14-3-3ζ结合aPKC-ι经上皮—间质细胞转化促进胆管癌侵袭转移

发布时间：2018-08-27 14:01

【摘要】：第一部分 14-3-3ξ和aPKC-ι在胆管癌组织中表达及其与临床预后的关系目的：检测14-3-3ξ和aPKC-ι在胆管癌组织中的表达,明确二者间的相互关系,并进一步探讨二者的表达与胆管癌临床病理特征的相关性。方法：运用免疫组织化学(IHC)、实时定量聚合酶链反应(qRT-PCR)和免疫印迹法(WB)的方法检测64例临床胆管癌患者的手术标本中14-3-3ξ、aPKC-ι和E-cadherin在癌组织与癌旁组织的差异表达,另外,取10例胆管囊肿组织做阴性对照。卡方检验和t检验检测14-3-3ξ和aPKC-ι与胆管癌病理分化和TNM分期的关系；Spearman相关性分析法分析二者之间相关性；Kaplan-Meier法计算二者的表达与胆管癌患者总体生存得关系；Cox多因素回归分析分析胆管癌患者预后独立影响因素。结果：在mRNA和蛋白质水平,14-3-3ξ在胆管癌组织中显著高表达,在癌旁组织和癌旁组织呈低表达；14-3-3ξ和aPKC-ι在胆管癌组织中协同高表达,二者呈正相关,并且与E-cadherin表达相反；14-3-3ξ和aPKC-ι二者的高表达与胆管癌的病理分化和TNM分期密切相关,二者共同低表达的患者总体生存时间较共同高表达者或单一低表达者生存时间长,Cox多因素回归分析14-3-3ξ是胆管癌预后的独立影响因素。结论：14-3-3ξ和aPKC-ι协同促进胆管癌的发生、发展和侵袭转移,14-3-3ξ是胆管癌预后的独立影响因素,可作为预测胆管癌预后的指标。第二部分14-3-3ξ与aPKC-ι结合并相互调节目的：在第一部分研究基础上进一步明确14-3-3ξ与aPKC-ι之间的相互关系.方法：通过CO-IP实验初步检测14-3-3ξ与aPKC-ι是否存在特异性的直接结合关系。并运用小分子干扰技术,合成14-3-3ζ-siRNA,aPKC-ι-siRNA和aPKC-ι过表达载体,构建稳定的14-3-3ξ低表达.aPKC-ι氏表达和aPKC-ι高表达人胆管癌细胞系。通过qRT-PCR、WB和IF检测靶向沉默14-3-3ξ或aPKC-ι以及aPKC-ι-siRNA拯救实验中,相应aPKC-ι或14-3-3ξ的表达情况。结果：CO-IP结果提示,经14-3-3ξ与aPKC-ι抗体沉淀的人胆管癌组织和细胞蛋白中,WB可检测出相应的aPKC-ι和14-3-3ξ蛋白条带；在体外,靶向沉默人胆管癌细胞中14-3-3ξ或aPKC-ι表达后,相应的出现aPKC-ι或14-3-3ξ表达下降现象；并且在aPKC-ι-siRNA拯救实验中,伴随着aPKC-ι表达恢复的同时,14-3-3ξ表达亦出现升高。结论：在人胆管癌中,14-3-3ξ与aPKC-ι存在特异性的直接结合关系,并且二者通过结合而互相调节。第三部分14-3-3ζ-aPKC-ι复合物促进胆管癌细胞上皮-间质细胞转化目的：进一步探讨14-3-3ξ对人胆管癌细胞EMT转化的调控机制。方法：通过TGF-β1诱导构建人胆管癌细胞EMT体外模型,并运用小分子干扰技术和siRNA拯救实验,靶向调控胆管癌14-3-3ξ和aPKC-ι的表达,通过显微镜观察细胞形态学改变,以及qRT-PCR、WB和IF检测EMT表型标志物。结果：通过TGF-β1诱导构建人胆管癌细胞EMT体外模型,检测细胞形态学变化有短梭形或三角形以及多边形变为细长的纤维状或长梭形；EMT标志物：上皮样表型E-cadherin和β-catenin表达减少,而间质样表型N-cadherin和Vimentin表达增多；以及transwell侵袭实验检测细胞EMT转化后细胞侵袭移动能力增强。然后当靶向沉默人胆管癌细胞中14-3-3ξ或aPKC-ι的表达, TGF-β1刺激引起转变为细长的纤维状或长梭形的细胞数目减少,EMT标志物无明显变化。另外,aPKC-ι-siRNA拯救实验提示,伴随aPKC-ι的表达恢复,14-3-3ξ的表达亦得以升高,同时EMT标志物的变化再次出现典型的EMT转化表型标志物变化。结论：在体外,14-3-3ξ通过与aPKC-ι结合形成复合物,促进人胆管癌细胞EMT转化。第四部分靶向沉默14-3-3ξ在体内外抑制胆管癌侵袭转移及增殖目的：通过体外、体内实验验证靶向沉默14-3-3ξ对胆管癌增殖和转移的作用。方法：通过Transwell侵袭实验、划痕实验和克隆平板实验体外验证靶向沉默14-3-3ξ对胆管癌细胞的增殖和转移作用；通过裸鼠皮下移植瘤实验、肺转移模型的构建,并且皮下移植瘤和肺转移瘤行HE染色、IHC染色检测,以及皮下移植瘤行qRT-PCR和WB检测,体内验证靶向沉默14-3-3ξ对胆管癌细胞的增殖和转移作用。结果：在体外,靶向沉默14-3-3ξ后,Transwell侵袭试验中胆管癌细胞穿透基底膜的细胞数目减少；划痕实验中细胞移动的速率下降；克隆平板实验中细胞克隆数目减少。在体内,靶向沉默14-3-3ξ后,皮下移植瘤成瘤能力显著下降；而肺转移瘤的数目亦显著减少；经IHC、qRT-PCR和WB检测证实实验组中14-3-3ξ和aPKC-ι的表达下降。结论：在体内、外靶向沉默14-3-3ξ能抑制胆管癌细胞的侵袭转移和增殖,有可能成为胆管癌治疗的新靶点。
