摘要目的:探讨极光激酶A(AURKA)抑制剂对胶质母细胞瘤(GBM)的细胞增殖和免疫检查点B7-H3表达的影响。方法:基于中国胶质瘤基因组图谱(CGGA)计划数据库(共325例患者)，分析AURKA mRNA的表达水平与肿瘤的病理学类型、世界卫生组织(WHO)级别、原/复发状态、患者的生存期、O 6-甲基鸟嘌呤-DNA-甲基转移酶(MGMT)甲基化、异柠檬酸脱氢酶(IDH)突变及染色体1p19q共缺失的相关性。比较AURKA mRNA在不同病理学类型、不同WHO级别，以及原发与复发性脑胶质瘤中表达的差异。绘制Kaplan-Meier生存曲线以比较不同AURKA mRNA表达水平的脑胶质瘤患者生存期差异。通过单因素和多因素Cox回归模型分析探讨AURKA mRNA表达水平、IDH突变、染色体1p19q共缺失、发病年龄、放疗、化疗、WHO级别及原/复发状态对脑胶质瘤患者生存期的影响。绘制受试者工作特征(ROC)曲线并计算曲线下面积(AUC)，以确定AURKA mRNA表达水平对脑胶质瘤患者生存期的预测价值。收集首都医科大学附属北京天坛医院神经外科学中心接受手术治疗患者的脑胶质瘤组织样本，其中低级别胶质瘤(LGG)4例，GBM 4例；通过蛋白质免疫印迹(WB)法检测AURKA在肿瘤组织样本中的表达情况。采用AURKA抑制剂alisertib处理人GBM细胞株U87-MG，将其分为对照组(以DMSO处理)和alisertib处理组(以5 μmol/L alisertib处理)。采用CCK-8法和结晶紫染色方法分别检测alisertib对细胞活性和克隆形成的影响。采用WB法检测alisertib对AURKA、磷酸化AURKA和B7-H3蛋白表达的影响。应用流式细胞术检测alisertib对GBM细胞膜蛋白B7-H3表达的影响。 结果:CGGA计划数据库分析结果表明，AURKA mRNA在GBM中的表达水平高于星形胶质细胞瘤和少突胶质细胞瘤(均 P<0.001)；在WHO 2、3及4级胶质瘤组间，AURKA mRNA的表达水平随WHO级别的升高而增加( P<0.001)；AURKA mRNA在复发性胶质瘤中的表达水平高于原发性胶质瘤( t＝4.50， P<0.001)。Kaplan-Meier生存曲线分析结果显示，AURKA mRNA高表达组的患者比低表达组生存期短(均 P<0.05)。单因素Cox回归分析结果显示，AURKA mRNA的表达水平、IDH突变、染色体1p19q共缺失、发病年龄、放疗、化疗、WHO级别及原/复发状态均为脑胶质瘤患者生存期的影响因素(均 P<0.05)；多因素Cox回归模型分析结果显示，AURKA mRNA高表达、复发状态、WHO高级别、发病年龄偏高、未接受化疗及无染色体1p19q共缺失均为脑胶质瘤患者生存期的独立危险因素(均 P<0.05)。ROC曲线分析显示，AURKA mRNA表达水平预测脑胶质瘤患者1年、3年和5年生存期的AUC(95% CI)分别为0.78(0.72～0.83)、0.85(0.80～0.89)、0.84(0.79～0.89)。临床样本的WB结果显示，人GBM组织中AURKA蛋白的表达水平高于LGG( t＝2.62， P＝0.040)。CCK-8实验结果显示，alisertib处理24、48、72 h后均显著抑制了U87-MG细胞的活性(均 P<0.05)。克隆形成实验结果显示，alisertib明显抑制了U87-MG细胞的克隆形成( t＝9.30， P<0.001)。WB实验结果表明，alisertib处理组的磷酸化AURKA表达水平明显低于对照组( t＝10.98， P<0.001)，而B7-H3蛋白表达水平高于对照组( t＝7.55， P＝0.002)。流式细胞术检测结果显示，与对照组比较，alisertib处理组膜蛋白B7-H3的表达水平升高( t＝20.04， P<0.001)。 结论:AURKA抑制剂alisertib可能能够抑制GBM的细胞增殖，并增强免疫检查点B7-H3的表达。

abstractsObjective:To investigate the effects of aurora kinase A (AURKA) inhibitor on the proliferation of glioblastoma (GBM) cells and expression of immune checkpoint B7-H3.Methods:The Chinese Glioma Genome Atlas (CGGA) dataset (325 cases) was used to analyze the correlation between expression levels of AURKA mRNA and clinical data including histopathology, World Health Organization(WHO) grade, primary/recurrent status, overall survival (OS), O 6-methylguanine-DNA-methyltransferase (MGMT) methylation, isocitrate dehydrogenase (IDH) mutation and chromosome 1p19q co-deletion. The expression levels of AURKA mRNA in different histopathology types, WHO grades, and primary/recurrent status gliomas were analyzed. We compared the OS of glioma patients with different expression levels of AURKA mRNA using Kaplan-Meier survival curves. The effects of AURKA mRNA expression level, IDH mutation, chromosome 1p19q co-deletion, age, radiotherapy, chemotherapy, WHO grade and primary/recurrent status on the OS of glioma patients were assessed through univariate and multivariate Cox regression analyses. Receiver operating characteristic (ROC) curve was plotted and area under the curve (AUC) was used to evaluate the performance of AURKA mRNA expression level in predicting the OS of glioma patients. Tissue samples of gliomas were collected from