highly potent morphogens and induce differentiation and/or apoptosis of NB cells in vitro. In addition, retinoids induce neuronal differentiation of different other cell types [23, 24]. Nevertheless, some patients relapse after differentiation therapy, indicating that some NB cells survive retinoid treatment without terminal differentiation. Therefore, the identification of new targets seems necessary.
Treatment of cells with retinoids induces massive changes in the gene expression profile of these cells, including down-regulation of stem cell-specific transcription factors . Promotor elements responsible for retinoic acid induced gene expression in NB cells have been identified . ASCL1 is one important gene which is down-regulated after treatment of NB cells with retinoic acid . However, the consequences of ASCL1 down-regulation have not been clarified. Here, we show that down-regulation of ASCL1 resulted in up-regulation of IGF2. Up-regulation of IGF2 has also been observed after treatment of NB cells [27, 28] and other cell types  with retinoic acid. Our data suggest that down-regulation of ASCL1 during (retinoid induced) NB differentiation is directly involved in up-regulation of IGF2.
The gene for IGF2 is located on chromosome 11 at position 11p15.5. Usually, IGF2 shows monoallelic expression from the paternal chromosome. Similarly, H19, which is located in the same chromosomal region, is expressed only from the maternal chromosome [30, 31]. Differential expression of H19 has been observed in tumors . Interestingly, our DNA microarray analysis indicated not only up-regulation of IGF2 but also a similar up-regulation of H19 after knock-down of ASCL1 (Fig. 2). Again, different probe sets for H19 showed a highly similar signal intensity pattern. The simultaneous up-regulation of IGF2 and H19 is in agreement with the observation of a single regulatory region controlling expression of both genes [30, 31]. IGF2 can act as autocrine growth factor for neuroblastoma cells . However, differentiating neuroblastoma cells did not longer respond to this growth promoting activity of IGF2 . Interestingly, IGF2 expression has been observed in a subgroup of neuroblastomas with favorable prognosis and also in paraganglia that are prone to postnatal involution . UP-regulation of IGF2 in ASCL1-suppressed cells might be an important factor for differentiation and regression of NB cells during differentiation therapy.