Astrocyte elevated gene-1 (AEG-1) was originally characterized as a human immunodeficiency virus (HIV)-1-inducible gene in primary human fetal astrocyte [7, 8], which is a downstream target molecule of Ha- ras and c- myc mediating their tumor promoting effects [9]. AEG-1 is ubiquitously expressed in numerous cell types, elevated levels have also been observed in some solid tumors including those of breast, brain and prostate [9, 10]. Intriguingly, AEG-1 expression is elevated in diverse neoplastic conditions, it cooperates with Ha- ras to promote transformation,
and its overexpression in Hela cells induces increased anchorage-independent growth and invasiveness and increase expression ABT-263 cost of adhesion molecules by activating the NF-κB pathway [11]. However, such studies are lacking in neuroblastoma. Recently, we found that AEG-1 is also frequently overexpressed in neuroblastoma (submitted). In patients with advanced neuroblastoma, poor clinical outcome were observed related to AEG-1 overexpression, highlight a potential role of AEG-1 in promoting tumor progression GSK-3 inhibitor and metastasis of neuroblastoma. In the present study, we hypothesize
that overexpressed AEG-1 enhances tumorogenic properties of neuroblastoma cells in the same manner as observed in cultured HeLa cells [11]. The inhibition of AEG-1 expression could be a new adjuvant therapy for neuroblastoma. Methods Cell lines and culture Human neuroblastoma cell lines M17 and SK-N-SH (Chinese Type Culture Collection, Beijing, China)
were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen, Carlsbad, CA, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Gibco, AUS) at 37°C in an atmosphere of 5% CO2 with humidity. AEG-1 -siRNA transfection Knockdown of AEG-1 expression was achieved using transfection of AEG-1 -siRNA. AEG-1 -siRNA1 and AEG-1 -siRNA2 Idoxuridine targeting nucleotides 971–991 and 1355–1375 of human AEG-1 mRNA sequence (GenBank Accession No. NM_178812.3) were synthesized by Genepharma (Shanghai, China) as shown in Table 1 and annealed to form siRNA duplexes according to manufacturer’s instructions. Non-targeting siRNA was used to control for non-specific effects. Cells were transfected 24 hours under standard culture conditions with 100 nM siRNA duplexes using Lipofectamine™ 2000 (Invitrogen, Carlsbad, CA) following manufacturer’s protocols. Table 1 Targeted AEG-1 sequences and the control siRNA were chemically synthesized by Genepharma (Shanghai, China). Name Senquences AEG-1 siRNA 1 s: 5′-GACACUGGAGAUGCUAAUAUU-3′ as: 5′-UAUUAGCAUCUCCAGUGUCUU-3′ AEG-1 siRNA 2 s: 5′-GGUGAAGAUAACUCUACUGUU-3′ as: 5′-CAGUAGAGUUAUCUUCACCUU-3′ Control siRNA s: 5′-UUCUCCGAACGUGUCACGUTT-3′ as: 5′-ACGUGACACGUUCGGAGAATT-3′ Real-time RT-PCR Fourty-eight hours after transfection, cells were harvested in TRIzol Reagent (Invitrogen) and total RNA was isolated following the manufacturer’s instructions.