FK506 Binding Protein 65 Expression In Activated Hepatic Stellate Cells And The Invasion And Metastasis Of Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is one of the most prevalent tumor types, and both the incidence and mortality rates of HCC have increased in recent years. Newly diagnosed cases of HCC in China account for 55% of the total cases wordwide per year. Hepatic stellate cells (HSCs), as the important stromal cells in liver, when stimulated by pathology factors such as virus infection, are activated and produce extracellular matrix including collagens, participating in the development of hepatic fibrosis. It has been found that HCC cells can induce the activation of HSCs, and activated HSCs involves in tumor microenvironment formation and remolding and promote HCC progression by increasing secretion of various cytokines and extracellular matrix proteins. FK506 binding protein 65, an endoplasmic reticulum localized protein, is found to play a critical role in the folding and trafficking of various collagens and elasticins, and be associated with some tumors progression. The study was aimed to show FKBP65 was expressed in activated HSCs, and determined if it was associated with HCC invasion and metastasis.Part Ⅰ. FKBP65 expression in human HCC tissues and clinical significanceObjective:To show the expression of FKBP65 in HCC tissues and investigate the clinical significance.Methods:147 cases of HCC tissue were accumulated and made into tissue microarrays. Expression of FKBP65 and a-SMA was detected by immunohistochemical staining, and then clinical significance of FKBP65 expression and the correlation between FKBP65 and a-SMA were analyzed by SPSS 19.0.Results:FKBP65 was mainly expressed in the cytoplasm of the fibroblast like cells in the tumor stroma. The high expression of FKBP65 was significantly associated with alanine aminotransferase level and tumor size. Kaplan Meier analysis showed that high FKBP65 expression groups with decreased overall survival and recurrence free survival compared with low FKBP65 expression groups (P=0.052, P=0.023). And a strong positive correlation between FKBP65 and a-SMA levels was observed by Pearson correlatin analysis (r=0.288, P<0.001). Conclusion:High FKBP65 expression in HCC tissue is associated with poor clinical outcomes, and FKBP65 was mainly expressed in activated HSCs.Part Ⅱ. The role of FKBP65 in activated HSCs promoting HCC cells invasion and metastasis potential and related mechanismObjective:To study the role of FKBP65 in activated HSCs promoting HCC cells invasion and metastasis potential and explore the related mechanism.Method:Western blot was applied to detect FKBP65 expression in different HCC cell lines and different HSCs (primaty cells or cell line), and immunofluorescence was used to detect FKBP65 expression in activated human hepatic stellate cells (HHSteCs). A co-culture system with HCC cell lines (MHCC97H and HepG2) and HHSteCs was used to study the effect of activated HSCs on HCC invasion and metastasis, and lentivirus-mediated micro RNA was used to attenuate the expression of FKBP65 in HHSteCs. The alterations of morphology, cell invasion and moleculars expression related metastasis potential were detected by microscopy, transwell migration assay, western blot and RT-PCR. Then the ultrastructure of HHSteCs with FKBP65 knockdown was observed by transmission electron microscope, and procollagen type Ⅰ in HHSteCs with FKBP65 knockdown and in the condition medium was assessed by western blot and ELISA, respectively.Results:FKBP65 was mainly expressed in activated HSCs. After co-cultured with HSCs, HCC cells acquired epithelial mesenchymal teansition and increasing invasion ability, with E-cadherin downregulation and vimentin、MMP2、MMP9、VEGFA、VEGFB upregulation, and knockdown of FKBP65 expression in HHSteCs could attenuate these alterations, significantly. Ultrastructure observation found some aggregates under the cell membrane in HHSteCs with FKBP65 knockdown; procollagen type Ⅰ increasing in cells and reducing in the condition medium was found by western blot and ELISA.Conclusion:FKBP65 was expressed in activated HSCs; activated HSCs could promote HCC cells invasion and metastasis potential, and knockdown of FKBP65 in activated HSCs could attenuate the effect; knockdown of FKBP65 in activated HSCs could block the secretion of some proteins, such as procollagen type Ⅰ, which may affect HCC invasion and metastasis potential.Novelty1. We firstly found the FKBP65 expression in HCC tissue was closely related to HCC prognosis.2. We demonstrated the FKBP65 was expressed in activated HSCs, and it played an important role in activated HSCs promoting HCC cells invasion and metastasis potential, for the first time.Potential merits for clinical application1. FKBP65 may be a potential therapeutic target involving HCC microenvironment promoting HCC recurrence and metastasis.2. Our study provides experimental data to research further for mechanism of activated HSCs promoting HCC invasion and metastasis potential.