The Role Of Peptidylarginine Deiminase 4 In The Growth And Invasion Of Ovarian Cancer Cells With Different P53 State

ObjectiveWe performed this study in two ovarian cancer cell lines A2780 and SKOV3 to examine the function of PADI4 in p53 signaling pathway, cell proliferation, apoptosis, migration and invasion, and select the related genes in these signaling pathways when PADI4 expression was inhibited.MethodsThe ovarian cancer cells were A2780, a cell line with wild-type p53, and SKOV3, a p53-null cell line. The cells were transfected with PADI4-siRNA and negative control siRNA in vitro respectively. Proliferation rate, survival and gene expression pattern of the cells transfected with PADI4-siRNA were compared among different groups. The mRNA and protein expression of PAD 14 were confirmed with real-time PCR and western blot, then cell proliferation, apoptosis, migration and invasion were measured with CCK-8 assay, Annexin V-PE/7-AAD assay and transwell assay. We also utilized p53 signaling pathway, apoptosis and cell motility RT2 profiler PCR array to determine how PAD 14 is involved in tumorigenic signaling pathway. Finally, we confirmed the results of PCR array using real time PCR.Results1. The mRNA and protein expression were inhibited by PADI4-siRNA in two ovarian cancer cell lines using real time PCR and western blot.2. The proliferation of both A2780 and SKOV3 cells following PADI4-siRNA treatment decreased significantly (PA2780<0.05; Pskovs<0.001). Apoptotic rate of A2780 cells after transfection with PADI4-siRNA were increased, but there was no such increase in SKOV3 cells (P>0.05). The migration and invasion ability of A2780 cells after PADI4-siRNA transfection also showed significant decrease, but the p53-null SKOV3 cells showed no such decrease (P<0.001).3. In A2780 cells transfected with PADI4-siRNA, RT 2 profiler PCR array revealed up-regulated or down-regulation expression in 13 genes. The data from the p53 signaling pathway array showed that 2 genes were significantly up-regulated more than two fold in the treated cells [estrogen receptor 1 (ESR1) and tumor necrosis factor (TNF)], and 4 genes were down-regulated [sestrin 2 (SESN2), (tumor protein p63 (TP63), glycosylphosphatidylinositol anchored molecule-like protein (GML) and interleukin 6 (IL6)]. There were 2 up-regulated genes (tumor necrosis factor receptor superfamily, member 9 (TNFRSF9) and cell death-inducing DFFA-like effector a (CIDEA)] and 4 down-regulated genes [BCL2-like 2 (BCL2L2), BCL2-antagonist/killer 1 (BAK1), tumor necrosis factor receptor superfamily, member 1 lb (TNFRSF1 IB) and TNF superfamily, member 6 (FASLG)] in the apoptosis array. The results of the cell motility array showed that 3 genes were significantly up-regulated more than two fold [matrix metallopeptidase 14 (MMP14), WAS/WASL-interacting protein family, member 1 (WIPF1), insulin-like growth factor 1 (IGF1)], and there was 1 gene significantly down-regulated [integrin, beta 3 (ITGB3)].ConclusionPADI4 mRNA and protein expression were significantly inhibited in the PAD 14 siRNA treated cells. PADI4 silence inhibited cell proliferation rate in both A2780 cells with wild type-p53 (wt-p53) and p53-null SKOV3 cells. However, PADI4 silence induced cell apoptosis in ovarian caner cells with p53 gene expression, but didn’t in p53-null ovarian cancer cells. Meanwhile, down-regulated PADI4 could inhibite the ability of migration and invasion in ovarian caner cells with p53 gene expression, but didn’t in p53-null ovarian cancer cells. PADI4 silence altered proliferation and survival of ovarian cancer cells demonstrating that PADI4 plays an important role in the tumorigenesis of ovarian cancer. The mechanism may be related to the genes such as p53, TNF, ESR1 et al, and it may have great significance in antineoplaston therapy targeted PADI4 gene.