The Effect And Mechanism Study Of BDNF On The Cell Proliferation,invasion And Metastasis Of Human Esophageal Squamous Cell Carcinoma Stimulated By TrkB

Background and purposeEsophageal cancer is one of the malignant tumors of the digestive system and is a major health challenge in the world.In China,esophageal cancer ranks sixth and fourth in the incidence and mortality of malignant tumors,respectively.There are two major pathological types of esophageal cancer,namely esophageal squamous cell carcinoma(ESCC)and esophageal adenocarcinoma.In our country,ESCC is accounting for about 90% of esophageal cancer,53% of cases worldwide.ESCC is highly aggressive with high morbidity and mortality,and the 5-year survival rate of cancer patients is about 20%.At present,ESCC is an advanced cancer characterized by metastasis and invasion,and its prognosis has not been significantly improved.Early intervention and appropriate treatment can effectively inhibit the progression of ESCC.Therefore,it is urgent to improve the diagnosis and treatment of ESCC to find the molecular target that causes the malignant transformation of ESCC.Brain-derived neurotrophic factor,belonging to the same gene family as nerve growth factor(NGF),neurotrophin-3(NT-3)and neurotrophin-4(NT-4),is a kind of neurotrophic factor and plays important roles in the nerve cell proliferation,differentiation and apoptosis.Many studies showed that BDNF abnormally expressed in many non-neural tissues and is closely related to a variety of malignant tumors.Tyrosine kinase receptor B(Trk B),a tyrosine protein receptor kinase encoded by the Trk family of proto-oncogenes,is a potent oncogene and a specific receptor for BDNF.Both BDNF and Trk B are generally up-regulated and involved in the regulation of cell proliferation,differentiation,and apoptosis in many malignant tumors,such as liver cancer,non-small cell lung cancer,prostate cancer,colorectal cancer,and so on.The combination of BDNF and Trk B can activate Wnt/β-catenin,MAPK/ERK,PI3 K,and PLC-γ signaling pathways.Abnormal activation of those signaling pathways has a direct correlation with the occurrence,development,proliferation,differentiation,migration and invasion,angiogenesis,apoptosis,radiotherapy,chemotherapy and prognosis of tumors.However,there are few studies on the role and mechanism of BDNF binding with Trk B in ESCC.Researchers have demonstrated that BDNF is closely related to the poor prognosis of ESCC with bioinformatics and immunohistochemical analysis.In this study,we explored the expression of BDNF in ESCC tissues and the effects and mechanism on the proliferation,migration and invasion of ESCC cell.Firstly,immunohistochemistry and western bloting were used to detect the expression level of BDNF in ESCC tissue and cells;western bloting experiment was used to screened ESCC cell lines,and the poorly differentiated KYSE150 and highly differentiated KYSE30 cells were selected for later experiment;The CCK-8 assay,plate cloning formation,transwell assay,flow cytometry and tubule formation experiments were used to study the effects of BDNF and Trk B inhibitor K252 a on the ESCC cell proliferation,clone formation,migration,invasion,apoptosis,and we also study the effects of conditional media on lumen formation,migration and invasion of Human umbilical vein endothelial cells(HUVECs);finally,bioinformatics and Western bloting were used to explore the molecular mechanism of BDNF regulating the proliferation,apoptosis,invasion and metastasis of ESCC cells through Trk B.Through the above studies,we intended to reveal the role and mechanism of BDNF on regulating the cell proliferation,invasion and metastasis of ESCC through Trk B,and then provides a theoretical basis for the application of targeted therapy for ESCC.Methods(1)Explore the correlation between the expression of BDNF and the development of esophageal squamous cell carcinoma.Analyze the expression of BDNF in normal esophageal epithelium,adjacent tissues and esophageal cancer tissues with bioinformatics;Detect the expression of BDNF in normal esophageal epithelium,adjacent tissues and ESCC tissues with immunohistochemical staining.Using Western blotting,the expression of BDNF in normal esophageal epithelial cells and ESCC cell lines were checked.(2)Explore the effects of BDNF on regulating the cell proliferation,invasion and metastasis of ESCC through Trk B.After treated ESCC cells of KYSE150 and KYSE30 with BDNF and K252 a,the cytotoxicity test was used to detect the effect of BDNF and K252 a on the toxicity of ESCC cells;the plate clone formation test was used to check the effect of BDNF and K252 a on the proliferation ability of ESCC cells;the apoptosis test was used to test effect of BDNF and K252 a on the apoptotic ability of ESCC cells;detect the effect of BDNF on ESCC cells and induced HUVECs cell migration and invasion by cell scratch and Transwell experiment;detect the effect of BDNF on angiogenesis of ESCC cells by tubule formation experiment.(3)Explore the molecular mechanism of BDNF on regulating cell proliferation,invasion and metastasis of ESCC through Trk B.Analyze the signal pathways regulated by BDNF/Trk B with bioinformatics;The expression levels of signaling pathway related proteins regulated by BDNF/Trk B in ESCC cells were determined by immunohistochemical assay;Using Western blotting to detect the expression proteins associated with signal pathways regulated by BDNF through Trk B in ESCC cells.Results(1)The results of immunohistochemistry,bioinformatics and Western blotting showed that BDNF was highly expressed in ESCC tissues,but almost no or low in normal esophageal epithelium and adjacent cancer tissues;BDNF was low expressed in normal esophageal epithelial cells,while high expressed in ESCC cells.(2)Treated of ESCC cells of KYSE150 and KYSE30 with exogenous BDNF,we found that BDNF not only promoted the proliferation,clone formation,migration and invasion of ESCC cells,but also increased the expression of cell proliferation related proteins of PCNA and Cyclin D1,and cell invasion and metastasis related proteins of MMP9 and N-cadherin.Treated of ESCC cells of KYSE150 and KYSE30 with Trk B inhibitor of K252 a,the results showed that K252 a inhibited the proliferation,clone formation,migration and invasion,tubule formation and promoted apoptosis of ESCC cells.Meanwhile,K252 a down-regulated the expression of PCNA,Cyclin D1,MMP9 and N-cadherin,while the expression of E-cadherin increased.(3)Bioinformaticsanalysis,showed that BDNF/Trk B may regulate the occurrence and development of ESCC through the MAPK/ERK and PI3K/AKT pathways;after treating ESCC cells with the Trk B inhibitor K252 a,the expression level of total and phosphorylated ERK1/2 and Akt proteins were down-regulate.Conclusion1.The high expression of BDNF is closely related to the progression of esophageal squamous cell carcinoma.2.BDNF can promote the proliferation,migration and invasion,angiogenesis and inhibit apoptosis of ESCC cells.3.K252 a can inhibit the proliferation,migration,invasion,angiogenesis and promote apoptosis of ESCC cells.4.The BDNF/Trk B axis may regulate the proliferation,migration,invasion,angiogenesis and promote apoptosis of ESCC through the MAPK/ERK and PI3K/AKT signaling pathways.