14-3-3ζ結(jié)合aPKC-ι經(jīng)上皮—間質(zhì)細(xì)胞轉(zhuǎn)化促進(jìn)膽管癌侵襲轉(zhuǎn)移
[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.
【學(xué)位授予單位】:華中科技大學(xué)
【學(xué)位級別】:博士
【學(xué)位授予年份】:2015
【分類號】:R735.8
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