Aurora-A通过抑制细胞衰老介导卵巢癌化疗抵抗的机制研究

Both of Aurora-A kinase and cell senescence regulate cell division and cell cycle, which plays great roles in cancer, included in origin, development and therapy. As is shown in literatures and our previous studies, Aurora-A is highly expressed in about half of neoplastic ovarian tissue samples, and inhibits the expression of senescence promoting proteins such as p53/p21, p16/Rb, and BRCA1/2. Cell senescence promotes tumor chemo-sensitivity, which is opposed to Aurora-A-induced tumor chemo-resistance. We infer an important association among Aurora-A, cell senescence and ovarian cancer chemo-sensitivity. Then, as expected, we found that Aurora-A decreased ovarian cancer cell senescence and chemo-sensitivity of paclitaxel obviously accompanied by changed senescence-associated proteins in our pre-experiment. Therefore, we hypothesize that Aurora-A induces ovarian cancer chemo-resistance by inhibition cell senescence. The program is planned to investigate the association among Aurora-A , cell senescence and chemo-resistance, and to explore the mechanisms via the study of molecules, cells, animals and tissues so as to find new targeted molecules for the comprehensive therapy of Aurora-A high expressed ovarian cancers.

Aurora-A激酶与细胞衰老均可调节细胞有丝分裂和周期，并对肿瘤发生、发展、治疗等具有重要影响。申请人在文献报道以及前期研究基础上，发现Aurora-A在约半数卵巢癌组织中高表达，且可抑制p53/p21，p16/Rb，BRCA1/2等促衰老蛋白表达。细胞衰老增加肿瘤化疗敏感性，此与Aurora-A介导多种肿瘤化疗抵抗的作用相反。我们推断Aurora-A与细胞衰老以及卵巢癌化疗敏感性之间存在重要关联。预实验结果表明，Aurora-A明显降低卵巢癌细胞衰老水平以及紫杉醇化疗敏感性，并影响衰老相关蛋白表达。最终本申请者提出假说 “Aurora-A可通过抑制细胞衰老介导卵巢癌化疗抵抗”。本项目拟在卵巢癌中从分子、细胞、动物和组织临床水平上，探讨Aurora-A与细胞衰老，化疗敏感性之间的关系以及其机理，并寻求新的药物靶向分子，为Aurora-A高表达卵巢癌的全面治疗找到新的方法。

背景和目的.Aurora-A 是丝/苏氨酸蛋白激酶家族的成员之一，在包括卵巢癌在内的多种肿瘤组织中均高表达。Aurora-A与细胞衰老均可调节细胞有丝分裂和周期，并参与肿瘤发生、发展以及化疗抵抗。我们拟通过研究Aurora-A与细胞衰老的关系揭示其诱导卵巢癌化疗抵抗的机制，为卵巢癌治疗寻找新的治疗靶点，以期得到更充分的治疗。.方法.通过反转录病毒转染/感染Aurora-A高表达卵巢癌细胞系OVCA 429以及SKOV3，建立Aurora-A基因沉默细胞系，以空白载体转染的细胞系为对照。Western Blot检测目的蛋白Aurora-A表达水平。应用Cell Counting Kit-8 (CCK-8)试剂盒进行细胞增殖试验及其奥拉帕尼处理细胞后的细胞毒性测定。流式细胞术检测细胞凋亡水平变化。β-半乳糖苷酶染色检测奥拉帕尼处理卵巢癌细胞后Aurora-A对卵巢癌细胞衰老的影响。Western Blot检测衰老相关蛋白的变化，发现干细胞因子八聚体结合转录因子（OCT4）在Aurora-A沉默后低表达。将OCT4在卵巢癌细胞系中基因沉默检测其对卵巢癌细胞增殖、化疗敏感性和细胞衰老的影响。构建裸鼠皮下移植瘤模型，绘制肿瘤生长速度曲线，处死裸鼠后对组织标本进行免疫组化、β半乳糖苷酶染色、免疫荧光。最后进行相关临床资料分析，Kaplan-Meier 生存分析检测Aurora-A表达情况与卵巢癌患者生存期关系。.结果.CCK8实验结果显示，Aurora-A基因沉默明显抑制卵巢癌细胞增殖。流式细胞术及CCK8实验结果显示，Aurora-A基因沉默增加卵巢癌细胞凋亡，抑制细胞活力。β-半乳糖苷酶染色结果显示，Aurora-A基因沉默促进卵巢癌细胞衰老，并下调干细胞因子OCT4表达。CCK8试验结果显示OCT4基因沉默明显抑制卵巢癌细胞增殖，增加奥拉帕尼细胞毒性。流式细胞技术结果显示OCT4基因沉默增加化疗敏感性。β-半乳糖苷酶染色结果显示，OCT4基因沉默促进卵巢癌细胞衰老。动物实验显示，Aurora-A基因沉默抑制卵巢癌增殖，促进肿瘤衰老，且OCT4表达低水平。Kaplan-Meier生存分析显示Aurora-A高表达卵巢癌患者总生存期及无进展生存期短。.结论.Aurora-A或可通过干细胞因子OCT4抑制卵巢癌细胞衰老，最终引起化疗抵抗。OCT4或可成为卵巢癌治疗新的靶向分子。