The Mechanisms Of Epithelial-mesenchymal Transition And Chemotherapy Resistance In Cholangiocarcinoma Regulated By APKC-ι

Part 1 Altered Expression of aPKC-i and E-Cadherin Correlate with Clinical Features in CholangiocarcinomaObjective:To investigate the correlations of aPKC-i and E-cadherin expression with clinicopathological characteristics, invasion, and metastasis, as well as with the prognosis of patients with cholangiocarcinoma (CCC).Methods:Carcinomatous and peritumoral tissues were obtained from 64 patients with CCC after resection without neoadjuvant, radiotherapy, or chemotherapy from January, 2008 to June,2013. And the normal tissues were obtained from 10 patients with Choledochalcyst. Both aPKC-τ and E-cadherin gene and protein expression were determined using reverse transcription polymerase chain reaction, immunohistochemistry, and western blotting. The relationship of aPKC-τ and E-cadherin expression with CCC clinical features was analyzed by Cox multivariate analysis. The data were analysed using the software package SPSS (version 19.0) for Windows supplied by the Statistics Teaching Room of Tongji Medical Collage, HUST. A value of P less than 0.05 was considered significant.Results:The expression of aPKC-τ was reduced in 100% of normal tissues (10/10) and 76.56% of peritumoral tissues (49/64), and increased aPKC-τ expression was detected in tumor tissues (33/64,51.56%). aPKC-τ expression were higher in tumor than in peritumoral (x2=10.8, P=0.001) and normal tissue (x2=9.306, P=0.002). In contrast, high E-cadherin expression was found in 90% of normal tissues (9/10) and 32.8% of peritumoral tissues (21/64), and E-cadherin expression was reduced in 67.2% of tumor tissues (43/64). E-cadherin expression were lower in tumor than in peritumoral (x2=16.949, P<0.001) and normal tissue (x2=10.614, P<0.001). Multivariate analysis showed tumor differentiation, TNM stage, aPKC-τ and E-cadherin were significant independent prognostic factors for overall survival in patients with CCC.Conclusion:This study demonstrates that abnormal expression of aPKC-τ and E-cadherin was closely related to tumor differentiation, TNM stage, as well as to patient survival.Part 2 aPKC-i regulates epithelial-mesenchymal transition in cholangiocarcinoma dependent on snailObjective:To study the molecular mechanisms of cholangiocarcinoma cells EMT process regulated by aPKC-τ.Methods:Cholangiocarcinoma cell line TFK-1 was cultured in vitro,and divided into 4 groups: Blank control、Negative control、aPKC-i-Si-RNA-TFK-1 and aPKC-i-SaRNA-TFK-1. aPKC-τ、E-cadherin、Vimentin、Snail and Snai2 were detected by Western Blot, the gene were detected by qRT-PCR. Then, we transfected Si-Snail to the aPKC-i-SaRNA-TFK-1, and detected the gene and protein of aPKC-τ、E-cadherin、 Vimentin、Snail.All data were expressed as the mean ±EM. Between group and among-group comparisons were conducted using Student t tests and ANOVA, respectively. Differences were considered statistically significant when P value was less than 0.05.Results:Cells overexpressing aPKC-τ became fibroblast-like in appearance and exhibited decreased levels of the epithelial marker E-cadherin but increased the expression levels of the mesenchymal markers Vimentin. Snail and Snai2 were the key transcription factors in EMT progression. Our data also showed that the expression of Snail was enhanced with aPKC-τ overexpression, whereas there was no significant change in Snai2 expression. In the aPKC-τ-Si-RNA-TFK-1 cell, Silencing of aPKC-τ induced an increase of E-cadherin,and a decrease of Vimentin, and Snail. Snai2 levels remained unaltered. To confirm the effects of Snail, we silenced Snail by transfecting Snail SiRNA in aPKC-i-SaRNA-TFK-1, and we found the downregulation of Snail reverses this process.Conclusion:In this study, we found that aPKC-τ plays an important role in the regulation of the process of cholangiocarcinoma cancer EMT, and the regulation dependent on Snail.Part 3 The mechanisms of chemotherapy resistance in cholangiocarcinoma regulated by aPKC-τObjective:To explore the chemotherapy resistance in cholangiocarcinoma cell of TFK-1 regulated by aPKC-τ.Methods:Detected the survival rates of cholangiocarcinoma cell lines TFK-1 treated with various doses of Gemcitabine or cisplatin by CCK-8.Detected the apoptosis rates of cholangiocarcinoma cell lines TFK-1 treated with various doses of Gemcitabine or cisplatin by flow cytometry. On the basis of the experimental results of flow and CCK-8, we test the protein expression of P-gp and Caspase3.Between group and among-group comparisons were conducted using Student t tests. Differences were considered statistically significant when P value was less than 0.05.Results:Under the concentration of 0.1μg/ml、lμg/ml、5μg/ml、10μg/ml Gemcitabine or 1μg/ml、10μg/ml、20μg/ml、50μg/ml Cisplatin treated, cell growth was inhibited and showed a dose-dependent manner. In Gemcitabine, the IC50 of Blank, aPKC-τ-siRNA-TFK-1, aPKC-t-saRNA-TFK-1 is 1.52ug/ml,0.56μg/ml,2.78μg/ml (P<0.001).In Cisplatin, the IC50 is 9.3μg/ml、5.7μg/ml、16.3μg/ml(P<0.001). After Gemcitabine(lμg/ml) or Cisplatin(10μg/ml) treated 48 hours, the apoptosis of aPKC-τ-siRNA-TFK-1 was higher than TFK-1, but the aPKC-τ-saRNA-TFK-1 was resistant to the apoptosis. Western blot analysis showed the expression of P-gp was the least in the aPKC-τ-siRNA-TFK-1, and the Caspase3 was the highest.Conclusion:Taken together, our results suggest that aPKC-t could regulated chemoresistance in cholangiocarcinoma cells dependent on upregulated P-gp. And, aPKC-τ could regulated apoptosis dependent on downregulated Caspase3.