PPA1通過促進上皮—間質(zhì)轉(zhuǎn)化影響上皮性卵巢癌的轉(zhuǎn)移
發(fā)布時間：2018-10-18 21:28
【摘要】：第一部分:不同類型的卵巢上皮性腫瘤和正常卵巢組織PPA1的表達目的:明確焦磷酸酶1(PPA1)在上皮性卵巢癌(EOC)、交界性腫瘤、卵巢良性上皮性腫瘤及正常卵巢組織中的表達差異,探討PPA1表達和與EOC的發(fā)生發(fā)展及預后的關(guān)系。方法:1.應用免疫組化法檢測PPA1蛋白在55例EOC、20例交界性卵巢腫瘤、24例卵巢良性上皮性腫瘤和23例人正常卵巢石蠟組織中的表達情況;分析EOC患者PPA1的表達水平與其臨床病理參數(shù)的關(guān)系;對55例EOC患者進行隨lx,分析其五年生存率和PPA1蛋白表達的關(guān)系。2.Real-time PCR和Western blot法檢測人新鮮EOC組織和正常卵巢組織PPA1 m RNA及蛋白的表達水平。結(jié)果:1、免疫組化檢測顯示,PPA1在正常卵巢和良性卵巢腫瘤組織中低表達,而在卵巢交界性上皮腫瘤和EOC中高表達,并呈逐漸增加的趨勢。2、EOC的組織學類型及患者年齡不同,PPA1的表達無顯著差異。而EOC的不同臨床分期和病理分級各組間,PPA1的表達有顯著差異,PPA1的表達與臨床分期和病理分級相關(guān),并隨臨床分期和病理分級升高而呈升高趨勢。與PPA1低表達的患者比較,PPA1表達高的患者五年生存率明顯降低。3、人新鮮EOC腫瘤組織的PPA1蛋白及RNA表達均顯著高于正常卵巢組織。結(jié)論:1.PPA1的表達與人EOC的分級和分期相關(guān)�？赡茉贓OC的發(fā)生和進展中起重要作用。2.PPA1的表達高低對EOC的預后有一定的預測價值。第二部分:用RNA干擾的方法建立PPA1基因敲除的細胞系目的:建立PPA1基因敲除的穩(wěn)定轉(zhuǎn)染的EOC細胞系,用于進一步研究。方法:1.應用RNA干擾技術(shù),設計合成2個特異性靶向PPA1基因的shRNA載體(PPA1-sh RNA表達載體),同時將空載體作為對照穩(wěn)定轉(zhuǎn)染至人卵巢癌ES2和SKOV3細胞,并對不同靶點進行有效篩選。2.應用Realtime PCR及Western blot檢測目的基因PPA1的敲除效率。結(jié)果:1.本研究應用RNA干擾技術(shù),成功建立了PPA1基因敲除的ES2和SKOV3細胞系。2.檢測結(jié)果顯示:和空白對照組比較,實驗組的2個靶向PPA1-sh RNA干擾序列中,PPA1在RNA水平和蛋白水平均顯著下調(diào)達50%以上,2個靶點敲除效率理想。結(jié)論:1.RNA干擾技術(shù)在在腫瘤學研究中具有重要的價值,同樣也可以應用于EOC的研究。2.在本研究中,我們成功建立了PPA1基因敲除的ES2和SKOV3細胞系,這兩組細胞系均可用于下一步實驗研究。第三部分:PPA1對EOC細胞增殖、侵襲、遷移和EMT的影響目的:觀察PPA1對EOC細胞的增殖、侵襲、遷移和上皮-間質(zhì)轉(zhuǎn)化(EMT)的影響從而觀察PPA1對腫瘤生物學行為的影響。方法:1.CCK8法檢測PPA1基因?qū)OC細胞ES2和SKOV3增殖的影響。2.Transwell法檢測PPA1對EOC細胞ES2和SKOV3侵襲的影響。3.劃痕實驗檢測PPA1對EOC細胞ES2和SKOV3遷移的影響。4.Western blot法檢測PPA1對EOC細胞ES2和SKOV3 EMT相關(guān)蛋白:E-鈣粘素(E-cadherin)、N-鈣粘素(N-cadherin)和波形蛋白(Vimentin)表達的影響。5.免疫熒光法檢測PPA1對EOC細胞ES2和SKOV3 EMT相關(guān)蛋白α平滑肌肌動蛋白(α-SMA)和Vimentin表達的影響。結(jié)果:1.CCK8法檢測發(fā)現(xiàn),PPA1基因敲除后對于EOC細胞增殖的影響不大。2.Transwell侵襲實驗表明,PPA1能夠增強EOC細胞的侵襲能力。