PPA1通过促进上皮—间质转化影响上皮性卵巢癌的转移
发布时间：2018-10-18 21:28
【摘要】：第一部分:不同类型的卵巢上皮性肿瘤和正常卵巢组织PPA1的表达目的:明确焦磷酸酶1(PPA1)在上皮性卵巢癌(EOC)、交界性肿瘤、卵巢良性上皮性肿瘤及正常卵巢组织中的表达差异,探讨PPA1表达和与EOC的发生发展及预后的关系。方法:1.应用免疫组化法检测PPA1蛋白在55例EOC、20例交界性卵巢肿瘤、24例卵巢良性上皮性肿瘤和23例人正常卵巢石蜡组织中的表达情况;分析EOC患者PPA1的表达水平与其临床病理参数的关系;对55例EOC患者进行随lx,分析其五年生存率和PPA1蛋白表达的关系。2.Real-time PCR和Western blot法检测人新鲜EOC组织和正常卵巢组织PPA1 m RNA及蛋白的表达水平。结果:1、免疫组化检测显示,PPA1在正常卵巢和良性卵巢肿瘤组织中低表达,而在卵巢交界性上皮肿瘤和EOC中高表达,并呈逐渐增加的趋势。2、EOC的组织学类型及患者年龄不同,PPA1的表达无显著差异。而EOC的不同临床分期和病理分级各组间,PPA1的表达有显著差异,PPA1的表达与临床分期和病理分级相关,并随临床分期和病理分级升高而呈升高趋势。与PPA1低表达的患者比较,PPA1表达高的患者五年生存率明显降低。3、人新鲜EOC肿瘤组织的PPA1蛋白及RNA表达均显著高于正常卵巢组织。结论:1.PPA1的表达与人EOC的分级和分期相关。可能在EOC的发生和进展中起重要作用。2.PPA1的表达高低对EOC的预后有一定的预测价值。第二部分:用RNA干扰的方法建立PPA1基因敲除的细胞系目的:建立PPA1基因敲除的稳定转染的EOC细胞系,用于进一步研究。方法:1.应用RNA干扰技术,设计合成2个特异性靶向PPA1基因的shRNA载体(PPA1-sh RNA表达载体),同时将空载体作为对照稳定转染至人卵巢癌ES2和SKOV3细胞,并对不同靶点进行有效筛选。2.应用Realtime PCR及Western blot检测目的基因PPA1的敲除效率。结果:1.本研究应用RNA干扰技术,成功建立了PPA1基因敲除的ES2和SKOV3细胞系。2.检测结果显示:和空白对照组比较,实验组的2个靶向PPA1-sh RNA干扰序列中,PPA1在RNA水平和蛋白水平均显著下调达50%以上,2个靶点敲除效率理想。结论:1.RNA干扰技术在在肿瘤学研究中具有重要的价值,同样也可以应用于EOC的研究。2.在本研究中,我们成功建立了PPA1基因敲除的ES2和SKOV3细胞系,这两组细胞系均可用于下一步实验研究。第三部分:PPA1对EOC细胞增殖、侵袭、迁移和EMT的影响目的:观察PPA1对EOC细胞的增殖、侵袭、迁移和上皮-间质转化(EMT)的影响从而观察PPA1对肿瘤生物学行为的影响。方法:1.CCK8法检测PPA1基因对EOC细胞ES2和SKOV3增殖的影响。2.Transwell法检测PPA1对EOC细胞ES2和SKOV3侵袭的影响。3.划痕实验检测PPA1对EOC细胞ES2和SKOV3迁移的影响。4.Western blot法检测PPA1对EOC细胞ES2和SKOV3 EMT相关蛋白:E-钙粘素(E-cadherin)、N-钙粘素(N-cadherin)和波形蛋白(Vimentin)表达的影响。5.免疫荧光法检测PPA1对EOC细胞ES2和SKOV3 EMT相关蛋白α平滑肌肌动蛋白(α-SMA)和Vimentin表达的影响。结果:1.CCK8法检测发现,PPA1基因敲除后对于EOC细胞增殖的影响不大。2.Transwell侵袭实验表明,PPA1能够增强EOC细胞的侵袭能力。3.划痕实验表明,在体外,PPA1同样能够促进EOC细胞的迁移能力。4.Western blot法检测发现,敲除PPA1基因后卵巢癌ES2和SKOV3细胞上皮性标志物E-cadherin的表达显著上调,间质性标志物Vimentin的表达显著下降。间质性标志物N-cadherin在ES2细胞的表达显著下降,而在SKOV3细胞中检测不到。5.免疫荧光法检测发现,敲除PPA1基因后卵巢癌ES2和SKOV3细胞间质性标志物α-SMA和Vimentin的表达均显著下降。结论:1.PPA1具有促进EOC细胞侵袭和迁移的能力,在体外,对于EOC细胞增殖的影响不大。2.PPA1能够促进EOC细胞的EMT。PPA1有可能通过促进EOC细胞的EMT而影响EOC的转移。第四部分:PPA1在体内对EOC细胞转移的影响目的:使用动物模型观察PPA1在体内对EOC转移的影响。方法:1.建立NOD/SCID小鼠卵巢癌腹腔移植瘤模型,观察PPA1敲除前后卵巢癌腹腔移植瘤转移能力的变化。2.活体成像法检测PPA1敲除前后卵巢癌腹腔移植瘤转移能力的变化。3.免疫组化法检测PPA1敲除前后卵巢癌腹腔移植瘤E-cadherin、Vimentin的表达变化。结果:1.本研究成功建立NOD/SCID小鼠卵巢癌腹腔移植瘤模型,并发现PPA1敲除前可见腹腔内器官及腹膜转移结节,而PPA1敲除后,转移结节少见。2.活体成像法检测也证实了PPA1有促进卵巢癌腹腔移植瘤转移的作用。3.免疫组化法检测表明,在形成的腹腔移植瘤组织中,相比对照组,敲除PPA1者EMT相关蛋白E-cadherin表达升高,而Vimentin表达降低。结论:1.本研究发现PPA1敲除后小鼠卵巢腹腔移植瘤的转移能力降低。2.在体内,PPA1具有促进卵巢癌细胞EMT的能力,从而促进EOC细胞的转移。第五部分:PPA1影响EOC转移机制的探讨目的:初步探讨PPA1影响EOC转移的机制。方法:1.Western blot法检测PPA1敲除后EOC细胞ES2和SKOV3蛋白β-catenin表达的变化。2.免疫荧光法检测PPA1敲除后ES2和SKOV3细胞β-catenin表达的变化。结果:1.Western blot法检测发现,PPA1敲除后总蛋白及核蛋白中β-catenin的表达下降。2.免疫荧光法也证实,PPA1基因敲除后的EOC细胞核的β-catenin的表达下降。结论:Wnt/β-catenin信号通路是肿瘤EMT的重要信号通路之一,PPA1可能通过调节Wnt/β-catenin信号通路影响EOC细胞的EMT,从而促进EOC的转移。
[Abstract]:The expression of PPA1 in epithelial ovarian cancer (EOC), borderline tumors, ovarian benign epithelial tumors and normal ovarian tissues was determined. To investigate the relationship between the expression of PPA1 and the development and prognosis of EOC. Method: 1. The expression of PPA1 protein in 55 EOC, 20 borderline ovarian tumors, 24 ovarian benign epithelial tumors and 23 normal ovarian paraffin tissues were detected by immunohistochemistry; the relationship between the expression level of PPA1 and its clinical pathological parameters was analyzed in 55 patients with EOC, and 55 patients with EOC were treated with lx, The relationship between the five-year