Study On The Mechanism Of Aurora-B Kinase Phosphorylated CENP-U Regulating The Proliferation Of Cholangiocarcinoma Cells

Part I Study on the expression of Aurora-B and CENP-U in intrahepatic cholangiocarcinoma and their clinicopathological relationship ObjectiveThe purpose of this study is to detect the expression of Aurora-B and CENP-U in intrahepatic cholangiocarcinoma,to clarify the clinicopathological relationship between Aurora-B and CENP-U and intrahepatic cholangiocarcinoma,and to understand the correlation between the expression of CENP-U and Aurora-B.MethodsDetecting the expressions of Aurora-B and CENP-U in 40 cases of intrahepatic cholangiocarcinoma and paracancerous tissue tissues were using immunohistochemistry.The relationship between Aurora-B,CENP-U and clinicopathological factors of intrahepatic cholangiocarcinoma was analyzed,and the relationship between them was analyzed by Spearman correlation analysis.Result1.The results showed that the expression of Aurora-B and CENP-U in intrahepatic cholangiocarcinoma was significantly higher than that inadjacent tissues(P < 0.05).2.The intensity of Aurora-B expression was related to the degree of differentiation,tumor size,lymph node metastasis and clinical stage of cholangiocarcinoma(P = 0.024,0.012,0.026,0.024).The expression of CENP-U was closely related to tumor size,lymph node metastasis and clinical stage(P = 0.037,0.048,0.013).There was a positive correlation between the expression of Aurora-B and CENP-U in intrahepatic cholangiocarcinoma(r = 0.647,P < 0.0001).ConclusionTherefore,Aurora-B and CENP-U,as important molecules of mitosis,are highly expressed in intrahepatic cholangiocarcinoma,and their expression level is closely related to the clinical stage of intrahepatic cholangiocarcinoma.Aurora-B and CENP-U may participate in the development of intrahepatic cholangiocarcinoma.Part II Study on the mechanism of Aurora-B regulating the effect of CENP-U on the proliferation of intrahepatic cholangiocarcinoma cells ObjectiveThe purpose of this study is to investigate the role and mechanism of Aurora-B/CENP-U signaling pathway in the proliferation of cholangiocarcinoma.MethodsThe cholangiocarcinoma cell line QBC939 was established to silence Aurora-B by constructing Aurora-B-shRNA plasmid and transferring it into QBC939 cells with lipo2000.The knockout efficiency of Aurora-B gene was verified by qPCR.The expression of total CENP-U protein and phosphorylated CENP-U protein(p-CENP-U protein)was detected by WB.To construct a lentivirus vector with inactivated mutation composed of CENP-U phosphorylation sites(Ser349 and Ser350)and infect the cholangiocarcinoma cell line QBC939,obtain the cholangiocarcinoma cell line QBC939 with inactivated mutation composed of CENP-U,detect the expression level of p-CENP-U by WB,and detect the proliferation of cholangiocarcinoma QBC939 by CCK8 and Clone formation test.Result1.The results showed that the expression of QBC939 CENP-U and P-CENP-U protein in aurora-b-silenced cholangiocarcinoma cells decreased.2.The inactivated lentivirus vector composed of the phosphorylation sites of CENP-U(Ser349 and Ser350)decreased the proliferation of cholangiocarcinoma cell line QBC939 after infection with QBC939.ConclusionTherefore,Aurora-B kinase may regulate the expression of CENP-U by phosphorylation,thus affecting the proliferation of cholangiocarcinoma cells.