demonstrated that IRX1 overexpression weakened the adhesive ability of cell-collagen IV and cell-ﬁbronectin adhesion. As to blood supply of tumor, much study focused on the ‘classic’ genes, such as vascular endothelial growth factor (Altundag et al., 2004). In our studies, vascular endothelial growth factor or other metastasis-associated factor matrix metalloproteinases were not changed so much after IRX1 overexpression (Supplementary Figure 1). We found that BDKRB2 and its effector PAK1 are downregulated by IRX1 overexpression. BDKRB2 is a 9 amino acids bradykinin peptide receptor, which is constitutively expressed in vasculature. Bradykinin functions as a positive regulator for angiogenesis. BDKRB2 is overexpressed in various types of human cancer (Ishihara et al., 2002; Krankel et al., 2008). In addition, BDKRB2 interacts with integrin a5 b1 to transactivate epidermal growth factor receptor in kidney cells (Kramarenko et al., 2010). Ikeda et al. (2004) found that antagonizing bradykinin can reduce angio- genesis and tumor growth in rats. BDKRB2 knockout mice showed decreased growth of tumor xenografts due to inhibited angiogenesis. BDKRB2 mediated induction of cyclooxygenase-2 and endothelin-1, which function as stimulators of vascular formation (Basu et al., 2006; Zhang et al., 2008; Andoh et al., 2010). We evaluated the roles of BDKRB2 in tumor-associated angiogenesis on cell model. RNA interference (RNAi) is a process in which double-strand RNA directs the speciﬁc degrada- tion of a corresponding target mRNA. The mediators of this process are small double-strand RNAs, of B21 bp in length, called siRNAs (Calderon and Lavergne, 2005; Oleinikov et al., 2005; Bessard et al., 2008). siRNAs can be transfected into cells and direct the degradation of corresponding mRNAs. Hence, siRNAs represent a powerful tool for studying gene functions, as well as having the potential of being highly speciﬁc pharma- ceutical agents (Gartel and Kandel, 2006; Gu et al., 2008). PAK, a mammalian homologs of Ser/Thr protein kinase, is the effector of bradykinin peptide receptors pathway, which has been implicated in a variety of intracellular signaling events, including cell cytoskeleton rearrangement, proliferation, cell-cycle progression, cell migration and angiogenesis (Tang et al., 2000; Fryer and Field, 2005; Galan Moya et al., 2009). Overexpression and activation of PAK has been identiﬁed in the development of cancers (Kumar and Vadlamudi, 2002; Liu et al., 2009). Recently, targeting PAKs have been proposed as a potential therapeutic strategy for gastric cancer (Cai et al., 2009; Li et al., 2010). In this study, protein levels of BDKRB2 and its downstream effector PAK1 were obviously downregu- lated by overexpression of IRX1 gene in gastric cancer cells. We knocked down the BDKRB2 or PAK1 by RNAi and got an obvious inhibitory effect on tube formation, cell proliferation, cell migration and inva- sion. Our results suggested that BDKRB2 promote tumor proliferation and invasion by increased blood supply combined with other survival signals. Because BDKRB2 antagonist has been developed (Ishihara et al., 2002), the administration of BDKRB2 antagonist IRX1 inﬂuences peritoneal spreading and metastasis J Jiang et al may provide a promising method for anti-cancer therapy. In conclusion, our data, for the ﬁrst time, implicate that IRX1 suppresses peritoneal spreading and long distance metastasis via inhibiting BDKRB2-dependent angiogenesis and VM mechanisms on gastric cancer. When we inhibited BDKRB2 by speciﬁc RNAi, a remarkable reduction of neovascularization was ob- served. BDKRB2 might be a potential target for anti- cancer strategy. Materials and methods Cell culture and transfection of IRX1 gene Human gastric cancer cell SGC-7901 and HUVECs were preserved in our institute. Brieﬂy, cells were grown in RPMI1640 supplemented with 10% fetal calf serum, and 2 mmol L-glutamine. Cells were maintained at 37 1C with 5% CO2. pEGFP-N1-IRX1 eukaryotic expression vector was constructed as previously described (Guo et al., 2010). pReceiver-M03-BDKRB2 eukaryotic expression vector was purchased from FulenGen Company (Guangzhou, China). Transfectant of the empty vectors was used as controls. After 48 h incubation of SGC-7901 with transfectants, the super- natants were collected for further studies. Endothelial tube formation assay Angiogenesis in vitro was assessed by the endothelial tube formation assay kit (Cell Biolabs, San Diego, CA, USA). The tube formation assay is based on the ability of endothelial cells to form 3D capillary-like tubular structures when cultured on a gel of basement membrane extract. Brieﬂy, each well of prechilled 96-well culture plates was coated with a thin layer of the ECM gel prepared from Engelbreth-Holm-Swarm tumor cells (50 ml/well), which was left to polymerize at 37 1C for 1 h. HUVECs were resuspended in collected supernatants from controls (SGC-7901), mock (SGC-7901/vector), SCG-7901/ IRX1, SGC-7901/siRNA-BDKRB2, respectively. HUVECs (2 104 cells/well) were added to the polymerized ECM gel with 300 ml of the supernatants. After 18 h incubation at 37 1C with 5% CO2, the tube formation ability was evaluated by counting the tubular number, the tubular length and tubular intersecting nods in ﬁve random ﬁelds using Image Pro Plus software (Media Cybernetics Inc., Bethesda, MD, USA; Serial No. 45N51000-33993) according to Mirshahi’s method (Mirshahi et al., 2009). Each experiment was performed at least three times. VM assay on 3D culture 3D type I collagen gel and matrigel mixture were produced as following: 25 ml of type I collagen (BD Bioscience, Bedford, MA, USA; 3.7 mg/ml) and matrigel (BD Bioscience) were mixed according to 1:1, dropped onto 16 mm glass cover slides in six-well culture plate. Absolute ethanol of 100 ml was added to each well until the gel was polymerized at room temperature. After a wash with PBS, 1.5 105 cells of SGC- 7901, SGC-7901/vector and SCG-7901/IRX1 were plated onto the gel plate. After 4-day culture without changing culture media, the cells were ﬁxed with 4% formaldehyde in PBS for 10 min, then stained with PAS stain and counterstained with hematoxylin. The vascular mimicry formation ability was evaluated by counting the average vascular mimicry length and intersecting nods. Each experiment was performed at least three times. 4505 Oncogene