1.湖南省人民医院/湖南师范大学附属第一医院 肝胆外科/肝胆肿瘤研究室，湖南 长沙 410005;2.湖南省胆道疾病防治临床医学研究中心，湖南 长沙 410005
毛先海， Email: firstname.lastname@example.org
1.Department of Hepatobiliary Surgery/Research Laboratory of Hepatobiliary Tumor, Hunan Provincial People’s Hospital/the First-affiliated Hospital of Hunan Normal University, Changsha 410005, China;2.Clinical Medical Research Center for Biliary Disease of Hunan Province, Changsha 410005, China
背景与目的 有丝分裂激酶Aurora-B是胆管癌原癌基因，着丝粒蛋白U（CENPU）参与有丝分裂受Aurora-B激酶磷酸化调控，笔者团队前期在胆管癌中筛选出CENPU并证实CENPU表达水平明显高于其相应的癌旁组织，从而推测Aurora-B可能通过调控CENPU的磷酸化参与胆管癌的肿瘤生物学过程。因此，本研究探讨Aurora-B与CENPU在胆管癌中的关系及作用。方法 采用免疫组化检测10对胆管癌组织和癌旁组织中Aurora-B和CENPU以及磷酸化CENPU（p-CENPU）的表达。采用TCGA数据库的数据分析胆管癌中Aurora-B和CENPU的表达及相关性。构建Aurora-B基因敲减的胆管癌QBC939细胞系，用Western blot检测CENPU和p-CENPU的表达，CCK8法检测细胞增殖活性。构建CENPU磷酸化位点突变的胆管癌QBC939细胞系，用DMSO或Aurora-B抑制剂处理后，观察Aurora-B、CENPU和p-CENPU表达以及细胞增殖活性的变化。结果 免疫组化结果显示，胆管癌组织中Aurora-B和CENPU表达阳性率分别为22.61%和12.34%，而两者在癌旁组织中几乎无表达，同时胆管癌组织中p-CENPU的表达也明显高于癌旁组织。TCGA数据显示，胆管癌中Aurora-B和CENPU的上调（均P<0.05），且Aurora-B和CENPU的表达水平呈正相关（r=0.7322，P<0.05）。敲减Aurora-B表达后，QBC939细胞CENPU表达无明显变化，但p-CENPU表达降低、增殖活性明显减弱（P<0.05）。QBC939细胞在突变CENPU磷酸化位点或加入Aurora-B抑制剂后，前者Aurora-B表达无明显变化，后者明显降低，两者CENPU表达均无明显变化，但p-CENPU表达明显降低；突变CENPU磷酸化位点后，Aurora-B介导的QBC939细胞增殖活性较野生型明显下降（P<0.05）。结论 在胆管癌中，CENPU是Aurora-B的磷酸化底物，Aurora-B可能通过磷酸化CENPU促进胆管癌细胞的增殖。
Background and Aims The mitotic kinase Aurora-B is considered to be a proto-oncogene of cholangiocarcinoma. The action of centrosome protein U (CENPU) in mitosis is governed by phosphorylation regulation of Aurora-B kinase. The previous study conducted by the authors’ group has identified CENPU in cholangiocarcinoma and confirmed that the expression level of CENPU is significantly higher in cholangiocarcinoma tissue than that in its corresponding adjacent tissue. So, it is speculated that Aurora-B probably participates the tumor biological process of cholangiocarcinoma through regulation of the CENPU phosphorylation. Therefore, this study was performed to investigate the relationship between Aurora-B and CENPU and their effects in cholangiocarcinoma.Methods The expressions of Aurora-B and CENPU as well as the phosphorylated CENPU (p-CENPU) in 10 pairs of specimens of cholangiocarcinoma and para-carcinoma tissue were determined by immunohistochemical staining. The expressions of Aurora-B and CENPU and their correlation in cholangiocarcinoma were analyzed in TCGA database. The cholangiocarcinoma QBC939 cells with Aurora-B gene knockout were constructed, and then, the expressions of CENPU and p-CENPU were determined by Western blot analysis and the proliferative activity was detected by CCK8 assay. QBC939 cells with mutated phosphorylation site of CENPU were constructed, in which, the changes in expressions of Aurora-B, CENPU and p-CENPU as well as the proliferation activity were observed.Results The results of immunohistochemical staining showed that positive expression rates of Aurora-B and CENPU in cholangiocarcinoma tissue were 22.61% and 12.34%, respectively, but nearly none of them expressed in adjacent tissue. Meanwhile, the expression level of p-CENPU in cholangiocarcinoma tissue was remarkably higher than that in adjacent tissue. TCGA data showed that both expressions of Aurora-B and CENPU were up-regulated in cholangiocarcinoma (both P<0.05), and there was a positive correlation between the expressions of Aurora-B and CENPU (r=0.7322, P<0.05). In QBC939 cells after knockdown of Aurora-B expression, there was no significant change in CENPU expression, but the p-CENPU expression was down-regulated, and the proliferation activity was significantly decreased (P<0.05). In QBC939 cells after mutating the phosphorylation site of CENPU or adding Aurora-B inhibitor, Aurora-B expression showed no change in the former, but was significantly reduced in the latter, and in both conditions, the CENPU expressions showed no changes, but the p-CENPU expressions were decreased; after mutating the phosphorylation site of CENPU, the proliferation activity of QBC939 cells mediated by Aurora-B was significantly lower than that of the wild-type (P<0.05).Conclusion CENPU is the phosphorylation substrate of Aurora-B in cholangiocarcinoma cells. Aurora-B promotes the proliferation of cholangiocarcinoma cells probably by phosphorylation of CENPU.