survival rate and the expression of PPA1 protein was analyzed. The expression level of PPA1 mRNA and protein was detected by Real-time PCR and Western blot. Results: 1. Immunohistochemically, PPA1 was expressed in normal ovary and benign ovarian tumor tissues. The expression of PPA1 in ovarian borderline epithelial tumors and EOC was gradually increased. The histological types and age of EOC were different, but there was no significant difference in PPA1 expression. There was a significant difference in PPA1 expression between different clinical stages and pathological grades of EOC, and the expression of PPA1 was correlated with clinical stage and pathological grade, and the expression of PPA1 increased with clinical stage and pathological grade. Compared with PPA1 low-expression patients, the 5-year survival rate of PPA1 expression was significantly lower than that of normal ovarian tissue. Conclusion: 1. PPA1 expression is related to the classification and stage of human EOC. It is possible to play an important role in the occurrence and progression of EOC. The expression of PPA1 has a certain predictive value for the prognosis of EOC. Part 2: To establish a stable transfected EOC cell line for PPA1 gene knockout using RNA interference method for further study. Method: 1. RNA interference technique was applied to design a vector (PPA1-sh RNA expression vector) which specifically targeted the PPA1 gene, and the empty vector was stably transfected into human ovarian cancer ES2 and SKOV3 cells as control, and the different target sites were effectively screened. The knock-out efficiency of PPA1 was detected by Realtime PCR and Western blot. Result: 1. In this study, RNA interference technique was applied to successfully establish the ES2 and SKOV3 cell lines of PPA1 knockout mice. The results showed that in the two target PPA1-sh RNA interference sequences of the experimental group, PPA1 significantly decreased by over 50% in RNA level and protein level, and 2 target knock-out efficiency was ideal. Conclusion: 1. RNA interference technology has important value in the research of oncology, and it can also be applied to the study of EOC. In this study, we successfully established the ES2 and SKOV3 cell lines of the PPA1 gene knockout, both of which can be used for the next experiment study. The effects of PPA1 on the proliferation, invasion, migration and epithelial-to-mesenchymal transition of EOC cells were investigated. Methods: 1. CCK8 was used to detect the effect of PPA1 gene on ES2 and SKOV3 proliferation of EOC cells. The effect of PPA1 on the migration of ES2 and SKOV3 in EOC cells was tested by scratch test. The expression of ES2 and SKOV3, E-cadherin, N-cadherin and Vimentin was detected by Western blot. Immunofluorescence assay was used to detect the effects of PPA1 on the expression of ES2 and SKOV3 in EOC cells and the expression of Vimentin. Results: 1. The effect of PPA1 gene knockout on the proliferation of EOC cells was not greater than that in the 1. CCK8 assay. The invasion of PPA1 showed that PPA1 could enhance the invasive ability of EOC cells. The scratch test showed that PPA1 could promote the migration ability of EOC cells in vitro. The expression of ES2 and SKOV3 cell epithelial markers E-cadherin was significantly upregulated after knock-out of PPA1 gene