4502 IRX1 inﬂuences peritoneal spreading and metastasis J Jiang et al Figure 5 Effect of enforcing IRX1 on angiogenesis in chick embryo CAM. (a) Schematic feature of how to open a window on fertilized chicken egg. (b) Dissecting microscope graphs of chick embryos. A remarkable reduction of blood vessels was observed in IRX1 transfectant, compared with mock or controls. (c) The bar chart of vessel numbers around ﬁlter. The blood vessel numbers in SCG-7901/IRX1 (6.41±2.59) group were signiﬁcantly reduced, compared with mock (9.18±1.99) or control (10.27±2.64, *Po0.001). expression of BDKRB2 signiﬁcantly (Figure 6c). Mean- while, we assayed the protein expression of BDKRB2 and IRX1 on peritoneal tumor tissues from nude mice by immunohistochemical stain. We found a signiﬁcant reduction of BDKRB2 expression in tumor tissues at IRX1 overexpressing group, compared with control (Figure 6d). In wound healing assay, overexpression of IRX1 alone suppressed the cell migration signiﬁcantly (Figure 6e, lane 2) and overexpression of BDKRB2 alone signiﬁcantly enhanced the cell migration (Figure 6e, lane 3). Co-expression of BDKRB2 and IRX1 revealed the effect observed as in control (Figure 6e, lane 4). The similar effects on cell migration and invasion were also observed by transwell assay (Figure 6f), along with the cell viability examination. The cell viability in SGC-7901, SCG-7901/IRX1, SGC- 7901/BDKRB2 and SGC-7901/IRX1/BDKRB2 at in- itial point was 97.475±0.502, 97.235±0.728, 96.08± 0.438 and 96.065±0.120, respectively (P ¼ 0.09). The cell viability of above different groups in 24 h point was 93.765±0.771, 91.88±3.295, 93.595±0.813 and 92.15±1.753, respectively (P ¼ 0.71), and in 48 h point was 84.465±0.134, 81.655±1.746, 83.515±1.972 and 82.75±0.296, respectively (P ¼ 0.32). To examine the reproducibility of the ﬁnding, we compared the wound healing ability after BDKRB2 and IRX1 overexpression on NCI-N87 gastric cancer cells. We got the similar result as described in SGC-7901 cancer cells. The result is presented in Supplementary Figure 1. By KEGG pathway analysis, p21-activated kinase (PAK) is identiﬁed as a key effector in bradykinin pathway. To validate the association of IRX1 and BDKRB2 pathway, we transfected IRX1, siRNA-PAK1 or both IRX1 and siRNA-PAK1 to SGC-7901 cells, respectively. We found that overexpression of IRX1 Oncogene downregulated PAK1 expressing level, whereas knock- down of PAK1 level did not alter the IRX1 level signiﬁcantly (Figure 7a). We examined the cell prolifer- ating activity after IRX1 overexpression or interfering PAK1 by siRNA. We found that the cell proliferation was obviously downregulated in IRX1 overexpressing group or PAK1 interfering group, compared with controls. Whereas the cell proliferation activity in 7901/IRX1/siRNA-PAK1 group was nearly the same as that observed in IRX1-overexpressed cells (Figure 7b). By transwell assays, overexpression of IRX1 or knockdown of PAK1 downregulated the transferred cell numbers. Whereas, in 7901/IRX1/ siRNA-PAK1 group, the transferred cell numbers did not change so much relative to IRX1-overexpressed cells (Figure 7c). These ﬁndings suggested that IRX1 gene can regulate downstream BDKRB2-PAK1 levels as well as their associated functions. Discussion Tumors require a blood supply for growth and hematogenous dissemination. Much attention has been focused on the role of angiogenesis, a recruitment of new vessels from pre-existing vessels. However, angio- genesis may not be the only mechanism by which tumors acquire a microcirculation. Highly aggressive tumors are capable of forming highly patterned VM that is composed of a basement membrane that stains positive with the PAS histochemical reagent in the absence of endothelial cells and ﬁbroblasts. IRX1 is a newly identiﬁed tumor suppressor gene. Hitherto, the tumor suppressor function of IRX1 transcription factor has been reported by us as well as by Bennett and colleagues