title: Lentivirus-Mediated Knockdown of Astrocyte Elevated Gene-1 Inhibits Growth and Induces Apoptosis through MAPK Pathways in Human Retinoblastoma Cells.
abstract: OBJECTIVE
  To explore expression and function of astrocyte elevated gene-1 (AEG-1) in human retinoblastoma (RB).
  
  
  METHODS
  The 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.
  
  
  RESULTS
  AEG-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.
  
  
  CONCLUSIONS
  Expression 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.
authors.affiliation: Beijing Institute of Ophthalmology, Beijing TongRen Eye Center, Beijing TongRen Hospital, Capital Medical University, Beijing Ophthalmology and Visual Sciences Key Laboratory, Beijing, China.
authors.forename: Ying
authors.initials: Y
authors.lastname: Chang
authors.affiliation: Beijing Institute of Ophthalmology, Beijing TongRen Eye Center, Beijing TongRen Hospital, Capital Medical University, Beijing Ophthalmology and Visual Sciences Key Laboratory, Beijing, China.
authors.forename: Bin
authors.initials: B
authors.lastname: Li
authors.affiliation: Beijing Institute of Ophthalmology, Beijing TongRen Eye Center, Beijing TongRen Hospital, Capital Medical University, Beijing Ophthalmology and Visual Sciences Key Laboratory, Beijing, China.
authors.forename: Xiaolin
authors.initials: X
authors.lastname: Xu
authors.affiliation: Beijing Institute of Ophthalmology, Beijing TongRen Eye Center, Beijing TongRen Hospital, Capital Medical University, Beijing Ophthalmology and Visual Sciences Key Laboratory, Beijing, China.
authors.forename: Ling
authors.initials: L
authors.lastname: Shen
authors.affiliation: Beijing Institute of Ophthalmology, Beijing TongRen Eye Center, Beijing TongRen Hospital, Capital Medical University, Beijing Ophthalmology and Visual Sciences Key Laboratory, Beijing, China.
authors.forename: Haixia
authors.initials: H
authors.lastname: Bai
authors.affiliation: Beijing Institute of Ophthalmology, Beijing TongRen Eye Center, Beijing TongRen Hospital, Capital Medical University, Beijing Ophthalmology and Visual Sciences Key Laboratory, Beijing, China.
authors.forename: Fei
authors.initials: F
authors.lastname: Gao
authors.affiliation: Beijing Institute of Ophthalmology, Beijing TongRen Eye Center, Beijing TongRen Hospital, Capital Medical University, Beijing Ophthalmology and Visual Sciences Key Laboratory, Beijing, China.
authors.forename: Zhibao
authors.initials: Z
authors.lastname: Zhang
authors.affiliation: Department of Ophthalmology, Medical Faculty Mannheim of the Ruprecht-Karls-University Heidelberg, Mannheim, Germany.
authors.forename: Jost B
authors.initials: JB
authors.lastname: Jonas
mesh_terms: D017209:Apoptosis / Q000235:genetics*; D053148:Caspase 3 / Q000235:genetics / Q000378:metabolism; D015815:Cell Adhesion Molecules / Q000235:genetics* / Q000378:metabolism; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D055785:Gene Knockdown Techniques; D020868:Gene Silencing; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism; D016086:Lentivirus / Q000235:genetics; D020935:MAP Kinase Signaling System; D008297:Male; D008565:Membrane Proteins; D060787:Neoplasm Grading; D009367:Neoplasm Staging; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D016601:RNA-Binding Proteins; D012175:Retinoblastoma / Q000235:genetics* / Q000378:metabolism* / Q000473:pathology
chemical_list: D015815:Cell Adhesion Molecules; C483039:MTDH protein, human; D008565:Membrane Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D034741:RNA, Small Interfering; D016601:RNA-Binding Proteins; D048049:Extracellular Signal-Regulated MAP Kinases; D048031:JNK Mitogen-Activated Protein Kinases; D053148:Caspase 3