in vitro, and the expression of Vimentin was significantly decreased. The expression of N-cadherin in ES2 cells was significantly decreased and no. 5 was detected in SKOV3 cells. Immunofluorescence assay showed that the expression of ER-SMA and Vimentin in ovarian cancer ES2 and SKOV3 cells decreased significantly after knock-out of PPA1 gene. Conclusion: 1. PPA1 has the ability to promote the invasion and migration of EOC cells. In vitro, the effect of PPA1 on the proliferation of EOC cells is not large. PPA1 can promote the proliferation of EOC cells. PPA1 may influence the transfer of EOC cells by promoting the proliferation of EOC cells. Part four: The effect of PPA1 on EOC cell metastasis in vivo was studied: the effect of PPA1 on EOC metastasis was observed in vivo. Method: 1. To observe the change of metastasis ability of ovarian cancer after PPA1 knock-out. In vivo imaging method to detect the change of metastasis ability of ovarian cancer after PPA1 knock-out. The expression of E-cadherin and Vimentin in ovarian carcinoma after PPA1 knockout was detected by immunohistochemistry. Result: 1. In this study, we successfully established a model of intraperitoneal transplantation of NOD/ DBM mouse ovarian cancer, and found that the intra-abdominal organs and peritoneal metastatic nodules were seen before PPA1 knockout, whereas after PPA1 knockout, metastatic nodules were uncommon. In vivo imaging, PPA1 has been proved to have an important role in promoting the metastasis of ovarian cancer. Compared with the control group, the expression of E-cadherin in knock-out PPA1 was increased and the expression of Vimentin decreased compared with that in the control group. Conclusion: 1. In this study, the metastatic ability of ovarian peritoneal transplanted tumor in mice after PPA1 knockout was decreased. In vivo, PPA1 has the ability to promote the proliferation of ovarian cancer cells, thereby promoting the transfer of EOC cells. The fifth part: PPA1 influences the mechanism of EOC transfer: preliminary study of PPA1 affects the mechanism of EOC metastasis. Methods: 1. Western blot was used to detect the changes of the expression of ES2 and SKOV3 protein p27-catenin in PPA1 knockout mice. The expression changes of ES2 and SKOV3 cells after PPA1 knockout were detected by immunofluorescence. Results: 1. Western blot showed that the expression of p27-catenin was decreased after PPA1 knockout. The immunofluorescence assay also confirmed that the expression of caspase-catenin in the EOC nucleus following the knock-out of PPA1 gene was decreased. Conclusion: The PPA1 signal pathway is one of the important signaling pathways in the tumors, and PPA1 may influence the transfer of EOC cells by modulating the extracellular domain of EOC cells.
【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R737.31
本文编号：2280403
