These authors contributed equally to this work.
Version of Record online: 6 SEP 2011
Copyright © 2011 American Association for the Study of Liver Diseases
Volume 54, Issue 6, pages 2149–2158, December 2011
How to Cite
Urtasun, R., Latasa, M. U., Demartis, M. I., Balzani, S., Goñi, S., Garcia-Irigoyen, O., Elizalde, M., Azcona, M., Pascale, R. M., Feo, F., Bioulac-Sage, P., Balabaud, C., Muntané, J., Prieto, J., Berasain, C. and Avila, M. A. (2011), Connective tissue growth factor autocriny in human hepatocellular carcinoma: Oncogenic role and regulation by epidermal growth factor receptor/yes-associated protein–mediated activation. Hepatology, 54: 2149–2158. doi: 10.1002/hep.24587
Potential conflict of interest: Nothing to report.
Work in the authors' laboratory is supported by the agreement between FIMA and the “UTE project CIMA.” Red Temática de Investigación Cooperativa en Cáncer RD06 00200061 (to C.B. and M.A.A.), CiberEhd (to J.P.), Fundación Echébano, Fundacion Barrié de la Maza y Condesa de Fenosa, and grants FIS PI070392, PI070402, PI10/00038, and PI10/02642 from Instituto de Salud Carlos III. M.U.L. and R.U. were supported by a “Ramón y Cajal” and a “Torres Quevedo” contract from Ministerio de Educación, respectively. M.I.D. and S.B. were supported by the Erasmus program. M.E. was supported by a fellowship from Gobierno de Navarra.
- Issue online: 30 NOV 2011
- Version of Record online: 6 SEP 2011
- Accepted manuscript online: 28 JUL 2011 09:59AM EST
- Manuscript Accepted: 16 JUL 2011
- Manuscript Received: 7 APR 2011
Additional Supporting Information may be found in the online version of this article.
|HEP_24587_sm_SuppFig1.tif||9040K||Supporting Information Figure 1. Expression of CTGF and YAP in healthy and diseased human liver. (A) CTGF and YAP mRNA levels were measured in healthy livers (HL, n-9), healthy liver tissue from organs harbouring a hepatocellular carcinoma (HL+HCC, n-12), cirrhotic liver tissue from organs harbouring a hepatocellular carcinoma (Cirr+HCC, n-17) and hepatocellular carcinoma (HCC, n-20). “+” indicates the mean value of CTGF or YAP expression for each set of samples. For CTGF expression statistical significance was: HL vs HL+HCC p-0.002; HL vs Cirr+HCC p- 0.0002; HL vs HCC p-0.026. For YAP expression statistical significance was: HL vs HL+HCC p-0.0003; HL vs Cirr+HCC p-0.0001; HL vs HCC p-0.0001. (B) Immunohistochemical detection of CTGF and YAP proteins in sections from HL, Cirr+HCC and HCC liver tissues. Representative photomicrographs are shown (magnification × 200).|
|HEP_24587_sm_SuppFig2.tif||4972K||Supporting Information Figure 2. EGFR activation stimulates CTGF and YAP expression in MCF-10A cells. (A) CTGF mRNA levels in cells treated with AR (50 nmol/L) or HB-EGF (50 nmol/L) for 3h. (B) YAP mRNA levels in cells treated with AR (50 nmol/L) or HB-EGF (50 nmol/L) for 3h. (C) YAP protein levels in cells treated with AR (50 nmol/L) or HB-EGF (50 nmol/L) for 12h analyzed by Western blotting. (D) Immunofluorescent detection of YAP protein in control and AR (50 nmol/L, 12h) treated MCF-10A cells.|
|HEP_24587_sm_SuppFig3.tif||85K||Supporting Information Figure 3. Effect of AR on miRNA-375 expression. Levels of miRNA-375 in control and AR (50 nmol/L, 3h) stimulated Hep3B cells (A) and primary human hepatocytes (B).|
|HEP_24587_sm_SuppFig4.tif||788K||Supporting Information Figure 4. Effect of CTGF knockdown on anchorage-independent cell growth in soft agar and on in vivo growth in nude mice. (A) PLC/PRF/5 cells were transfected with control (siGL) or CTGF specific (siCTGF) siRNAs. After 48h cells were harvested, counted, resuspended in 0.2% soft agar, and seeded onto 0.4% soft agar in DMEM supplemented with 10% fetal calf serum (104 cells per plate). After 3 weeks, colonies were stained with crystal violet and counted. (B) PLC/PRF/5 cells were transfected with control (siGL) or CTGF specific (siCTGF) siRNAs. After 48h cells were harvested and subcutaneously injected into nude mice (10 × 106 cells per mouse). Tumor volumes (mm3) were monitored over time. Data are means ± SEM (n-10 mice per group). Asterisks indicate p<0.05 vs. siCTGF.|
|HEP_24587_sm_SuppFig5.tif||185K||Supporting Information Figure 5. Validation of selected genes identified by microarray analysis as differentially expressed in Hep3B cells upon CTGF knockdown. Quantitative real-time PCR analysis of gene expression in total RNA samples from Hep3B cells 48h after transfection with siCTGF or control siGL siRNAs. Values are expressed in relative transcript levels as compared with control siGL siRNA. (A) Genes downregulated upon CTGF knockdown included G protein-coupled receptor 160 (GPR160), connexin 43/GJA1 (GJA), lysyl oxidase (LOX), orosomucoid 1 and 2 (ORM1, ORM2) and fibroblast growth factor receptor 2 (FGFR2). (B) Genes upregulated upon CTGF knockdown included bile acid CoA: amino acid N-acyltransferase (BAAT), the PR-Set7 histone methyltransferase (SETD8), latent TGFβ binding protein-1 (LTBP1), p21-activated protein kinase-2 (PAK2), lysine acetyltransferase 2B (KAT2B/PCAF), UDP-glucuronosyltransferase-2B15 (UGT2B15), tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor 2 (TNFRSF10B or TRAIL-R2) and tryptophan dioxygenase (TDO). Differences were statistically significant (p values at least <0.05) with respect control transfections for all genes shown.|
|HEP_24587_sm_SuppTabS1.doc||57K||Supporting Information Table 1. Selection of genes with differential expression in Hep3B cells upon CTGF knockdown identified by microarray analysis.|
|HEP_24587_sm_SuppTabS2.doc||71K||Supporting Information Table 2. Primers used in this study to measure gene expression by quantitative RT-PCR.|
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