These two authors contributed equally to this work.
Oncogenic activation of glypican-3 by c-Myc in human hepatocellular carcinoma†
Article first published online: 4 OCT 2012
Copyright © 2012 American Association for the Study of Liver Diseases
Volume 56, Issue 4, pages 1380–1390, October 2012
How to Cite
Li, L., Jin, R., Zhang, X., Lv, F., Liu, L., Liu, D., Liu, K., Li, N. and Chen, D. (2012), Oncogenic activation of glypican-3 by c-Myc in human hepatocellular carcinoma. Hepatology, 56: 1380–1390. doi: 10.1002/hep.25891
Potential conflict of interest: Nothing to report.
- Issue published online: 4 OCT 2012
- Article first published online: 4 OCT 2012
- Accepted manuscript online: 18 JUN 2012 04:58AM EST
- Manuscript Accepted: 22 MAY 2012
- Manuscript Received: 21 DEC 2011
- National Natural Science Foundation of China. Grant Number: 30910103915, 81071843
- Beijing Natural Science Foundation. Grant Number: 7101005;
- Twelve-fifth Key Science and Technology Five Year Plan of China. Grant Number: 2012ZX10001-003, 2012ZX10001-004
Additional Supporting Information may be found in the online version of this article.
|HEP_25891_sm_SuppFig1.tif||2397K||Supporting Information Figure 1. The expression of c-Myc upregulates GPC3 and accelerates HCC cell proliferation. A, Western blotting showed increased levels of GPC3 in HepG2 cells that were transfected with the c-Myc expression plasmid. β-actin was used as a loading control. B, HepG2 cells were seeded into 6-well plates and were then transfected with the pcDNA3.1 empty plasmid or the pcDNA3.1-c-Myc expression plasmid. The cells were next stained with antibodies against GPC3 (green) and c-Myc (red). GPC3 exhibited localization both at the cell surface and within the cytoplasm after transfection with the pcDNA3.1-c-Myc expression plasmid. C, Flow cytometric cell cycle analysis revealed that both the overexpression of GPC3 and c-Myc significantly induced more S phase accumulation of Huh-7 cells, which suggestes increased cell proliferation. Western blotting confirmed that the transient overexpression of c-Myc stimulates GPC3 expression. D, Flow cytometric cell cycle analysis demonstrated more cells G1 phase-arrested Huh-7 cells upon co-transfection with the c-Myc overexpression plasmid and the siRNA-GPC3 plasmid. Consistent with the G1 arrest, this treatment decreased the proliferation rate of the HCC cells. The western blotting results indicated that GPC3 expression was decreased in the co-transfected cells. Three independent experiments were performed. E, The characterization of the expression of GPC3, c-Myc or c-Myc/siRNA-GPC3 by HCC cell proliferation assays. The pcDNA3.1 empty plasmid, GPC3, c-Myc or c-Myc/siRNA-GPC3 plasmids were transfected into Huh-7 cells, and the MTT assay was performed. The cell growth rate was significantly increased in cells that were transfected with the GPC3 or c-Myc expression plasmids compared with those transfected with the pcDNA3.1 empty plasmid and c-Myc/siRNA-GPC3 plasmid, there was no significant difference between the cells transfected with the pcDNA3.1 empty plasmid and c-Myc/siRNA-GPC3 plasmid (*P < 0.05, #P < 0.05 at days 2 and 3). F, The effects of GPC3 knockdown on the apoptotic rate of c-Myc-overexpressing cells. We co-transfected EGFP/c-Myc/siRNA-GPC3 (1:2:2) into Huh-7 cells, isolated for GFP-positive cells, and assessed the alterations in cell cycle and apoptosis. The results show that the co-transfection of c-Myc and siRNA-GPC3 synergistically enhanced apoptosis compared with c-Myc-transfected cells and the empty plasmid -transfected cells alone.|
|HEP_25891_sm_SuppFig2.tif||291K||Supporting Information Figure 2. The following plasmid combinations were co-transfected into the Huh-7 or HepG2 cells: EGFP/c-Myc (1:2), EGFP/GPC3 (1:2), EGFP/c-Myc/siRNA-GPC3 (1:2:2). The GFP-positive cells were isolated, and analyzed for sub-G0/G1 (apoptosis peak), G1, S, G2-M. The result shows that cells co-transfected with EGFP/c-Myc/siRNA-GPC3 plasmid exhibited significantly increased apoptosis.|
|HEP_25891_sm_SuppTab1.doc||22K||Supporting Information Table 1. Correlation between c-Myc expression and the differentiation of HCC|
|HEP_25891_sm_SuppTab2.doc||22K||Supporting Information Table 2. Correlation between GPC3 expression and the differentiation of HCC|
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