The Study Of Mortalin Promotes Epithelial-mesenchymal Transition Of Intrahepatic Cholangiocarcinoma Cells Through PI3K/Akt/mTOR Signaling Pathway

Mortalin is a member of the heat shock protein 70 family,which is involved in multiple cellular processes and over-expressed in multiple cancers.However,its expression and role in intrahepatic cholangiocarcinoma remain unclear.In the present study,the role of Mortalin in three sections for clinical significance,cell invasion and migration,epithelial-mesenchymal transition,and PI3K/Akt/mTOR signaling pathways was demonstrated using clinical tissues,cell lines in vitro,and nude mouse experiments.Part Ⅰ:Mortalin expression in intrahepatic cholangiocarcinoma andits clinical implicationsObjectives:The objectives of the current study were to determine the expression of Mortalin in intrahepatic cholangiocarcinoma(ICC)tissues,to investigate the relationship between Mortalin expression and clinicopathological features of ICC,and to assess the value of Mortalin in the prognosis of patients with ICC.Methods:qRT-PCR and Western blot were used to detect the expression of mRNA and protein levels in 10 pair ICC tumor tissues and normal liver tissues.Immunohistochemistry was used to detect the expression of Mortalin in 2 sets of tissue microarrays and eollected clinical tissues.The relationship between Mortalin and clinicopathological features of ICC was analyzed according to clinical pathological data and Mortalin staining.Based on follow-up data,survival prognosis analysis was used to determine the prognosis of Mortalin in patients with ICC.Moreover,we further evaluated the combined effect Mortalin and clinical pathological factors on the prognosis of patients with ICC.Results:The results of qRT-PCR and Western blot showed that the expression of Mortalin in tumor tissues was significantly higher than that in adjacent normal liver tissues both at mRNA and protein level.Based on immunohistochemical staining in different clinical tissues,the expression of Mortalin in tumor tissues was significantly higher than that in normal liver tissues and normal cholangiocarcinoma tissues in the liver.High expression of Mortalin was correlated significantly with TNM staging,poor differentiation and lymph node metastasis according to analysis of clinicopathological features.Kaplan-Meier analysis revealed that ICC patients with high levels of Mortalin had a significantly shorter overall survival rate than those with low levels of Mortalin.We further examined the combined role of Mortalin expression with malignant phenotypes,including high TNM staging,lymphatic metastasis in overall survival rate.Multivariate analysis revealed that Mortalin overexpression was an independent prognostic indicator for patients with ICC.Conclusion:Overexpression of Mortalin was implicated in invasion,metastasis and poor prognosis of ICC.Part Ⅱ:Relationship of Mortalin expression and epithelial mesenchymaltransition of intrahepatic cholangiocarcinomaObjectives:The aim of this study is to investigate the role of Mortalin in intrahepatic cholangiocarcinoma(ICC)migration and invasion and to explore the relationship between Mortalin and epithelial mesenchymal transition(EMT)in ICC.Methods:Mortalin expression in multiple ICC cell lines was examined using Western blot.Lentivirus overexpression transfection kit was used to up-regulation Mortalin expression in HCCC9810 cell.The expression of Mortalin in QBC939 cell was inhibited by a Lentiviral short hairpin RNA(shRNA)transfection kit.Role of Mortalin in migration and invasion of ICC cells was assessed by transwell and wound-healing assay.We constructed a subcutaneous xenograft model using HCCC9810-Mortalin cells,and QBC939-Mortalin shRNA cells their controls Moreover,we performed Western blotting to determine the expression of Mortalin and EMT-related markers in subcutaneous xenograft.The expression of EMT-related markers in ICC cells was assessed by Western blot.The EMT-related protein snail was detected by immunohistochemistry in tissues array and the survival curve was analyzed with snail and Mortalin staining.Results:Transwell assay showed the improved invasion of HCCC9810 cells after Mortalin over-expression.Wound-healing assay also showed that up-regulation of Mortalin in HCCC9810 cell dranatically enhanced the ability of migration.After successful transfection with Mortalin-shRNA in ICC cell line 939 with a high level of Mortalin,the decreased Mortalin expression significantly impaired the ability of invasion and migration of tumor cells by transwell and wound-healing assay.Western blott showed an up-regulated expression of Slug,Snail,Vimentin,and N-cadherin,and a down-regulated expression of E-cadherin in ICC cells after over-expression of Mortalin expression,vice verse.Tumor growth curve and weight in suhcutaneous xenograft model showed that tumors derived from HCCC9810-Mortalin and QBC939-nc cells grew faster than those in HCCC9810-nc and QBC939-Mortalin shRNA cells,respectively.In addition,increased expression of Mortalin and N-cadheirn and decreased expression of E-cadheirn were detected in tumors derived from ICC cells transfected with Mortalin over-expression,vice verse.We further analyzed overall survival rate by Mortalin expression and expression of EMT－transcription factor snail in the ICC tissues.The results showed that a high level of Mortalin and snail expression in ICC patients has lower overall survival rate.Conclusion:The high level of Mortalin in ICC cells promoted migration and astasis of tumor cells and induced EMT.Part Ⅲ:Mortalin promoted invasion and migration of ICC cells and induction of EMT by activation of PI3K/AKT/mTOR signalingObjectives:In this section,our aim is to investigate the relationship between Mortalin and PI3K/AKT/mTOR signaling in ICC cells and the role of Mortalin through PI3K/AKT/mTOR signaling on epithelial-mesenchymal transition(EMT).Methods:The expression of PI3K/AKT/mTOR signaling was assessed in ICC cells transfected with Mortalin over-expression/interference using Western blot.The activation of PI3K in an HCCC9810-Mortalin cell line was inhibited using a specific agent with LY294002.Subsequently,the capacity for invasion,migration,PI3K/AKT/mTOR signaling EMT-related markers in HCCC9810-Mortaiin ICC cells were assessed after LY294002 treatment.Results:Decreased expression of p85,akt and mTOR phosphorylation was detected in QBC939 ICC cell transfected with Mortalin interference.The level of p85,akt and mTOR phosphorylation was upregulated in HCCC9810-Mortalin cell.We found a down-regulation of Akt and mTOR phosphorylation and down-regulation of slug,vimentin and N-cadheirn and an up-regulation of E-cadherin after the disruption of PI3K by LY294002:in HCCC9810-Mortalin cell compared with the HCCC9810-Mortalin cell.Howerver,above changes were not observed in HCCC9810 cells that were treated with LY294002 compared with the HCCC9810-nc cells.Conclusion:The high level of Mortalin in ICC cells induced EMT and promoted invasion and metastasis of tumor cells through activation of PI3K/AKT/mTOR signaling.