Ye et al. BMC Cancer 2010, 10:611 http://www.biomedcentral.com/1471-2407/10/611 members bind their ligands independently [3]. The majority of semaphorins are not assigned to a specific receptor [4]. To date, it is known that plexin-B1 binds Sema 4D, plexin-B2 binds Sema 4C and plexin-B3 binds Sema 5A [5]. Plexin-semaphorin interactions typically play impor- tant roles in processes such as neuron extension, axon growth guidance and the maintenance of established neuronal pathways. Recent studies have further revealed that plexins-semaphorins are also expressed outside the nervous system and that their interactions are involved in the regulation of immune responses [6-10], morpho- genetic lung development [11], the cardiovascular sys- tem [12-15], the skeleton [16-18], and tumor growth and metastasis [19,20]. It is unique that Plexin-B1, a transmembrane receptor, interacts directly with Rho family GTPases through a cytoplasmic Rho GTPase binding domain (RBD) [21,22]. Rho GTPases play important roles in regulating the actin cytoskeleton during developmental processes [23] such as cell adhesion [24], cell migration, axon guidance, cell cycle events and membrane transport [25], suggest- ing potential involvement of Plexin-B1 in cancer pro- gression and metastasis. Swiercz et al. recently reported that Plexin-B1 activates or inactivates RhoA in the pre- sence of ErbB-2 or Met, respectively, and leads to pro- or anti-migration effects [26]. Furthermore, recent stu- dies have shown that loss of Plexin-B1 expression in breast cancer correlates closely with ER status and indi- cates a more aggressive tumor phenotype [27]. In renal cell carcinomas, Plexin-B1 was down-regulated and growth of cancer cells was inhibited [28]. However, overexpression of Plexin-B1 promoted invasiveness in prostate cancer [29]. These different and even opposing results reflect the fact that the exact biological function of Plexin-B1 in tumorigenesis and cancer progression is not yet clear. In the present study, the expression levels of Plexin-B1 protein were found to be higher in serous ovarian carci- nomas than in normal ovaries or benign ovarian neoplasms. We then characterized the expression of Plexin-B1 in several ovarian cancer cell lines, and SKOV3 showed highest level of Plexin-B1. To examine the role of Plexin-B1 in SKOV3 cells, Plexin-B1 was knocked down by using three different siRNA duplexes. The results showed that silencing of Plexin-B1 inhibited migration and invasion in SKOV3 cells. Even though Plexin-B1 has been previously reported to be involved in mouse ovary follicular growth and development [4], and it might be a marker predicting unfavorable outcome in ovarian serous carcinomas [30], only a limited number of studies have investigated the role of Plexin-B1 in ovarian carcinogenesis. Page 2 of 11 Methods Tissue specimens and cell lines A collection of tissue specimens including 20 normal and benign ovarian samples (the benign ovarian tissues were histologically diagnosed as serous cystadenoma), 20 serous borderline ovarian tumors, and 80 serous ovarian cystadenocarcinomas, were obtained from the Department of Pathology of Tongji Hospital with Insti- tutional Review Board approval. Histological type and grade were diagnosed according to the World Health Organization (WHO) criteria [31]. Staging was based on TNM classification of the International Union Against Cancer (UICC) [32]. The human epithelial ovarian ade- nocarcinoma cell line SKOV3 was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). The A2780 human ovarian epithelial carci- noma cell line was obtained from the European Collec- tion of Cell Cultures (ECACC, Salisbury, UK). The OV2008 and C13* ovarian cancer cell lines were gifts from Dr. Rakesh Goel in the Ottawa Regional Cancer Center, Ottawa, Canada. SKOV3 cells were cultured in McCOY’s 5A containing 10% fetal bovine serum. The other cell lines were cultured in RPMI1640 containing 10% fetal bovine serum. Cell cultures were maintained in a humidified atmosphere containing 5% CO2 at 37°C, and the growth medium was renewed every 2-3 days. All culture medium and supplements were purchased from Invitrogen, Carlsbad, CA, USA. Immunohistochemistry Immunohistochemistry was performed on formalin-fixed, paraffin-embedded sections (5 μm thick) according to standard procedures. Briefly, after dewaxing and rehydra- tion, antigen retrieval was done with 0.01 M citrate buffer at pH 6.0 for 15 min in a microwave oven. Then endogen- ous peroxidase activity was inhibited by incubating sec- tions with 0.3% hydrogen peroxide for 30 min at 37°C, non-specific binding was blocked with normal goat serum for 30 min at 37°C, and this was followed by incubating the sections with mouse monoclonal anti-human Plexin- B1 (Santa Cruz Biotechnology, sc-28372, 1:100 dilution) at 4°C overnight. The primary antibody was detected using biotinylated anti-mouse IgG. The signal was amplified by avidin-biotin complex formation and developed with dia- minobenzidine followed by counterstaining with hematox- ylin. Finally, the slides were dehydrated and mounted. Plexin-B1 immunoreactivity was determined using the well-standardized H-Score system (H = I × P) [33]. I is the staining intensity, which was evaluated according to the following criteria: 0 (no staining), 1 (weak immunostain- ing), 2 (moderate immunostaining), 3 (strong immunos- taining), and P is the percentage of positively stained cells in each sample. A sample with an H score ≥20 was