[Abstract]:Objective: To detect the expression of 14-3-3x2 and aPKC-_in cholangiocarcinoma and their relationship with clinical prognosis. Quantitative real-time polymerase chain reaction (qRT-PCR) and immunoblotting (WB) were used to detect the differential expression of 14-3-3, aPKC-_and E-cadherin in the tumor tissues and adjacent tissues of 64 patients with cholangiocarcinoma. In addition, 10 cases of cholangiocyst tissues were taken as negative control. Chi-square test and t-test were used to detect 14-3-3 and aPKC-_and cholangiocarcinoma. Correlation between pathological differentiation and TNM staging was analyzed by Spearman correlation analysis; Kaplan-Meier method was used to calculate the relationship between their expression and overall survival of patients with cholangiocarcinoma; Cox multivariate regression analysis was used to analyze independent prognostic factors in patients with cholangiocarcinoma. The expression of 14-3-3x2 and aPKC-_was positively correlated with the expression of E-cadherin, while the expression of 14-3-3x2 and aPKC-_was correlated with the pathological differentiation and TNM stage of cholangiocarcinoma. Cox multivariate regression analysis showed that 14-3-3_was an independent predictor of the prognosis of cholangiocarcinoma. Conclusion: 14-3-3_and aPKC-_co-promoted the occurrence, development, invasion and metastasis of cholangiocarcinoma, and 14-3-3_was an independent predictor of the prognosis of cholangiocarcinoma. The second part, 14-3-3 zeta and aPKC-_binding and mutual regulation purposes: Based on the first part of the study to further clarify the relationship between 14-3-3 zeta and aPKC-_. Methods: CO-IP experiment was used to detect whether 14-3-3 zeta and aPKC-_had specific direct binding relationship. Small molecule interference technology was used to synthesize 14-3-3 zeta. 3_-siRNA, aPKC-_-siRNA and aPKC-_overexpression vectors were constructed to construct stable 14-3-3_low expression vectors. The expression of aPKC-_and aPKC-_high expression human cholangiocarcinoma cell lines. Targeted silencing 14-3-3_or aPKC-_or aPKC-_-siRNA rescue experiments were performed by qRT-PCR, WB and IF detection. The results suggested that the expression of aPKC-_or 14-3-CO-IP 3_in the corresponding aPKC-_or aPKC-siRNA rescue experiments. WB could detect the corresponding aPKC-_and 14-3-3_protein bands in human cholangiocarcinoma tissues and cell proteins precipitated by 14-3-3_and aPKC-_antibodies; in vitro, the expression of aPKC-_or 14-3-3_decreased after targeted silencing of 14-3-3_or aPKC-_expression in human cholangiocarcinoma cells; and in aPKC-_-siRNA rescue experiment, the expression of aPKC-_-siRNA was accompanied by the decrease of aPKC-_-3-3 The expression of aPKC-_was restored and the expression of 14-3-3 was also elevated. Conclusion: 14-3-3 and aPKC-_were specifically and directly bound to each other in human