[Abstract]:The expression of PPA1 in epithelial ovarian cancer (EOC), borderline tumors, ovarian benign epithelial tumors and normal ovarian tissues was determined. To investigate the relationship between the expression of PPA1 and the development and prognosis of EOC. Method: 1. The expression of PPA1 protein in 55 EOC, 20 borderline ovarian tumors, 24 ovarian benign epithelial tumors and 23 normal ovarian paraffin tissues were detected by immunohistochemistry; the relationship between the expression level of PPA1 and its clinical pathological parameters was analyzed in 55 patients with EOC, and 55 patients with EOC were treated with lx, The relationship between the five-year survival rate and the expression of PPA1 protein was analyzed. The expression level of PPA1 mRNA and protein was detected by Real-time PCR and Western blot. Results: 1. Immunohistochemically, PPA1 was expressed in normal ovary and benign ovarian tumor tissues. The expression of PPA1 in ovarian borderline epithelial tumors and EOC was gradually increased. The histological types and age of EOC were different, but there was no significant difference in PPA1 expression. There was a significant difference in PPA1 expression between different clinical stages and pathological grades of EOC, and the expression of PPA1 was correlated with clinical stage and pathological grade, and the expression of PPA1 increased with clinical stage and pathological grade. Compared with PPA1 low-expression patients, the 5-year survival rate of PPA1 expression was significantly lower than that of normal ovarian tissue. Conclusion: 1. PPA1 expression is related to the classification and stage of human EOC. It is possible to play an important role in the occurrence and progression of EOC. The expression of PPA1 has a certain predictive value for the prognosis of EOC. Part 2: To establish a stable transfected EOC cell line for PPA1 gene knockout using RNA interference method for further study. Method: 1. RNA interference technique was applied to design a vector (PPA1-sh RNA expression vector) which specifically targeted the PPA1 gene, and the empty vector was stably transfected into human ovarian cancer ES2 and SKOV3 cells as control, and the different target sites were effectively screened. The knock-out efficiency of PPA1 was detected by Realtime PCR and Western blot. Result: 1. In this study, RNA interference technique was applied to successfully establish the ES2 and SKOV3 cell lines of PPA1 knockout mice. The results showed that in the two target PPA1-sh RNA interference sequences of the experimental group, PPA1 significantly decreased by over 50% in RNA level and protein level, and 2 target knock-out efficiency was ideal. Conclusion: 1. RNA interference technology has important value in the research of oncology, and it can also be applied to the study of EOC. In this study, we successfully established the ES2 and SKOV3 cell lines of the PPA1 gene knockout, both of which can be used for the next experiment study. The effects of PPA1 on the proliferation, invasion, migration and epithelial-to-mesenchymal transition of EOC cells were investigated. Methods: 1. CCK8 was used to detect the effect of PPA1 gene on ES2 and SKOV3 proliferation of EOC cells. The effect of PPA1 on the migration of ES2 and SKOV3 in EOC cells was tested by scratch test. The expression of ES2 and SKOV3, E-cadherin, N-cadherin and Vimentin was detected by Western blot. Immunofluorescence assay was used to detect the effects of PPA1 on the expression of ES2 and SKOV3 in EOC cells and the expression of Vimentin. Results: 1. The effect of PPA1 gene knockout on the proliferation of EOC cells was not greater than that in the 1. CCK8 assay. The invasion of PPA1 showed that PPA1 could enhance the invasive ability of EOC cells. The scratch test showed