CRBN泛素化修饰AMPK调控骨髓瘤细胞增殖与凋亡的分子机制研究

CRBN is a component of an E3 ubiquitin ligase, which recently been identified as the direct target of immunomodulatory drugs for their anti-myeloma effects. AMPK is a key regulator of cellular energy metabolism, regulating the proliferation and apoptosis of myeloma cells.It is not clear whether AMPK can be ubiquitined by CRBN and thereby regulates the proliferation and apoptosis of myeloma cells. This study intends to use bioinformatics methods to predict the potential ubiquitination sites among AMPK, and then knock in or out CRBN gene in myeloma cells by TALEN technology, co-precipitation immunization, WB, flow cytometry, cell proliferation assays will be taken to detect its impact on the ubiquitination status of myeloma cells with wild-type and mutated AMPK ubiquitination sites. The consiquential effects on AMPK/mTOR pathway and the proliferation and apoptosis of myeloma cells will also be evaluated. Finally,we will use animal models and clinical data to validate the effects of AMPK ubiquitination by CRBN on the proliferation and apoptosis of myeloma cells via CRBN/AMPK/mTOR pathway. This finding will not only help to reveal the resistance mechanism of immunomodulatory drugs in MM, but also to enhence and extend the signal transduction mechanisms of MM cells, providing new insights into exploring new therapeutic targets novel and therapeutic strategies for myeloma.

CRBN具有E3泛素连接酶功能。该蛋白最近被鉴定为免疫调节类药物抗骨髓瘤作用的直接靶点。AMPK是细胞能量代谢的关键调控酶，与骨髓瘤细胞的异常增殖、凋亡关系密切。CRBN能否泛素化修饰AMPK并调控其下游mTOR信号转导通路，从而影响骨髓瘤细胞的增殖与凋亡尚不清楚。本课题拟采用生物信息学技术预测出AMPK的潜在泛素化位点，再借助TALEN技术使骨髓瘤细胞过表达或低表达CRBN，通过免疫共沉淀、WB、流式细胞术、细胞增殖实验等方法检测其对野生型和突变型AMPK泛素化状态以及mTOR通路乃至骨髓瘤细胞增殖与凋亡的影响。最后，通过动物模型和临床数据验证，阐明CRBN泛素化修饰AMPK，调控CRBN/AMPK/mTOR通路，导致骨髓瘤细胞异常增殖和凋亡的分子机制。研究结果将不仅有助于揭示MM中免疫调节类药物的耐药机制，也为拓展MM信号转导机制研究，研发新的治疗靶点，提出新的治疗策略提供理论依据。

CRBN是免疫调节类药物抗骨髓瘤作用的直接靶点，具有E3泛素连接酶作用。AMPK是细胞能量代谢的关键调控酶，与骨髓瘤细胞的异常增殖、调控关系密切。目前骨髓瘤细胞中，CRBN如何通过AMPK影响骨髓瘤细胞的生物学特性尚不清楚。我们在骨髓瘤细胞中通过改变CRBN的表达，研究其对骨髓瘤细胞增殖、周期、凋亡等生物学功能的影响。通过Western blot在骨髓瘤原代细胞中检测CRBN, AMPK, p-AMPK，mTOR等重要通路蛋白，研究其作用机制。实验结果表明，原代骨髓瘤细胞中显著高表达CRBN。敲低骨髓瘤细胞中CRBN抑制细胞增殖，阻滞细胞周期由S期向G2期转化，促进细胞凋亡。骨髓瘤细胞中CRBN的高表达抑制了AMPK的磷酸化，从而解除对mTOR通路活性的抑制，而促进骨髓瘤细胞的生长。在上游泛素化调控机制的探索中，我们发现去泛素化酶USP15通过去除NF-κBp65的泛素化而上调其表达量，促进骨髓瘤的发生发展。结论：骨髓瘤细胞中异常表达的CRBN能够影响AMPK的磷酸化，改变下游mTOR通路活性，参与调控骨髓瘤细胞的增殖、凋亡等生物学过程。