Journal Article

Osteopontin silencing by small interfering RNA suppresses <i>in vitro</i> and <i>in vivo</i> CT26 murine colon adenocarcinoma metastasis

Philip Y. Wai, Zhiyong Mi, Hongtao Guo, Shiva Sarraf-Yazdi, Chengjiang Gao, Junping Wei, Carlos E. Marroquin, Bryan Clary and Paul C. Kuo

in Carcinogenesis

Volume 26, issue 4, pages 741-751
Published in print April 2005 | ISSN: 0143-3334
Published online April 2005 | e-ISSN: 1460-2180 | DOI:
Osteopontin silencing by small interfering RNA suppresses in vitro and in vivo CT26 murine colon adenocarcinoma metastasis

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Hepatic metastasis is a primary cause for failure of locoregional therapy in colorectal cancer. Increased expression of osteopontin (OPN), a ligand for αvβ3 integrin and CD44 receptors, is associated with metastasis in several types of cancer. However, the mechanism by which OPN mediates metastasis in colorectal cancer remains unknown. We hypothesized that OPN mediates invasion of colon cancer cells through basement membrane and migration through extracellular matrix (ECM). In this study, we used CT26 murine colon adenocarcinoma cells syngeneic to BALB/c mice to generate cell lines (pS-OPN) in which OPN expression was suppressed through small interfering RNA (siRNA) plasmids. CT26 wild-type cells (WT) and CT26 cells stably expressing murine-mismatch siRNA (pS-MM) served as controls. Western blotting quantified OPN protein levels and our most downregulated clone, pS-OPN-A4, demonstrated a mean 3.0-fold decrease in OPN protein expression versus WT. In vitro cell motility and invasiveness were decreased in pS-OPN-A4 by 3.6-fold (P = 0.004 versus WT) and 4.1-fold (P = 0.01 versus WT), but proliferation was similar amongst cell lines. We demonstrated that OPN suppression significantly correlates with MMP-2 downregulation. In vivo hepatic metastasis was assessed by quantifying liver weights and surface tumor nodules in 33 BALB/c mice (11/group) subjected to intrasplenic injection of tumor cells. pS-OPN-A4 resulted in a 50.4% decrease in mean liver weight compared with WT (3.79 ± 1.49 g versus 1.88 ± 1.34 g, P = 0.009). Only 18% of pS-OPN-A4 livers had >20 metastatic surface nodules compared with 89% for WT and 75% for pS-MM-V6. This study demonstrates that RNA interference stably reduces CT26 tumor expression of OPN and significantly attenuates CT26 colon cancer metastasis by diminishing tumor cell motility and invasiveness.

Keywords: EGFR, epidermal growth factor receptor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; OPN, osteopontin; pS-OPN, plasmid vector expressing siRNA against OPN; pS-MM, plasmid vector expressing mismatch siRNA; PCNA, proliferating cell nuclear antigen; RNAi, RNA interference; siRNA, small interfering RNA; sR1, CT26 cells transiently transfected with OPN siRNA to target 1; sR2, CT26 cells transiently transfected with OPN siRNA to target 2; uPA, urokinase plasminogen activator; VGEF, vascular endothelial growth factor

Journal Article.  8461 words.  Illustrated.

Subjects: Clinical Cytogenetics and Molecular Genetics

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