Piwil2 Activates STAT3 and Inhibits P53 Figure 1. Piwil2 represses P53 and inhibits apoptosis. A, P53 expression was significantly repressed by Piwil2. HeLa cells were transfected with same amount of empty plasmid (+pcDNA3.1), pcDNA3.1-Piwil2 (+Piwil2) or Piwil2-specific siRNA (+siRNA), harvested and examined by western blot analysis (upper) and real-time qPCR (lower). Anti-GAPDH antibody and primers were applied as internal control, respectively. B, Western blot analysis of expression of HSP90, STAT3, P21 and MDM2 in HeLa cells, suggesting that P21 was downregulated by Piwil2. C, Apoptosis analysis of transfected HeLa cells with Annexin V/PI double staining detected by a flow cytometer. Western blot analysis of p53 expression was also performed, suggesting that Stat3 knockdown can rescue the expression of p53 inhibited by overexpressed Piwil2. D, Immunoprecipitation analysis showed that STAT3, rather than P53 or HSP90, can directly associated with Piwil2. doi:10.1371/journal.pone.0030999.g001 proteins that have potential roles in tumorigenesis. The results showed that the level of P21 significantly increased when Piwil2 was knockdowned, while slightly decreased when Piwil2 was overexpressed (Fig. 1B). Fluorescence activated cell sorter (FACS) analysis showed a significant decrease of apoptosis from 31.4% to 24.8%, while its counterpart, piwil2-knockdowned HeLa cells increased to 48.4%. Notably, when P53 specific siRNA was co-transfected into piwil2- knockdowned HeLa cells, apoptosis ratio significantly decreased to 33.9% (Fig. 1C), suggesting that P53 is essential for apoptosis pathway induced by Piwil2-knockdown. However, immunoprecipitation assay revealed that Piwil2 can not directly associate with P53 (Fig. 1D), suggesting the existence of a potential pathway through which Piwil2 can regulate P53 and inhibit apoptosis in tumor cells. Piwil2 suppresses P53 signaling by phosphorylating STAT3 Examination revealed that STAT3 knockdown can block inhibition of P53 expression and apoptosis induced by Piwil2 overexpression (Fig. 1C), indicating that STAT3 protein may play a role between piwil2 and P53 suppression. To determine the underlying mechanism, a western blot analysis was performed to detect the phosphorylation of STAT3, which represents the activation of this transcription factor [22]. A significant increase was observed in Piwil2-overexpressed HeLa Cells 12 hours after transfection. However, after 24 hours, the level of pYSTAT3 resumed its normal level approximately. In contrast, pYSTAT3 level decreased and then recovered after 24 hours in Piwil2- knockdown HeLa cells (Fig. 2A). Immunofluorescence assay also revealed that pYSTAT3 signal increased in cytoplasm 6 hours after pcDNA3.1-Piwil2 transfection (Fig. 2B). To evaluate the time-course of STAT3 activity and P53 expression, HeLa cells harvested from various time points after transfection was analyzed by western-blotting. The pYSTAT3 level had been boosted just 3 hours after transfection of pcDNA3.1-Piwil2 and continuously increased until 9 hours, whereas the level of P53 showed no more than a slight change until 9 hours. And the transfection of Piwil2-siRNA led to opposite effects in a parallel time-course profile. (Fig. 2C) These results demonstrated that phosphorylation of STAT3 protein occurred earlier than P53 regulation and suggested STAT3 activity may play as an upstream factor of P53 in Piwil2-induced anti-apoptosis. PLoS ONE | www.plosone.org 2 January 2012 | Volume 7 | Issue 1 | e30999