14-3-3ζ Binds With APKC-ι To Promote The Invasion And Metastasis Of Cholangiocarcinoma Cells Via Epithelia-Mesenchymal Transition

Part I The Synergetic Expression and Clinical Significance of 14-3-3ζ and aPKC-ι in Patients with CholangiocarcinomaObjective:To detected the expression of 14-3-3ζ and aPKC-ι in the cholangiocarcinoma (CCA) tissues for clearing the correlation between them. And to further assess the relationship between them and the clinical property of CCA.Methods:The expression of 14-3-3ζ, aPKC-ι and E-cadherin was examined in 64 cases of CCA tissues by immunohistochemistry, qRT-PCR and Western blot. Meanwhile, Chi-square test and t test were used to detect the relationship between the expression of 14-3-3ζ and aPKC-ι and the clinical property of CCA. Spearman correlation analysis was used to test the coordination between them. Kaplan-Meier was employed to evaluate the expression of them association with the overall survival. And Cox regression analyses was applied to assess the independent factors of CCA.Results:Clinically, opposite to E-cadherin, the expression of 14-3-3ζ and aPKC-ι was synergetic up-regulation in CCA tissues compared to peritumoural and normal tissues, and the relationship of them was positive correlation. Further,14-3-3ζ and aPKC-ι overexpression intimately associated with differentiation and tumour-node-metastasis (TNM) stage. The overall survival of patients with 14-3-3ζ and aPKC-ι high-expression was longer than those with 14-3-3ζ and aPKC-ι low-expression. Cox regression analyses suggested that 14-3-3ζ was an independent prognostic indicator for patients with CCA.Conclusion:The synergetic expression of 14-3-3ζ and aPKC-i promotes the initiation, development, invasion and metastasis of CCA. And 14-3-3ι is an independent prognostic indicator and can predict a poor prognosis of patients with CCA.Part Ⅱ The Mutual Regulation of 14-3-3ζ and aPKC-ι by Directly binding with each otherObjective:Further to study the correlation between 14-3-3ζ and aPKC-ι.Methods:CO-IP experiment was utilized to detect whether 14-3-3ζ can bind with aPKC-ι. Then 14-3-3ζ-siRNA, aPKC-i-siRNA and aPKC-ι over-expression vector were synthesized by small interfering technology, and transfected into the cholangiocarcinoma cells-TFK-1 and HuCCT1 for establishing the cell lines with lower expression of 14-3-3ζ and aPKC-ι, and with higher expression of aPKC-i. qRT-PCR, WB and IF were used to detect the expression of 14-3-3ζ and aPKC-i in those cells with targeted silencing the 14-3-3ζ or aPKC-ι expression, and within aPKC-ι-siRNA rescued experment.Results:The results of CO-IP showed that the protein bands of 14-3-3ζ or aPKC-i could be detected by WB in which immun precipitated by relevant antibodies. The expression of 14-3-3ζ and aPKC-ι was reduced after siRNA transfected in vitro, and was increased in aPKC-ι-siRNA rescue experiment.Conclusions:There is a specific binding relationship between 14-3-3ζ and aPKC-ι in cholangiocarcinoma cells, and they can regulate each other through this bond.Part Ⅲ 14-3-3ζ-aPKC-ι Compound Facilitates the Epithelial-Mesenchymal Transition of Cholangiocarcinoma Cells in vitroObjective:To further investigate the mechanism of 14-3-3ζ regulating the EMT of cholangiocarcinoma cells in vitro.Methods:The EMT model of cholangiocarcinoma cells was established by TGF-β1 inducing in vitro. The small interfering technology and siRNA rescue experiment were utilized to targeted silence the expression of 14-3-3ζ or aPKC-ι. Subsequently, the morphological changes of cells and the expression of EMT’s markers were observed.Results:The morphological changes of cells treated by TGF-β1(lOng/ml) were observed transition from a polarized, epithelial to depolarized, spindle-shaped and mesenchymal morphology under a phase-contrast microscope within 5 days. The some EMT markers, including E-cadherin and β-catenin, were decreased; and others EMT markers, including N-cadherin and Vimentin, were increased. And transwell invasion experiment were used to detect taht the invasion capability of cells treated by TGF-β1 was enhanced. However, targeted silencing 14-3-3ζ or aPKC-ι impaired the number of cells with depolarized, spindle-shaped and mesenchymal morphology. Meanwhile, the expression of EMT markers had no significantly changes. In addition, in aPKC-ι-siRNA rescue experiment, the expression of 14-3-3ζ was increased accompanying with the expression of aPKC-ι recovered, and the significantly changes of EMT markers expression were appeared again.Conclusions:In vitro,14-3-3ζ binds with aPKC-ι to promote the EMT of cholangiocarcinoma cells.Part IV Targeted Silence14-3-3ζ Can Suppress the Invasion, Metastasis and Proliferation of Cholangiocarcinoma Cells in vitro and in vivoObjective:To detect the effect on proliferation and metatasis of cholangiocarcinoma by targetting silence 14-3-3ζ in vitro and in vivo.Methods:Transwell cell invasion assay, Wound healing assay and Colony formation assay were utilized to assess the effect of the cholangiocarcinoma cells containing 14-3-3ζ-siRNA on proliferation and metastasis in vitro. Tumorigenicity assay and pulmonary metastasis tumor model were employed to assess the effect of the cholangiocarcinoma cells transfected 14-3-3ζ-siRNA on proliferation and metastasis in vivo.Results:Followed targetting silence 14-3-3ζ, the number of cells through basement decreased, the migration rate of cells slowed down, and the number of cell clons reduced in vitro. Further, the volume of xenograft and the number of pulmonary netastasis tumor were significantly decreased. The expression of 14-3-3ζ and aPKC-ι in the treatment groups was significantly decreased by IHC, qRT-PCR and WB.Conclusions:Targetted inhibition 14-3-3ζ could suppress the proliferation and metastasis of cholangiocarcinoma cells. And 14-3-3ζ is likely to be a new target for treatment of cholangiocarcinoma.