The Effect Of Abnormal B7-H4 On The Pathogenesis Of Intrahepatic Cholangiocarcinoma

Objective (s):Intrahepatic cholangiocarcinoma (intrahepatic cholangiocarcinoma,ICC) is that originated in two branches of the bile duct epithelium and adenocarcinoma. ICC primary liver cancer accounts for about 10%-15%. ICC is the incidence of hepatocellular carcinoma after liver primary malignant tumor in recent years, the incidence is rising. ICC no obvious early symptoms, symptoms hidden, difficult to detect early, most patients have lost the timing of surgery found, so early diagnosis and prompt treatment of the ICC put forward higher requirements.B7-H4 is the recent discovery of co-stimulatory factor B7 family member, by binding to receptors on T cells, inhibition of T cell proliferation, blocking the cell cycle and cell cycle arrest in GO/G1 phase, inhibition of cytokines such as IL-2, etc. release. Studies have shown that, B7-H4 in some tumor tissue expression and is associated with the clinical stage and grade. B7-H4 by inhibiting T cell-mediated anti-tumor immune response, escape immune surveillance. However, in addition to suppression of the immune response outside, B7-H4 abnormal expression in tumors whether a direct impact on the biological activity of tumor cells? Whether by influencing tumor cell signaling pathways regulate cell growth and survival, and thereby promote tumor progression? Related studies have reported little. This paper intends to focus on the B7-H4 mediated intrahepatic bile duct cancer cell malignant transformation of the development of pathological mechanisms, the use of cell biology, molecular biology, pathology and gene expression and regulation targeting methods and techniques to explore the B7-H4 whether a direct impact on the biological activity of tumor cells, involved in malignant transformation of intrahepatic bile duct epithelial cells and intrahepatic cholangiocarcinoma cancer, development of pathological processes, so as to further clarify the role of B7-H4 in cholangiocarcinoma pathological processes provide a theoretical basis and the experimental basis for clinical cholangiocarcinoma targeted therapy.Methods:1. Using a mouse to buy anti-B7-H4 monoclonal antibodies by Westemblotting, flow cytometry and immunohistochemistry (IHC) analysis methods such as B7-H4 expression of intrahepatic cholangiocarcinoma and features in a cell.2. Design and Synthesis of B7-H4-specific shRNAs and intrahepatic bile duct carcinoma cells transfected QBC939, RBE, RT-PCR and Western blotting, immunofluorescence method validation inhibiting B7-H4 mRNA expression and protein.3. In vitro experiments:observed under an optical microscope QBC939, RBE cell morphology, flow cytometry and Western blotting was used to detect cell surface molecules (or stem cell markers and epithelial mesenchymal phenotype marker) B7-H4 expression of silence after the change, CCK-8 assay cell proliferation change, colony assay or single cell colony formation change, scratch healing cell migration shift assay to detect, Transwell method test to detect cell invasion ability change, Annexin V-FITC/PI double staining cells withered death, Western blotting method, immunofluorescence assay cell signaling molecules (such as E-cad, Snail, Erk1/2, AKT, etc.) changes.4. In vivo tumor-bearing rats:After transfection QBC939, RBE cells in tumor-bearing nude mice were observed after B7-H4 shRNA treated tumor growth, tumor tissue sections H& E staining histopathological changes. While using IHC to detect tissue changes in intracellular signaling molecules, cells were analyzed by TUNEL apoptosis in situ tissue.5. Using tissue chip, combining shRNA technology, the second generation sequencing technology, in intrahepatic cholangiocarcinoma, initially screened with B7-H4 expression may target or pathway associated molecule is closely related to several characteristic; Furthermore through biological informatics methods to analyze these molecular targets and pathways in mediating the pathogenesis of intrahepatic bile duct carcinoma cell potential and its possible mechanisms and pathways.Results:1 B7-H4 expression in intrahepatic bile duct cell carcinoma was significantly higher than in adjacent intrahepatic cholangiocarcinoma cancer tissues, and the expression of B7-H4 with tumor grade and lymph node metastasis.2 design and synthesis of shRNA can effectively inhibit QBC939, the expression of mRNA and protein RBE intrahepatic bile duct cancer cell B7-H4.3 In vitro experiments found that:3.1 by shRNA-mediated silencing the expression of B7-H4 after, QBC939, RBE cells closely connected, less refractive pseudopodia significantly reduced. At the same time, E-cadherin expression in the epithelial cell phenotype marker of increased mesenchymal phenotype marker molecule Vimentin decreased expression, suggesting that B7-H4 may induce intrahepatic bile duct carcinoma cell morphological changes.3.2 B7-H4 expression QBC939 silence after, RBE cell proliferation, colony formation and scratches Trans well migration and invasion of decreased apoptosis increased significantly.3.3 Related studies suggest that signaling pathway, B7-H4 expression silencing QBC939, RBE cells Caspases 3 increased activity, increased expression of apoptosis-promoting factor Bax, apoptosis inhibitory factor reducing Bcl-2 expression, while reducing Erk1/2 phosphorylation level.4 in vivo tumor-bearing animal experiments:experiments in progress.Conclusion(s):The abnormal expression of B7-H4 in intrahepatic bile duct cell carcinoma, with tumor grade and lymph node metastasis, suggesting that B7-H4 may be involved in the pathogenesis of intrahepatic bile duct cell carcinoma development. Intrahepatic bile duct cancer cell targeting B7-H4 expression inhibition induces Caspases 3, inhibiting Erkl/2 phosphorylation, thus contributing to intrahepatic bile duct cancer cell apoptosis, while participating in the intrahepatic bile duct epithelial-mesenchymal transition (EMT) process, evidence of the B7-H4 occurred in the intrahepatic bile duct carcinoma development in rats. The project studies suggest that abnormal expression of B7-H4 may contribute to the growth of intrahepatic cholangiocarcinoma, targeting B7-H4 expression suppression technology can effectively inhibit the pathological progression of intrahepatic bile duct cell carcinoma, cholangiocarcinoma may become a potential target for therapy point.