JunB调节CD133/CD57双阳性胶质瘤干细胞样细胞自我更新和辐射抗拒的分子机制及临床意义

The hypothesis is based on the finding of previous international cooperation program: CD133/CD57 dual positive cell population with a strong self-renewal capacity and radiation resistance.We established orthotopic glioma mouse model by injecting fresh surgical specimens of human gliomas into mouse brain, and then simulated classic fractionated radiation in the treatment group. The data showed that most live cells are CD133/CD57 dual positve cells. Global gene expression profiles of control group, radiation group and relapsed group were then analyzed by whole genome microarrays, JunB was identified and verified to be upregulated in radiation and relapsed groups mouse models. In this study, base on the level of stem-like cells and orthotopic glioma mouse model, we plan to downregulate or overexpress the JunB gene by lenti-virus in CD133/CD57 dual positive glioma stem-like cells, then analyze self-renewal capacity and radiosensitivity in them. DNA damage repair capacity, cell cycle distribution, apoptosis and autophagy were detected as well; Also explore the impact of JunB expression on the ability of invasion by transwell assay and investigate the changes of stem cell related, DNA damage repair and cell cycle distribution pathways by Western-blot. On the other side, figure out the possible molecular mechanisms by gene array. Survive time of orthotopic glioma mouse model treated by JunB inhibitor combined radiotherapy will be monitored. At last, verify its impact on radiosensitivity and prognostic value in glioma patients by immunostaining 120 cases of clinical samples and 200 cases from prospective clinical trial. In this study, we want to represent JunB as a glioma therapeutic target.

前期国际合作课题申请者通过建立多例新鲜胶质瘤手术标本来源小鼠脑原位模型，模拟临床放射治疗，发现大量存活细胞为CD133/CD57双阳性细胞，基因芯片分析提示残留存活细胞共同高表达JunB。前期数据证明胶质瘤CD133/CD57双阳性细胞具备强大的自我更新能力和辐射抗拒性。本研究将在干细胞样细胞及原位模型水平分析慢病毒下调或过表达JunB基因后对CD133/CD57双阳性胶质瘤细胞自我更新和放射敏感性的影响，微侵袭改变；检测DNA损伤修复、细胞周期和凋亡自噬等阐述其机制，蛋白质印迹分析干细胞等相关信号通路改变；基因芯片筛选下游分子通路，探索可能的相互作用和调控关系；对JunB抑制剂联合放疗治疗荷瘤小鼠进行生存分析。在有完整预后资料的120例胶质瘤临床标本和前瞻性临床试验收集约200例标本中验证JunB蛋白与肿瘤分期、放射敏感性及预后的关系。本研究将阐明JunB作为脑胶质瘤治疗新靶点打下基础。

脑胶质瘤是最常见的原发性中枢神经系统肿瘤，而IV级胶质瘤母细胞瘤中位生存期只有14个月，是全世界医疗界的难点。近年来，放射治疗是治疗恶性脑胶质瘤最重要的辅助治疗手段，由于其浸润性生长特性导致无法切除干净，对放射治疗抗拒容易快速复发。如何克服脑胶质瘤辐射抗拒成为临床研究者的重要课题。我们通过大量来源于患者的新鲜手术标本建立原位小鼠模型（PDOX），从其细胞中筛选出CD133/CD57双阳性细胞证明其具备更加强大的干细胞特性，具有在颅内更强的成瘤能力及无血清培养状态下的成球能力。基因芯片筛选出经过放射治疗后PDOX模型残留复发胶质瘤细胞中共同高表达JunB，在胶质瘤细胞系中进行不同时间点，不同放射线剂量验证，发现其具有时间和剂量依赖性。CGGA数据库JunB基因与胶质瘤患者预后成反比（P<0.0001）,与病理级别成显著性正比。临床脑胶质瘤病理标本分析JunB蛋白高表达提示患者不良预后（P=0.017）。在高表达JunB的胶质瘤干细胞及细胞系水平慢病毒下调JunB基因后降低其辐射敏感性，而上调JunB基因以后可以增加胶质瘤细胞系的辐射抗拒性。通过划痕实验，我们证明JunB能够增强胶质瘤细胞的侵袭能力，下调其表达可以减弱其侵袭能力；γ-H2AX检测发现下调JunB基因能够延迟放射线诱导的DNA损伤修复，高表达JunB能加速脑胶质瘤细胞的放射线诱导的DNA损伤修复；下调JunB能够诱导胶质瘤细胞G2M周期阻滞，显著降低S期细胞数（p<0.05）；过表达JunB，逆转放射线诱导的G2M周期阻滞（p<0.05）。蛋白质印迹分析其机制可能与下调磷酸化cdc2, Cyclin-E2,p-Chk1也有相关性，下调JunB了联合放射线增强DNA损伤标志物Rad51的表达。通过对两株脑胶质瘤系进行下调JunB基因及放射线处理，进行全基因芯片扫描，筛选出一系列共同作用信号通路和基因，在下一步工作会做进一步分析验证。本研究在恶性脑胶质瘤中从根本阐述了CD133CD57双阳性细胞的强大肿瘤干性，JunB是脑胶质瘤重要的预后及病理恶性程度标记物，对于维持胶质瘤干细胞和细胞系自我更新、辐射抗拒有根本作用，为后续以其作为治疗和辐射增敏靶点开发打下了坚实基础。