基于PI3K/Akt/mTOR和AMPK/mTOR双信号通路探讨二甲双胍抗多发性骨髓瘤作用机制的研究

Multiple myeloma (multiple myeloma, MM) is a common hematological malignancy. Activated of PI3K/Akt/mTOR signaling pathways by IGF-1/IGF-1R is involved with proliferation of myeloma cell. Metformin is a biguanide antidiabetic, also known as AMPK agonist. It was reported that metformin had anti-tumor effect by inhibiting PI3K/Akt/mTOR signaling pathway. Our study also find that metformin can inhibit proliferation of MM cells and induce its apoptosis by this signal pathway. Meanwhile, we find that metformin can induce apoptosis of MM cells by inhibiting AMPK/mTOR pathway, knock out of AMPK of MM cells shows up regulation of Akt. It is supposed that there are other key factors play important roles in AMPK signaling or there exists certain relevance between PI3K and AMPK. In this study, we plan to explore the mechanism of metformin in inhibiting proliferation and inducing apoptosis of myeloma cells and the regulatory mechanism of PI3K/mTOR and AMPK/mTOR pathway by molecular biology methods to provide new ideas for the treatment of MM.

多发性骨髓瘤（multiple myeloma, MM）是常见的血液系统恶性肿瘤，已证实IGF-I/IGF-IR介导的PI3K/Akt/mTOR通路激活与MM细胞增殖相关。二甲双胍是传统双胍类降糖药，被认为是能量调控信号分子AMPK激动剂。最新研究表明，其能通过抑制PI3K/Akt/mTOR通路发挥抗实体瘤作用。我们前期研究发现二甲双胍能通过PI3K信号途径抑制MM细胞增殖并诱导细胞凋亡,同时抑制AMPK，而敲除AMPK的MM细胞可出现PI3K途径中Akt分子表达上调。因此，可能存在其他关键性信号分子参与AMPK信号通路，或AMPK通路与PI3K通路可能直接存在未知的关联。本课题拟采用分子生物学等技术，在体内外研究二甲双胍抑制MM细胞增殖并诱导凋亡的细胞内信号途径，深入探讨PI3K/Akt/mTOR和AMPK/mTOR途径的调控机制,为MM靶向治疗策略提供新的思路。

本项目体外研究发现二甲双胍能够显著激活PARP-1，caspase 3等促凋亡相关蛋白的表达，同时抑制Mcl-1，HIAP-1等抗凋亡蛋白的表达，进而诱导多发性骨髓瘤细胞（MM）凋亡，并使MM细胞周期阻滞于G1期，检测发现cyclin D1、CDK4、CDK6下调，p21、p27上调。进一步机制研究发现，二甲双胍能够抑制IGF-IR及其下游PI3K的表达，抑制AKT、mTOR的磷酸化，从而抑制下游4E-BP1及p70S6K的激活；加用重组人IGF-I可以减弱二甲双胍对MM细胞的抑制作用，伴随下游信号通路蛋白的重新激活，加用PI3K抑制剂LY294002则出现相反的结果。同时，研究还发现二甲双胍与地塞米松存在协同作用。小鼠体内实验也证实了以上结果和机制。本研究在能量代谢方面对二甲双胍抑制MM细胞增殖，诱导细胞凋亡的机制做了更深入的研究。通过高通量RNA-SEQ筛选二甲双胍相关的差异基因，结果发现二甲双胍可以显著降低酵解途径中重要限速酶PFKFB3基因的表达，进一步研究发现PFKFB3基因可以促进MM细胞的增殖，PFKFB3的抑制剂PFK15可以抑制PFKFB3/MAPKs/stat1信号通路诱导MM细胞凋亡。本项目阐明了二甲双胍诱导MM细胞凋亡及调控细胞周期的分子机制，为二甲双胍联合化疗治疗多发性骨髓瘤的临床治疗研究提供了新的思路和理论依据。