Nucleostemin非依赖p53信号通路的筛选及其关键节点作为p53缺失和突变型白血病治疗靶点的研究

Nucleostemin(NS), which is widely expressed in undifferentiated cells and kinds of tumor cells, could regulates cell proliferation and differentiation through p53 pathway.The pathological characteristics of tumor cells could be changed by downregulation of NS,so NS and its signal pathway could be new targets for leukemia therapy.But most leukemia belong to p53-null or p53-mutant type,and in this case, NS couldn't regulate cells through p53 pathway. There should be another NS p53-independent signal pathway according to our existing research results and information,but the details are still not clear.This subject will take p53-null, p53-mutant and p53-wild leukemia cell lines as materials,and screen some candidate genes and proteins closely related to NS p53-independent signal pathyway by gene chip,2-DE and mass spectrometry as well as bioinformatics technologies.Next, in order to propose the NS pathway in p53-null and p53-mutant leukemia in details,we will further screen and verify the candidate proteins through reaesrching the colocalization and interaction between possible upatream and downstream candidate proteins by using confocal laser technology and co-immunoprecipitation.Based on that,we will seek some possible key points,and observe the changes of cell biological characteristics after inhibiting the key points. Further,we will validate the feasibility of these points to be as therapeutic targets for p53-null and p53-mutant leukemia through experiments on animals.

核干细胞因子（NS）广泛表达于未分化细胞和肿瘤细胞，其通过p53信号通路影响细胞增殖与分化，抑制其表达可改变肿瘤细胞的病理学性质，NS及其作用通路有望成为白血病等肿瘤治疗的新靶向，但很多白血病属于p53缺失或突变型，NS无法通过p53途径影响细胞。根据我们的研究结果和资料，认为NS还应存在不依赖p53信号通路，但目前具体细节还是空白。本课题以p53缺失、突变、野生型白血病细胞系为材料，采用基因芯片、蛋白双向电泳、质谱分析以及生物信息学技术首先筛选出与NS非p53依赖信号通路有关系的候选基因和蛋白，进而采用激光共聚焦、免疫共沉淀技术研究可能的上下游候选蛋白间的相互结合及关系，进一步验证、确认候选蛋白，目标是提出p53缺失和突变型白血病NS信号通路的具体环节，在此基础上找出关键作用节点进行阻断观察白血病细胞生物学特性变化，进一步动物实验验证关键节点作为p53缺失和突变型白血病治疗靶点的可行性。

通过一年期的研究，本课题组顺利的完成了研究任务，成功构建了针对核干细胞因子（Nucleostemin，NS）基因的RNA干扰慢病毒表达载体，感染了人HL-60、NB4、K562三种白血病细胞株，运用基因芯片技术检测下调这三种细胞NS的表达后人全基因表达谱的变化情况，并通过生物信息学对结果进行分析。生物信息学分析包括GO分类法和Pathway分析。通过GO分类法分析发现，发生下调的基因在生物过程方面主要参与mRNA代谢过程及RNA加工等，在分子功能方面主要参与蛋白及RNA的结合等；上调基因在生物过程方面主要是参与了形态发生的解剖结构形成及多细胞生物过程的调节，在分子功能方面主要是溶菌酶活性及细胞骨架蛋白的锚定。而在Pathway分析中，重点研究了与细胞凋亡、细胞周期变化、DNA翻译和蛋白合成等有关的信号通路，并查阅大量文献，在下调基因中初步筛选出PI3K/AKT/mTOR、Jak/STAT和Ras/Raf/Mek/Erk通路；在上调基因中筛选出JUNK和P38/MAPK通路。之后我们对这几条通路中部分相关分子进行了qPCR测定，进一步确定了这些通路在基因水平上受NS基因所影响。本研究为NS非p53依赖信号通路具体机制的研究提供了参考，也为后续筛选靶向NS特异性小分子抑制剂打下了基础。本课题研究所获得的研究成果：待发表SCI论文两篇，题目分别为《Gene expression profiling of HL-60 cells after knocking down nucleostemin by DNA microarray technique》和《Gene expression profiling of NB4 cells after knocking down nucleostemin by DNA microarray technique》；发表国内核心论文两篇，题目分别为《核干细胞因子RNA干扰慢病毒载体的构建及鉴定》和《HL-60细胞Nucleostemin表达下调对PI3K/AKT/mTOR通路相关分子表达的影响》；硕士毕业论文两篇，题目分别为《HL-60细胞内核干细胞因子抑制后全基因组表达谱的变化》和《Screening of Nucleostemin p53-independent signal pathway in NB4 leukemia cell》