BDNF Expression And Its Regulation On Tumor Progression In Intrahepatic Cholangiocarcinoma

Background:Intrahepatic cholangiocarcinoma,derived from bile duct epithelium,is a kind of malignant tumor.It is second only to hepatocellular carcinoma,and ranks second in liver malignant tumors,accounting for 5%to 10%of primary liver malignant tumors.The incidence rate has been on the rise in recent years.But most patients with IHCC are diagnosed at an advanced stage because of lacking clinical symptoms.The overall survival rate of IHCC remains very poor even after radical surgery,partially because of the poor response of IHCC to adjuvant therapy.At present,surgery is still the first choice for the treatment of intrahepatic cholangiocarcinoma,but the reported 5-year survival rate is only 20%to 40%,and the resectable cases only account for 30%to 40%.The patients with IHCC usually suffer from early recurrence and lymphatic metastasis,leading to the unfavorable prognosis.Most patients who cannot undergo surgery were dead within 6-12 months after definite diagnosis.The need for more useful predictive or prognostic biomarkers is still urgent to push the treatment revolution of IHCC.Brain-derived neurotrophic factor(BDNF)is a member of the neurotrophin family primarily involved in the development and repair of the nervous system.BDNF regulates cell survival and differentiation by binding to the tropomyosin-associated receptor kinase B(TrkB)receptor or the pan-NT receptor p75(p75NTR)receptor.In addition to its function in neuronal development,BDNF also mediates RAS/MAPK or PI3K/AKT signaling pathways,suggesting that BDNF is involved in proliferation,differentiation,survival or apoptosis of neuronal and non-neuronal tumors.Moreover,ectopic expression or function of BDNF was demonstrated to promote cancer progression,response to chemotherapy or prognosis in a variety of cancers including glioblastoma,colorectal cancer,lung cancer,or hepatocellular carcinoma and so on.It's also reported that BDNF is an oncoprotein that promotes the progression of malignant tumors such as glioma,lung cancer and stomach cancer.However,the function of BDNF in IHCC has not been explored thoroughly until now.In this study,the expression and prognostic significance of BDNF in intrahepatic cholangiocarcinoma(IHCC)were studied in clinical specimens and intrahepatic cholangiocarcinoma cell lines.Furthermore,BNDF was explored to promote the progression of tumors by promoting the proliferation,migration and invasion of intrahepaticcholangiocarcinoma cells.Finally,we figured out that BDNF regulated VEGF expression and promoted angiogenesis in intrahepatic cholangiocarcinoma.Part I Expression and prognostic significance of brain-derived neurotrophic factor(BDNF)in intrahepatic cholangiocarcinomaObjective:BDNF was found to be associated with poor prognosis in a variety of epithelial carcinomas.The aim of the study was to explore BDNF expression and its prognostic significance and analyze the relationship between BDNF and clinicopathological features in intrahepatic cholangiocarcinoma.Methods:Western blotting and ELISA were used to detect BDNF expression in intrahepatic cholangiocarcinoma cell lines and corresponding culture medium.Immuohistochemistry were used to determine BDNF expression in surgical specimens from 96 intrahepatic cholangiocarcinoma patients receiving radical resection.Correlation between BDNF and clinicopathological features was assessed by Fisher's test and Cox regression model was used for determining its prognosis significance in intrahepatic cholangiocarcinoma.Results:BDNF expression is significantly higher in intrahepatic cholangiocarcinoma cell lines than normal bile duct epithelial cells.High expression of BDNF was found in 64.6%(62/96)and low expression of BDNF was found in 35.4%(34/96).BDNF high expression was significantly associated with neural invasion(P=0.007)and lower overall survival(P = 0.006).High expression of BDNF was an independent prognostic factor in intrahepatic cholangiocarcinoma(P = 0.032).Conclusion:High expression of BNDF was significantly correlated with neural invasion and lower overall survival rate in intrahepatic cholangiocarcinoma.BDNF was identified as an independent prognostic factor for intrahepatic cholangiocarcinoua.Part ? Effect of BDNF on Proliferation,Migration and Invasion of Intrahepatic Cholangiocarcinoma Cells and Its Mechanism on Tumor AngiogenesisObjective:The aim is to investigate the roles of BDNF in tumor progression and angiogenesis and explore the molecular mechanism in intrahepatic cholangiocarcinoma.Methods:We constructed pcDNA 3.1-BDNF plasmids and infected HCCC9810 cell line to increase BDNF expression.SiRNA-BDNF was used to transfected RBE cell line to knockdown BDNF expression in intrahepatic cholangiocarcinoma.MTT assay,wound healing assay and transwell chamber assay were used to measure the effects of BDNF on proliferation,migration,invasion in hilar cholangiocarcinoma respectively.Meanwhile,ELISA was used to detect VEGF level in culture medium of RBE cells after transfecting siRNA-BDNF.HUVEC tube formation assay were used to measure the effects of BDNF on angiogenesis in intrahepatic cholangiocarcinoma respectively.Results:BDNF protein was down-regulated significantly in siRNA-BDNF transfected RBE cell line and BDNF increased significantly in HCCC9810 trasfected with pcDNA 3.1-BDNF plasmids.MTT assay demonstrated that BDNF increased the cell proliferation.Wound healing assay and transwell chamber assay showed that BDNF knockdown inhibited migration and invasion in intrahepatic cholangiocarcinoma.Tube formation assay indicated that condition medium from BDNF knockdown RBE cells inhibited tube formation of HUVEC on Matrigel.BDNF overexpression could increase synthesis and secrection of VEGF in intrahepatic cholangiocarcinoma cell lines.Conclusion:BDNF knockdown inhibited cell proliferation,migration,invasion and HUVEC tube formation.ELISA data showed that BDNF knockdown could decrease biosynthesis of VEGF in intrahepatic cholangiocarcinoma.