that PPA1 could promote the migration ability of EOC cells in vitro. The expression of ES2 and SKOV3 cell epithelial markers E-cadherin was significantly upregulated after knock-out of PPA1 gene in vitro, and the expression of Vimentin was significantly decreased. The expression of N-cadherin in ES2 cells was significantly decreased and no. 5 was detected in SKOV3 cells. Immunofluorescence assay showed that the expression of ER-SMA and Vimentin in ovarian cancer ES2 and SKOV3 cells decreased significantly after knock-out of PPA1 gene. Conclusion: 1. PPA1 has the ability to promote the invasion and migration of EOC cells. In vitro, the effect of PPA1 on the proliferation of EOC cells is not large. PPA1 can promote the proliferation of EOC cells. PPA1 may influence the transfer of EOC cells by promoting the proliferation of EOC cells. Part four: The effect of PPA1 on EOC cell metastasis in vivo was studied: the effect of PPA1 on EOC metastasis was observed in vivo. Method: 1. To observe the change of metastasis ability of ovarian cancer after PPA1 knock-out. In vivo imaging method to detect the change of metastasis ability of ovarian cancer after PPA1 knock-out. The expression of E-cadherin and Vimentin in ovarian carcinoma after PPA1 knockout was detected by immunohistochemistry. Result: 1. In this study, we successfully established a model of intraperitoneal transplantation of NOD/ DBM mouse ovarian cancer, and found that the intra-abdominal organs and peritoneal metastatic nodules were seen before PPA1 knockout, whereas after PPA1 knockout, metastatic nodules were uncommon. In vivo imaging, PPA1 has been proved to have an important role in promoting the metastasis of ovarian cancer. Compared with the control group, the expression of E-cadherin in knock-out PPA1 was increased and the expression of Vimentin decreased compared with that in the control group. Conclusion: 1. In this study, the metastatic ability of ovarian peritoneal transplanted tumor in mice after PPA1 knockout was decreased. In vivo, PPA1 has the ability to promote the proliferation of ovarian cancer cells, thereby promoting the transfer of EOC cells. The fifth part: PPA1 influences the mechanism of EOC transfer: preliminary study of PPA1 affects the mechanism of EOC metastasis. Methods: 1. Western blot was used to detect the changes of the expression of ES2 and SKOV3 protein p27-catenin in PPA1 knockout mice. The expression changes of ES2 and SKOV3 cells after PPA1 knockout were detected by immunofluorescence. Results: 1. Western blot showed that the expression of p27-catenin was decreased after PPA1 knockout. The immunofluorescence assay also confirmed that the expression of caspase-catenin in the EOC nucleus following the knock-out of PPA1 gene was decreased. Conclusion: The PPA1 signal pathway is one of the important signaling pathways in the tumors, and PPA1 may influence the transfer of EOC cells by modulating the extracellular domain of EOC cells.
【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R737.31
【参考文献】
相关期刊论文 前1条
1 Christen L Walters Haygood;Rebecca C Arend;J Michael Straughn;Donald J Buchsbaum;;Ovarian cancer stem cells: Can targeted therapy lead to improved progression-free survival?[J];World Journal of Stem Cells;2014年04期
，本文编号：2280403
本文链接：https://www.wllwen.com/yixuelunwen/zlx/2280403.html