patients undergoing surgical treatment at the Neurosurgery Center of Beijing Tiantan Hospital, Capital Medical University, including 4 low-grade gliomas (LGG) and 4 GBM tissue samples. The expression of AURKA in the tumor tissue samples was detected by Western blot (WB). The human GBM cell line U87-MG was treated with AURKA inhibitor alisertib and U87-MG cells were divided into a control group (treated with DMSO) and an alisertib-treated group (treated with 5 μmol/L alisertib). The effects of alisertib on GBM cells proliferation and colony formation were detected by cell counting Kit-8(CCK-8) test and crystal violet staining, respectively. The effects of alisertib on the expression of AURKA, phosphorylation Thr288 of AURKA and B7-H3 were detected by WB. Flow cytometry was applied to detect the expression of membrane protein B7-H3 in GBM cells. Results:In the CGGA dataset, the expression level of AURKA mRNA in GBM was significantly higher than that in astrocytoma and oligodendroglioma (both P<0.001). The expression level of AURKA mRNA increased with WHO grades ( P<0.001). The expression level of AURKA mRNA was higher in recurrent gliomas than in primary gliomas ( t=4.50, P<0.001). Kaplan-Meier survival curve analysis showed that patients in the high AURKA mRNA expression group had shorter survival than those in the low expression group (all P<0.05). The univariate Cox regression analysis showed that AURKA mRNA expression level, IDH mutation, chromosome 1p19q co-deletion, age, radiotherapy, chemotherapy, WHO grade and primary/recurrent status were all influential factors in the survival (all P<0.05). The multivariate Cox regression analysis showed that high expression level of AURKA mRNA, recurrent status, high WHO grade, age, chemotherapy and chromosome 1p19q co-deletion were independent risk factors (all P<0.05). ROC curve analysis showed that the AUC of AURKA mRNA expression level to predict 1-year, 3-year and 5-year survival glioma patients was 0.78 (95% CI: 0.72-0.83), 0.85 (95% CI: 0.80-0.89) and 0.84 (95% CI: 0.79-0.89) respectively. WB results based on clinical samples showed that the protein level of AURKA in GBM was significantly higher than in LGG ( t=2.62, P=0.040). CCK-8 results indicated that alisertib significantly inhibited the activity of U87-MG cells after 24 h, 48 h and 72 h of treatment (all P<0.05). Similarly, colony formation experiment results showed that alisertib significantly inhibited the clone formation of U87-MG cells ( t=9.30, P<0.001). WB results showed that the phosphorylation level of AURKA in alisertib-treated group was lower than in control group ( t=10.98, P<0.001), while the expression level of B7-H3 protein was higher than in the control group ( t=7.55, P=0.002). Flow cytometry showed that the alisertib-treated group had increased B7-H3 expression on the cell membrane compared to control group ( t=20.04, P<0.001). Conclusion:AURKA inhibitor alisertib can effectively inhibit GBM cells proliferation and increase the expression of immune checkpoint B7-H3.