More importantly, the present study showed that Par3 overexpression triggers expression of EMT-related genes such as Zeb1, Snail1, Twist1 and MMP1/9, by inactivation of metastasis-associated Hippo pathway . phosphorylation of the Hippo pathway and YAP. Finally, by increasing nuclear translocation of non-phosphorylated YAP and activation of TEAD transcription factors, the transcription of pro-metastasis genes is usually enhanced, which promotes the PCa metastasis. Thus, repression of Par3 may offer a potential treatment approach to inhibit PCa metastasis by activating the Hippo pathway. Methods Cell lines and cell culture Prostate cancer cell lines PC3, DU145 and normal prostate epithelial cell line PNT1B were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA). Cells were cultured in Dulbeccos altered Eagles medium (DMEM, Gibco, Thermo Fisher Scientific, Waltham, MA, USA) with 10% fetal bovine serum (FBS, Gibco) and maintained at 5% CO2 at 37?C. Cell transfection and lentiviral vector contamination The Par3 knock down and control plasmids were obtained from Origene (Rockville, MD, USA). Plasmids were transfected with Lipofectamine3000 (Thermo Fisher Scientific). Puromycin (0.5?g/ml) was used for selecting stable Par3 knockdown and relevant control subclones, which were named as PC3-shPar3, PC3-con, DU145-shPar3 and DU145-con respectively. The lentiviral vector expressing one of the Par3 isoforms (“type”:”entrez-nucleotide”,”attrs”:”text”:”NM_019619.3″,”term_id”:”296278254″,”term_text”:”NM_019619.3″NM_019619.3, 150?kDa) or a non-phosphorylatable YAP mutant, YAP(S127A); and control lentiviral Eng vector were SNS-032 (BMS-387032) SNS-032 (BMS-387032) constructed and packaged by Genomeditech Comp (Shanghai, China). 2??106 cells were seeded in 6-well plates and infected using relevant lentiviral vector (MOI?=?10 for each) concomitant with 5?g/ml polybrene. Western blot was employed to detect Par3 expression levels. IF was employed to detect YAP subcellular location. MOI: multiplicity of contamination. In vitro SNS-032 (BMS-387032) migration and invasion assay In vitro migration and invasion assays were performed using 24-well Cell Migration and Invasion Assay kit (Cell BioLabs, San Diego, CA, USA), according to the manufacturers instructions. Briefly, after serum starvation for 24?h, 1??105 cells were suspended in 100?l DMEM basic medium and seeded in the upper chamber, and 700?l DMEM medium with 10% FBS was added to the lower chamber. After incubation for 6?h (for migration assay) or 8?h (for invasion assay), cells on the lower surface of the membrane were fixed with 4% paraformaldehyde, stained with 0.2% crystal violet, photographed and counted under a microscope in five random fields. Orthotopic transplantation of PCa cell lines Six-week-old male BALB/C athymic nude mice (SLAC, Shanghai, China) were housed and manipulated according to the protocols approved by the Renji Hospital Medical Experimental Animal Care Commission rate. For orthotopic inoculation, 1??106 cells were first injected under the subcutaneous and tumor mass was harvested after 4?weeks. Xenografts were digested with collagenase IV for 30mins, 0.05% Trypsin for 10mins and then normalized by DMEM medium with 10% FBS to collect cells for orthotopic transplantation. Cell suspension (1??105 cells in 20?l) was mixed with 20?l matrigel and injected into the left ventral anterior of mouse prostate. PET-CT (Siemens Inveon) detection was performed using 18FCFDG (750?Ci/100?g) by intravenous injection 7?weeks after orthotopic inoculation. Orthotopic tumor SNS-032 (BMS-387032) growth and potential tumor metastasis were evaluated in living animals by the absorption of 18FCFDG. Mice were sacrificed 1?week after PET-CT detection. Orthotopic tumor mass and metastatic nodes were collected for H&E and immunofluorescence staining. Clinical samples Investigation has been conducted in accordance with the ethical standards and according to the Declaration of Helsinki and national and international guidelines. Human tissues used in this study were reviewed and approved by the Committee for Ethical Review.
More importantly, the present study showed that Par3 overexpression triggers expression of EMT-related genes such as Zeb1, Snail1, Twist1 and MMP1/9, by inactivation of metastasis-associated Hippo pathway