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Downregulation of tissue factor by RNA interference in human melanoma LOX‐L cells reduces pulmonary metastasis in nude mice
Author(s) -
Wang Xiaolin,
Wang Mengyu,
Amarzguioui Mohammed,
Liu Fang,
Fodstad Øystein,
Prydz Hans
Publication year - 2004
Publication title -
international journal of cancer
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.475
H-Index - 234
eISSN - 1097-0215
pISSN - 0020-7136
DOI - 10.1002/ijc.20527
Subject(s) - small hairpin rna , transfection , tissue factor , microbiology and biotechnology , cell culture , biology , cancer research , downregulation and upregulation , small interfering rna , gene knockdown , medicine , coagulation , gene , biochemistry , genetics
Tissue factor (TF) is the membrane receptor of the serine protease coagulation factor VIIa (FVIIa). Formation of the TF/FVIIa complex initiates the coagulation cascade. We used short hairpin RNA (shRNA)‐mediated RNA interference to knock down TF expression in the human metastatic melanoma cell line LOX‐L. After transfection with the shRNA construct, 3 stable clones with significantly downregulated TF expression were established. They exhibited decreased proliferation in vitro as determined by 14 C thymidine incorporation and soft agar assay. The in vivo metastatic potential was assessed in an experimental pulmonary metastasis model in which cells from different clones were injected into the tail vein of nude mice. The incidence of pulmonary tumors was significantly lower in mice receiving shRNA‐expressing cells (33% ± 15%) than in control mice injected with wild‐type cells or cells stably transfected with empty expression vector (90% ± 10%). The mice injected with TF‐downregulated cells had markedly longer survival time (69 ± 17 days) compared to the control mice (35.6 ± 5 days; p = 0.03). Thus, reduction of TF levels in LOX‐L cells significantly delayed and reduced lung tumor formation. As a first step in elucidating the molecular basis for this effect, we compared the global gene expression profile in TF‐downregulated cells and control cells by using cDNA microarray analysis. Forty‐four known human genes were found to be significantly upregulated (> 2‐fold; p < 0.05) and 228 genes significantly downregulated (≥ 3‐fold; p < 0.05) in TF‐downregulated cells compared to control cells. The differentially expressed genes encode proteins functioning in transcription, translation, cell communication and cell growth/death. The results provide a basis for investigating molecular mechanisms underlying the effects of TF on the metastatic capacity of LOX‐L melanoma cells. © 2004 Wiley‐Liss, Inc.