PPA1 Regulates Metastasis Through Epithelial-mesenchymal Transition In Epithelial Ovarian Cancer

Part I :The expression of PPA1 in different kind of epithelial ovarian tumors and normal ovarian tissues.Objective: To evaluate the expression of protein PPA1 in different kind of epithelial ovarian tumors and normal ovarian tissues. To provide theoretical evidence for the application of PPA1 in the evaluation of development and prognosis of epithelial ovarian cancer.Methods: 1.Immuhisochemistry technology was used to detect the expression of PPA1 in 55 epithelial ovarian cancer, 20 borderline tumors, 24 benign tumors and 23 normal ovarian tissues in total.To analyze the relationship between the expression of PPA1 and the clinical pathological parameters in patients with EOC. 55 cases of EOC patients were followed up, then the correlation between PPA1 expression and patients’ survival was assessed. 2. Epithelial ovarian cancer and normal ovarian fresh tissues were harvested,total RNA and protein were extracted, RT-PCR and Western blot were carried out to indicate the amounts of RNA and protein of PPA1.Results: 1.Immunohistochemical studies showed that expression of PPA1 in normal ovarian tissues and benign ovarian tumor tissue is low or negative. When it comes to borderline tumors and epithelial ovarian cancer tissues, the PPA1 expression is high. 2. No correlations were observed between the clinical parameters(pathological type, age) and PPA1 expression. With the increase of stages and pathological grades, PPA1 expression was significantly increased. In EOC patients, those with high expression of PPA1 had significantly shorter overall survival than those with low PPA1 expression. 3. To further confirm the IHC results, fresh tissue samples were subjected to Real-time PCR and Western-blot analysis, the results showed that the PPA1 expression was higher in EOC than in normal ovarian tissues.Conclusion: 1.The expression of PPA1 was closely related to the stages and grades of EOC. PPA1 may play an important role in the occurrence and development of EOC. 2. The expression of PPA1 has a predictive value for the prognosis of EOC.Part II:Building up the PPA1 knowdown cell lines in EOC cells.Objective: To build up the PPA1 knockdown cell lines in EOC cell lines ES2 and SKOV3.Methods: 1. Successful building PPA1-sh RNA expression vectors, which has been verified by sequencing. After being transfected to ES2 and SKOV3 cells, cell fluorescent display were detected, endogenous targets were to be confirmed. 2. The m RNA and protein level of PPA1 were analyzed by real-time PCR and Western blot, and then determined the interference efficiency to get the effective target.Results: 1.We successfully built up PPA1 knowdown and control cell lines in EOC cell lines ES2 and SKOV3. 2. The efficiency of PPA1 knocking down is more than 50% confirmed by real-time PCR and Western blot.Conclusion: RNAi(RNA interfering) is essential to cancer research. We successfully built up PPA1 knockdown cell lines in ovarian cancer ES2 and SKOV3 cells for further use.Part III : Effects of PPA1 in proliferation, invasion,migration and epithelial-mesenchymal transition in EOC cells.Objective: To observe the effect of PPA1 on the proliferation, invasion, migration and epithelial-mesenchymal transition(EMT) of EOC cells in order to observe the effect of PPA1 on the biological behavior of EOC.Methods: 1. In the ES2 and SKOV3 cells, for the PPA1 knockdown cell lines and its control group, cell proliferation, invasion, migration were detected by CCK8, Transwell and Wound healing assay respectively. 2. Using Western blot we examined the expression of EMT-related proteins including E-cadherin, N-cadherin and Vimentin in the ES2 and SKOV3 cell lines. 3. Using immunofluorescence staining we examined the expression of EMT-related proteins including SMA and Vimentin for the PPA1 knockdown cell lines and its control group in the two cell lines.Results: 1. Silencing of PPA1 significantly inhibited the invasion and led to a reduction on the ability to close the gap introduced by a scratch wound in ES2 and SKOV3 cell lines, but have no effect on cell proliforation. 2. Silencing of PPA1 significantly increased E-cadherin protein levels, while it down regulated mesenchymal markers N-cadherin, and Vimentin.Conclusion: 1. PPA1 is correlated with invasion and migration in ovarian cancer cells, but have no effect on proliforation. 2. PPA1 probably promotes tumor metastasis by the EMT mechanism in epithelial ovarian cancer.Part IV :Effects of PPA1 on the metastasis of EOC in vivo. Objective: To observe the effect of PPA1 on the metastasis of EOC in NOD/SCID mice.Methods: 1.To establish the abdominal xenograft tumor model of ovarian cancer in NOD/SCID mice and to observe the changes of the metastatic ability of ovarian xenograft tumor before and after PPA1 knockdown. 2. Using vivo imaging to find the changes of metastatic ability of ovarian abdominal xenograft tumor before and after PPA1 knocking down. 3. Using immuhisochemistry staining we examined the expression of EMT-related proteins including E-cadherin, Vimentin in xenograft tumors before and after PPA1 was knocking down.Results: 1.In this study, we successfully established a tumor model of ovarian cancer in abdominal cavity, and found that before PPA1 was knocking down, metastasis nodules could be detected in the abdominal organs and peritoneal, while in the PPA1 knocking down mice metastatic nodules were rare. 2. This was confirmed by in vivo imaging detection. 3. Immunohistochemistry assay showed that in the xenograft tumors, compared with the control group, the expression of E-cadherin increased in the PPA1 knocking down group, while the expression of N-cadherin decreased.Conclusion: 1.In this study, we found that after PPA1 was knocking down the ability of metastasis of abdominal ovarian xenograft tumor decreased. 2. In vivo, PPA1 has the ability to promote EMT, thereby promoting the metastasis of EOC cells.Part V : Research of the effect of PPA1 on the mechanism of EOC metastasis.Objective: A preliminary study on the mechanism of PPA1 affecting the transfer of EOC.Methods: 1. Using Western blotting to dectect the expression of β-catenin in ovarian cancer cell lines ES2 and SKOV3 before and after PPA1 was knocking down. 2. Using immunofluorescence stanining to dectect the expression of β-catenin in ovarian cancer cell lines ES2 and SKOV3 before and after PPA1 was knocking down.Results: 1.After silencing of PPA1, in ES2 and SKOV3 cell lines the PPA1 expression in total protein and nuclear protein decreased obviously confirmed by Western blotting. 2. After silencing of PPA1, the PPA1 expression in total protein expecially in nuclear protein decreased obviously confirmed by immunofluorescence.Conclusion: In summary, PPA1 may induce the EMT of ovarian cancer cells through regulation of β-catenin nuclear translocation and activation of the Wnt/β-catenin signaling pathway to promote the metastasis of EOC cells.