(b) Western blots showing the depletion of PAX8 protein upon siRNA treatment in the cell lines used

(b) Western blots showing the depletion of PAX8 protein upon siRNA treatment in the cell lines used. this article. All original data are available upon request. Abstract Background Ovarian cancer is the third most common cause of death among gynecologic malignancies worldwide. Understanding the COH000 biology and molecular pathogenesis of ovarian epithelial tumors is key to developing improved prognostic indicators and effective therapies. We aimed to determine the effects of PAX8 expression around the migrative, adhesive and survival capabilities of high-grade serous carcinoma cells. Methods PAX8 depleted Fallopian tube secretory cells and COH000 ovarian cancer cells were generated using short interfering siRNA. resistance, cell migration and adhesion properties of PAX8 silenced cells were analyzed by means of specific assays. Chromatin immunoprecipitation (ChIP) was carried out using a PAX8 polyclonal antibody to demonstrate that PAX8 is able to bind to the 5-flanking region of the ITGB3 gene positively regulating its expression. Results Here, we report that RNAi silencing of PAX8 sensitizes non-adherent cancer cells to and affects their tumorigenic properties. We show that PAX8 plays a critical role in migration and adhesion of both Fallopian tube secretory epithelial cells and ovarian cancer cells. Inhibition of PAX8 gene expression reduces the ability of ovarian cancer cells to migrate and adhere to the ECM and specifically to fibronectin and/or collagen substrates. Moreover, loss of PAX8 strongly reduces ITGB3 expression and consequently the correct expression of the v3 heterodimer around the plasma membrane. Conclusions Our results demonstrate that PAX8 modulates the conversation of tumor cells with the extracellular matrix (ECM). Notably, we also highlight a novel pathway downstream this transcription factor. Overall, PAX8 could be a potential therapeutic target for high-grade serous carcinoma. or detachment-induced apoptosis leading to EMT. Interestingly, inhibition of PAX8 gene expression in ovarian cancer cells decreases tumor cell adhesion to Rabbit polyclonal to ASH2L fibronectin and collagen. Furthermore, loss of PAX8 strongly reduces ITGB3 expression and consequently the correct expression of the Integrin v3 heterodimer on the plasma membrane. Integrin 3 has been already implicated in a wide variety of functions, including platelet aggregation and thrombosis, implantation, placentation, angiogenesis, bone remodeling, and tumor progression [25].?Amongst integrins that have been identified as important mediators of ovarian cancer metastasis, the heterodimer Integrin v3 holds a significant position [26, 27]. This is the first study reporting the correlation between PAX8 and Integrins uncovering a novel functional pathway downstream of this transcription. Moreover, we suggest a possible role for PAX8 in the peritoneal dissemination of ovarian cancer cells by modulating cancer cells assay To assess the activity, 1??104 of both scramble and PAX8 silenced Kuramochi cells 24?h after transfection were plated in triplicate on ultra-low attachment 96-well plates under regular culture conditions and on adherent 96-well plates, as control. Cell viability was detected 24?h and 48?h later using the MTS reagent (Promega, G3580). The viability ratio of cells grown in the two different wells was calculated using ODanoikis well/ODcontrol well. Immunofluorescence and Confocal Laser Scanning Microscopy After 24? h of transfection with siCTR and siPAX8 as described before, 50??103 of Primary hFTSECs and KURAMOCHI cells were plated on glass coverslips and maintained in culture for 24?h at 37?C. Cells were fixed in 4% paraformaldehyde in PBS 1 for 20?min at RT and incubated for 30?min in 10% FBS in PBS 1. Coverslips were subsequently incubated for 1?h with COH000 mouse monoclonal anti-v3 LM609 (Millipore Corp, USA) and rabbit polyclonal anti-PAX8 diluted to 1 1:100 and 1:1000 in 4% FBS in PBS 1, respectively. After PBS washes, cells were incubated for 30?min with Alexa Fluor-546 goat anti-mouse IgG (Vinci Biochem) and Alexa Fluor-488 goat anti-rabbit IgG (Vinci Biochem) both diluted to 1 1:200 in 4% FBS in PBS 1. After the final washes with PBS 1, coverslips were mounted on microscope slides using a 50% solution of glycerol in PBS 1 with Hoechst (1:3000). Experiments were carried out on an inverted and motorized microscope (Axio Observer Z.1) equipped with a 63/1.4 Plan-Apochromat objective. The attached laser-scanning unit (LSM 700 4 pigtailed laser 405-488-555-639; Zeiss, Jena, Germany) enabled confocal imaging. For excitation, 405, 488 and 555?nm lasers were used. Fluorescence emission was revealed by Main Dichroic Beam Splitter and Variable.