Effect Of Parp-1on Angiogenesis And Proliferation Of Ovarian Cancer And The Underlying Mechanism

Background and ObjectiveEpithelial ovarian cancer is a highly invasive disease and has the highest mortality among gynecological malignancies. More than70%of patients at admission are at the late stage. Currently, the standard treatment for ovarian cancer is cytoreductive surgery followed by platinum-and paclitaxel-based chemotherapy. However, this modality has been challenged by severe chemotherapeutic resistance and tumor recurrence. Thus, molecular targeted therapy for ovarian cancer in recent years becomes a hot research field. Current molecular targeting agents are mostly monoclonal antibodies targeting to abnormal tumor molecules or small molecule protein kinases inhibitors, which regulate cell growth, inhibit angiogenesis and have high specificity and low toxicity.PARP-1is a protease with polyadenylation diphosphate ribose (PAR) catalytic activity. It is present in most eukaryotic cells and plays important roles in DNA damage repair, gene transcription, cell cycle, chromosome function, genomic stability, cell death and other aspects. We have previously shown that PARP-1inhibitors can enhance ovarian cancer cell sensitivity to chemotherapeutic drugs. Pyriochou et al. have confirmed that PARP-1inhibitor PJ34could inhibit angiogenesis of experimental chorioallantoic membrane. However, whether inhibition of PARP-1could impact angiogenesis of ovarian cancer cells has not been reported. In this study, we utilized small interfering RNA (siRNA) to silence PARP-1at transcriptional level and explored the following change of vascular endothelial growth factor (VEGF)-A expression, and we investigated its effects on angiogenesis and cell proliferation, hoping to provide a theoretical basis for PARP-1targeted gene therapy.Methods1. Expressions of PARP-1、VEGF-A and MVD in60cases of epithelial ovarian cancer tissues and expression of PARP-1in30cases of normal ovarian tissues were detected by immunohistochemical technology (SABC). Ovarian cancer tissues were divided into PARP-1positive group and PARP-1negative group according to the assessment criteria of immunohistochemical staining.2. SKOV3cells were transfected with either negative control siRNA or PARP-1siRNA, cells were divided into SKOV3group, NC-siRNA group and PARP1-siRNA group.3. Tube formation assay in vitro was used to determine the angiogenesis capacity.4. Cell proliferation was analyzed using3-[4,5-dimethylthiazol-2-yl]-2,5diphenyl tetrazolium bromide assay.5. The expressions of PARP-1and VEGF-A in three group cells were examined with Quantitative real-time PCR (qRT-PCR) and Western blot. Expressions of VEGF-A in supernatants were tested by ELISA.Results1. Positive rate of PARP-1in ovarian cancer tissues(44/60,73.3%) was significantly higher than that in normal ovarian tissues(8/30,26.7%)(P<0.05)2. In ovarian cancer, expression of PARP-1was associated with tumor size, histological grade and lymphatic metastasis.3.Expression of PARP-1had positive correlation with VEGF-A (P<0.05). MVD value was significantly higher in PARP-1positive group than in negative group (P<0.01)4. Lentiviral vector carries green fluorescent protein (GFP), which is used to screening successfully transfected cells. Observation of the cells using an inverted fluorescence microscope indicated that cells started to express GFP at12h after transfection and GFP expressed highest at72h after transfection. The efficiency of transfection in NC-siRNA group and PARP-1group were89%、86%, respectivey.5. Tube density in NC-siRNA group (14.67±1.21) was higher than that in PARP1-siRNA group (8.83±1.47)(P<0.01)6. Cell proliferation of PARP-1group was decreased compared with NC-siRNA group at48h,72h,96h.7. Compared with NC-siRNA group, PARP-1mRNA expression in PARP1-siRNA group was effectively reduced. The protein expressions of PARP-1in NC-siRNA groups PARP1-siRNA group were0.93±0.22、0.38±0.05, respectively.8. Compared with NC-siRNA group, VEGF-A mRNA expression in PARP1-siRNA cells was effectively reduced. The protein expressions of VEGF-A in NC-siRNA group、PARP1-siRNA group were0.90±0.18、0.41±0.08, respectively.9. The expressions of VEGF-A in the supernatants in NC-siRNA group (447.22±188.52) was higher than PARP1-siRNA group(248.12±82.74)(P<0.05).Conclusion1. PARP-1may promote the angiogenesis of ovarian cancer by up-regulating the expression of VEGF-A.2. PARP-1silencing could suppress proliferation of SKOV3cells.