3.劃痕實驗表明,在體外,PPA1同樣能夠促進EOC細胞的遷移能力。4.Western blot法檢測發(fā)現(xiàn),敲除PPA1基因后卵巢癌ES2和SKOV3細胞上皮性標志物E-cadherin的表達顯著上調(diào),間質(zhì)性標志物Vimentin的表達顯著下降。間質(zhì)性標志物N-cadherin在ES2細胞的表達顯著下降,而在SKOV3細胞中檢測不到。5.免疫熒光法檢測發(fā)現(xiàn),敲除PPA1基因后卵巢癌ES2和SKOV3細胞間質(zhì)性標志物α-SMA和Vimentin的表達均顯著下降。結(jié)論:1.PPA1具有促進EOC細胞侵襲和遷移的能力,在體外,對于EOC細胞增殖的影響不大。2.PPA1能夠促進EOC細胞的EMT。PPA1有可能通過促進EOC細胞的EMT而影響EOC的轉(zhuǎn)移。第四部分:PPA1在體內(nèi)對EOC細胞轉(zhuǎn)移的影響目的:使用動物模型觀察PPA1在體內(nèi)對EOC轉(zhuǎn)移的影響。方法:1.建立NOD/SCID小鼠卵巢癌腹腔移植瘤模型,觀察PPA1敲除前后卵巢癌腹腔移植瘤轉(zhuǎn)移能力的變化。2.活體成像法檢測PPA1敲除前后卵巢癌腹腔移植瘤轉(zhuǎn)移能力的變化。3.免疫組化法檢測PPA1敲除前后卵巢癌腹腔移植瘤E-cadherin、Vimentin的表達變化。結(jié)果:1.本研究成功建立NOD/SCID小鼠卵巢癌腹腔移植瘤模型,并發(fā)現(xiàn)PPA1敲除前可見腹腔內(nèi)器官及腹膜轉(zhuǎn)移結(jié)節(jié),而PPA1敲除后,轉(zhuǎn)移結(jié)節(jié)少見。2.活體成像法檢測也證實了PPA1有促進卵巢癌腹腔移植瘤轉(zhuǎn)移的作用。3.免疫組化法檢測表明,在形成的腹腔移植瘤組織中,相比對照組,敲除PPA1者EMT相關(guān)蛋白E-cadherin表達升高,而Vimentin表達降低。結(jié)論:1.本研究發(fā)現(xiàn)PPA1敲除后小鼠卵巢腹腔移植瘤的轉(zhuǎn)移能力降低。2.在體內(nèi),PPA1具有促進卵巢癌細胞EMT的能力,從而促進EOC細胞的轉(zhuǎn)移。第五部分:PPA1影響EOC轉(zhuǎn)移機制的探討目的:初步探討PPA1影響EOC轉(zhuǎn)移的機制。方法:1.Western blot法檢測PPA1敲除后EOC細胞ES2和SKOV3蛋白β-catenin表達的變化。2.免疫熒光法檢測PPA1敲除后ES2和SKOV3細胞β-catenin表達的變化。結(jié)果:1.Western blot法檢測發(fā)現(xiàn),PPA1敲除后總蛋白及核蛋白中β-catenin的表達下降。2.免疫熒光法也證實,PPA1基因敲除后的EOC細胞核的β-catenin的表達下降。結(jié)論:Wnt/β-catenin信號通路是腫瘤EMT的重要信號通路之一,PPA1可能通過調(diào)節(jié)Wnt/β-catenin信號通路影響EOC細胞的EMT,從而促進EOC的轉(zhuǎn)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.
【學位授予單位】：天津醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R737.31
本文編號：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.
【學位授予單位】：天津醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R737.31
【參考文獻】
相關(guān)期刊論文 前1條
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
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