Abstract
PurposeTo explore expression and function of astrocyte elevated gene-1 (AEG-1) in human retinoblastoma (RB).MethodsThe expression of AEG-1 in histological sections of human RBs and in RB cell lines was examined using immunohistochemical staining and RT-PCR and Western blotting respectively. We knocked down AEG-1 gene levels by AEG-1-siRNA lentivirus transfection of human RB cell lines SO-RB50 and Y79, and using an MTT assay, we assessed the role of AEG-1 on RB cell proliferation. The biological significance of lentivirus transfection induced AEG-1 down-regulation was examined by assessing the apoptosis rate in the transfected RB cells by Annexin V-APC staining and flow cytometry. We additionally measured the expression of Bcl-2, Bax, cleaved-caspase-3 and caspase-3, and the phosphorylation and non-phosphorylation alternation of MAPKs.ResultsAEG-1 expression was detected to be strongly positive in the histological slides of 35 out of 54 (65%) patients with RB. AEG-1 expression increased significantly (P<0.05) with tumor stage. In the RB cell lines SO-RB50, Y79 and WERI-RB1 as compared with retinal pigment epithelium cells, expression of AEG-1 mRNA and AEG-1 protein was significantly higher. In AEG-1-siRNA lentivirus transfected cell cultures as compared with negative control lentivirus transfected cell cultures, levels of AEG-1 mRNA and of AEG-1 protein (P<0.05) and cell growth rates (P<0.01) were significantly lower, and apoptosis rate (P<0.001), Bax/Bcl-2 ratio and cleaved-caspase-3 protein level were significantly increased. The P-ERK/ERK ratio was significantly decreased in the AEG-1-siRNA lentivirus transfected cell lines.ConclusionsExpression of AEG-1 was associated with RB, in histological slides of patients and in cell culture experiments. Lentivirus transfection induced knockdown of AEG-1 had a tumor suppressive effect, potentially by tumor cell apoptosis induction through inhibition of ERK.
Highlights
Retinoblastoma (RB), the most common primary intraocular malignant tumor in children, has been widely studied because of the genetic mechanism leading to is development [1,2,3,4,5]
The P-extracellular regulated kinase (ERK)/ERK ratio was significantly decreased in the astrocyte elevated gene-1 (AEG-1)-small interfering RNAs (siRNAs) lentivirus transfected cell lines
We conducted this study to explore the expression of AEG-1 mRNA and AEG-1 protein in three RB cell lines and in human RB samples, and to construct a lentivirus-mediated knockdown of AEG-1 to downregulate its expression in the human retinoblastoma Y79 and SO-RB50 cell lines in vitro
Summary
Retinoblastoma (RB), the most common primary intraocular malignant tumor in children, has been widely studied because of the genetic mechanism leading to is development [1,2,3,4,5]. Investigations enhanced our understanding of the molecular pathogenesis of RB and proposed a multi-step process for the progression of normal retinal cells to RB cells [1,6,7]. The details of this multi-step development have remained elusive so far [8]. Numerous studies established a functional role of AEG-1 in the pathogenesis and progression of tumors, the regulation of apoptosis, and the induction of metastasis by activating diverse oncogenic signaling pathways, such as NF-kappaB, Ha-ras, PI3K/AKT, MAPK, and WNT pathways [12,13,14]. In controlled transfected RB cell colonies, we examined the effect of AEG-1 silencing on the proliferation and apoptosis of RB cells and explored the potential mechanism
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