Role And Mechanism Of MSI2 In Epithelial-mesenchymal Transition Of Cholangiocarcinoma

Background and Purpose Cholangiocarcinoma is the most common malignancy of the biliary tract.Patients diagnosed with cholangiocarcinoma usually have a poor prognosis due to its late clinical presentation and lack of non-surgical treatment.Surgical resection is the only preferred treatment for cholangiocarcinoma.Unfortunately,most patients are not suitable for curative resection since the disease is generally too advanced at the time of diagnosis.Therefore,identifying effective prognostic biomarkers of diagnosis and mechanisms of distant dissemination are crucial for improving the prognosis of cholangiocarcinoma.The Musashi2(MSI2)gene belongs to the RNA-binding protein family,which participates in posttranscriptional regulation by binding to target m RNAs.Recently,researchers found that MSI2 might be an oncogene that involved in cancer development and progression,and is associated with poor prognosis in some kind of malignancies.However,there has been no report about MSI2 in bile duct cancer.Based on the biological functions of MSI2,we suspected that it might be involved in the development and progression of cholangiocarcinoma.In the present study,we sought to evaluate MSI2 expression in human specimens,determine the association between MSI2 expression and clinicopathological characteristics of patients,and identify the functional role of MSI2 in the growth and metastasis of cholangiocarcinoma cells.Methods(1)Western Blot and q RT-PCR were used to detect the protein and m RNA expression of MSI2 in twenty pairs of fresh frozen tumor tissues and adjacent normal tissues,and the differences between them were also analyzed.(2)Immunohistochemical staining was used to detect the protein expression of MSI2 in 78 pairs paraffin-embedded tumor tissues and adjacent normal tissues,and the association between MSI2 expression and the clinicopathological features of patients were also analyzed.(3)Western Blot was used to detect the expression of MSI2 protein in human normal bile duct cell(HIBEpi C)and human cholangiocarcinoma cells(QBC939 and RBE).Then the MSI2-sh RNA vectors were cotransfected with lentiviral packing vectors to downregulate the expression of MSI2 in QBC939 and RBE cell lines.(4)Colony formation assay,cell counting kit-8 assay,wound healing assay and cell migration and invasion assays were used to detect the effect of MSI2 gene on proliferation,migration and invasion of cholangiocarcinoma cells.(5)Western Blot and immunohistochemical staining were used to detect the protein expression of E-cadherin,N-cadherin and Vimentin,which were the key molecules regulating epithelial-mesenchymal transition(EMT)process.Results(1)Compared with normal bile duct tissues,MSI2 expression is significantly increased in tumor tissues at m RNA and protein level,and high expression of MSI2 is associated with TNM staging,lymph node metastasis and poor prognosis in patients with cholangiocarcinoma.(2)MSI2 expression was dramatically higher in the cholangiocarcinoma cell lines QBC939 and RBE than in the normal bile duct epithelial cell HIBEpi C.MSI2 downregulation inhibited the proliferation,migration,and invasion of QBC939 and RBE cells.(3)E-cadherin expression was markedly downregulated in tumor tissues compared with normal bile duct tissues.MSI2 knockdown increased E-cadherin expression and decreased N-cadherin and Vimentin expression.ConclusionOur result indicates that high expression of MSI2 is an independent risk factor for poor prognosis in patients with cholangiocarcinoma.Downregulation of MSI2 can inhibit the proliferation,migration and invasion of cholangiocarcinoma.MSI2 can promote cholangiocarcinoma matastasis through,at least partially,inducing EMT.