Tissue factor (TF), a transmembrane protein, initiates the blood coagulation cascade by complexing with its serine protease ligand factor VII(a) (FVIIa), ultimately leading to thrombin generation, fibrin deposition and platelet aggregation. TF is aberrantly expressed in many solid tumors and tumor cell lines 1-4 with higher expression levels in malignant tumor cells compared to their nonmalignant counterpart. Subsequent results suggested that TF participates in the tumor growth and dissemination of various cancers. 5 Clinically, TF expression strongly correlates with tumor progression. 6 -9 TF can promote tumor progression via coagulation-dependent or -independent mechanisms. 10,11 Activation of coagulation may trap tumor cells in a fibrin-platelet clot and allow local tumor growth, which is critically important for tumor dissemination and metastasis. 12,13 Moreover, several products of the coagulation system, including thrombin and fibrin, can stimulate angiogenesis, 13-15 which is necessary for tumor growth and metastasis. Independent of the downstream coagulation factors, FVIIa binding to TF can promote tumor-associated angiogenesis by upregulating angiogenic factors VEGF and IL-8. 16 -18 TF/FVIIa signaling resulting from the proteolytic activity of the complex is required for the upregulation of these angiogenic factors 19 -21 and for TF-dependent metastasis. 22 TF/FVIIa signaling has received widespread interest 10,[23][24][25][26] after the discovery of TF/FVIIa-induced calcium signals. 27 FVIIa/TF signaling, independent of the coagulation cascade, results in the upregulation of cytokines, growth factors 22,24 and induction of antiapoptosis, 28 which may contribute to tumor growth. Blocking TF function by administration of TF antibody or inactivated FVIIa has resulted in the reduced metastasis in murine metastasis models. 29,30 We have in this study used RNA interference specifically to knock down TF expression and investigated the effects on the metastatic capacity of human malignant melanoma cells.RNA interference (RNAi) is a process of sequence-specific posttranscriptional gene silencing triggered by double-stranded RNA. This phenomenon has been developed into a powerful technology to elucidate mammalian gene function by delivery of small interfering RNA (siRNA), duplexes of 19-21-nt RNA with 2-nt 3Ј overhangs, into cells. 31 Variable transfection efficiency and the transient nature of silencing by exogenously delivered siRNA limit the applicability of chemically synthesized siRNA in functional genomic studies. 32 Stable RNAi can be achieved in mammalian cells through RNA polymerase III-dependent expression of so-called short hairpin RNAs (shRNA) from integrated DNA constructs in stably transfected cells. 33,34 The transcribed shRNA is thought to be processed into siRNA-like molecules intracellularly. Studies from different cell types and against various genes have demonstrated the efficacy of endogenously expressed shRNA. 33,[35][36][37] In order to explore the potential role and possible mechanism o...