cholangiocarcinoma. Part 3 14-3-3_-aPKC-_complex promotes epithelial-stromal cell transformation of cholangiocarcinoma cells METHODS: Human cholangiocarcinoma cell EMT model was established by TGF-beta 1 induction, and the expression of 14-3-3 ZE and aPKC-_in cholangiocarcinoma was regulated by small molecule interference technique and siRNA rescue experiment. The morphological changes of cells were observed by microscope, and the phenotype of EMT was detected by qRT-PCR, WB and IF. OBJECTIVE. RESULTS: EMT model of human cholangiocarcinoma cells induced by TGF-beta 1 was established in vitro. The morphological changes of the cells were observed as short spindle or triangle and polygonal shape as slender fibrous or long spindle. EMT markers: epithelioid phenotype E-cadherin and beta-catenin expression decreased, while mesenchymal phenotype N-cadherin and Vimentin expression increased. Then, when the expression of 14-3-3x2 or aPKC-_in human cholangiocarcinoma cells was silenced, the number of cells transformed into slender fibrous or long spindle shape was reduced by TGF-beta 1 stimulation, and the EMT markers did not change significantly. The results showed that the expression of 14-3-3 Zeta increased with the recovery of aPKC-_expression, and the changes of EMT markers appeared again. Conclusion: 14-3-3 Zeta promotes EMT transformation of human cholangiocarcinoma cells in vitro and in vivo by binding to aPKC-_to form a complex. Objective: To verify the effect of targeted silencing 14-3-3x2 on the proliferation and metastasis of cholangiocarcinoma in vitro and in vivo. Methods: Transwell invasion test, scratch test and clone plate test were used to verify the effect of targeted silencing 14-3-3x2 on the proliferation and metastasis of cholangiocarcinoma cells in vitro and subcutaneous transplantation of tumor in nude mice. In vivo, the proliferation and metastasis of cholangiocarcinoma cells were tested by targeting silencing 14-3-3x2. Results: In vitro, after targeting silencing 14-3-3x2, cholangiocarcinoma cells penetrated in the Transwell invasion test. The number of transbasement membrane cells decreased; the rate of cell migration decreased in scratch test; and the number of cell clones decreased in clone plate test. Conclusion: Targeted silencing of 14-3-3_in vitro can inhibit the invasion, metastasis and proliferation of cholangiocarcinoma cells, and may become a new target for the treatment of cholangiocarcinoma.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R735.8

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