TNF-a通过HuR上调TRAF1表达的机制及作用研究

In chronic lymphocytic leukemia (CLL), abundant immature B lymphocytes are accumulated due to the disorder of anti-apoptosis genes. It was reported that the activation of non-canonical NF-κB pathway could induce the abnormal development and anti-apoptosis of B lymphocytes. Our previous results implied that the transmembrane domain of endogenous TNF-α is involved in the activation of non-canonical NF-κB pathway as receptor. The secretory TNF-α obviously up-regulates the expression of conjunction molecule TRAF1. However, the mechanism of regulation is not clear now. In our proposal, we will investigate if the TNF-α could induce the nuclear transfer of HuR protein and up regulate the TRAF1 expression by confocal laser scanning microscope, pull down and co-immunoprecipitation. Then, EMSA and polysomal profiling tests should be executed to probe into if the regulation mechanism through the extended mRNA half-life or the initiation of Cap-independent mechanism. Finally, to observe the effect on the activation of non-canonical NF-κB pathway by western blotting and cell cytotoxic test after inhibiting the translational up-regulation of TRAF1 by endogenous TNF-α. Our research is expected to provide a new approach for the immune treatment of chronic lymphocytic leukemia.

慢性淋巴细胞白血病（CLL）中，凋亡基因异常造成大量不成熟的B细胞堆积。文献报道，活化核转录因子（NF-κB）的非经典途径能使B淋巴细胞发育异常并抗凋亡。我们前期研究发现，TNF-α跨膜段作为受体介导NF-κB的非经典途径活化，而分泌型TNF-α能显著上调该受体在胞内的接头分子TRAF-1，但具体调节机制仍不清楚。本课题拟通过激光共聚焦、pull down及免疫共沉淀研究TNF-α能否诱导HuR蛋白核转位，在翻译水平上调TRAF1，并通过EMSA和polysomal profiling技术探讨TRAF1在翻译水平的调节是通过延长mRNA半衰期或启动了非Cap依赖性途径。最后，抑制HuR转位，阻断其对TRAF-1在翻译水平的上调，通过western blotting、细胞毒实验观察TRAF-1对NF-κB非经典途径的影响。以期为慢性淋巴细胞白血病的免疫治疗提供新思路。

结题.慢性淋巴细胞白血病（CLL）中，由于凋亡基因异常造成大量不成熟的B细胞堆积。文献报道，活化核转录因子（NF-κB）的非经典途径能使B淋巴细胞发育异常并抗凋亡。我们前期研究发现，TNF-α跨膜段作为受体介导NF-κB的非经典途径活化，而分泌型TNF-α能显著上调该受体在胞内的接头分子TRAF-1，但具体调节机制仍不清楚。本课题中，基于前期实验结果和文献，我收集了临床CLL患者血液样本，证实CLL患者的B细胞中NF-κB非经典途径被活化。同时证明TNF-α刺激可有效激活NF-κB非经典途径并在转录或者翻译水平上调TRAF-1。具体的调节机制为TNF-α刺激细胞的HuR蛋白核转位后，HuR蛋白在胞浆中与TRAF-1的mRNA结合后延长其半衰期，从而上调TRAF-1后活化NF-κB非经典途径。最后，抑制细胞中的HuR核转位后，TNF-α不能上调TRAF-1的表达，并且TNF-α诱导细胞发生凋亡能力增强。本研究阐明了内源性TNF-α上调TRAF1表达的机制，以及TRAF1上调后对NF-κB非经典途径的活化的影响。并且，以HuR为靶点，阻断上调TRAF1表达，进而阻断内源性TNF-α对NF-κB非经典途径的活化，使TNF-α转向启动DISC形成，肿瘤细胞发生凋亡增加。本研究为慢性淋巴细胞白血病的治疗提供了新的分子靶点和线索。