LKB1/AMPK调控有氧酵解信号途径诱导结肠癌细胞凋亡机制的研究

It is a hotspot in research to design new targeting agent as a strategy by exploiting aerobic glycolysis of cancer cell, one of the hallmarks for cancer in recent years. Our previous studies found that tumor suppressor LKB1 regulates the process of aerobic glycolysis of monocytic leukemia cells and colon cancer cells and induces apoptosis by activating its downstream protein AMP activated protein kinase(AMPK),but the mechanisms are elusive. It is reported that microRNA-415 regulates LKB1/AMPK signaling in glioma cells but it is still unknown whether and how microRNAs involved in regulating LKB1/AMPK signaling in colon cancer cells. In addition, our study found a significant suppression for Bcl-2 expression after intervening the aerobic glycolysis of colon cancer cells. Based on the above studies, we here propose a new signaling pathway initiating from microRNA molecules and LKB1 to control the aerobic glycolysis and apoptosis in colon cancer. In order to illuminate the mechanisms of how LKB1 regulates the aerobic glycolytic signaling pathways and pave a way for targeted therapy, we will try to identify the candidate microRNA molecules which involved in regulating LKB1 in aerobic glycolysis by microarray and study the apoptotic mechanisms in colon cancer.

利用肿瘤有氧酵解这一代谢特征设计靶向性治疗药物，是近年的研究热点。本课题组用柠檬酸钠干预包括单核细胞白血病、结肠癌细胞的有氧酵解时发现：LKB1可激活AMPK信号分子，调控肿瘤细胞的有氧酵解及凋亡。但在结肠癌细胞中LKB1激活AMPK及LKB1本身被调控的机制尚不清楚。研究表明，MicroRNA451调控胶质瘤细胞的LKB1/AMPK通路，但MicroRNAs分子如何参与结肠癌细胞的LKB1/AMPK信号的传导尚未见报道。课题组还发现，结肠癌细胞的有氧酵解被干预后，Bcl-2的表达受到明显抑制。我们设想：结肠癌细胞可能存在着一条始自MicroRNA及LKB1控制有氧酵解的信号通路。为此，本课题拟以Microarray筛选参与有氧酵解的MicroRNA分子，通过Western blot，IP及体内外实验，试图阐明LKB1调控有氧酵解的机制，为利用肿瘤的代谢特点干预治疗奠定理论基础。

项目背景：有氧糖酵解即Warburg效应为肿瘤细胞获取能量的主要方式，利用肿瘤细胞不同于正常细胞获取能量的特点设计特异性抗癌药物引起人们的广泛兴趣。本课题组前期研究发现柠檬酸钠、二氯乙酸能够通过激活AMPK而干预肿瘤细胞的糖酵解。研究表明miR-451调控胶质瘤细胞的LKB1/AMPK通路，但miR如何参与结肠癌细胞的LKB1/AMPK信号的传导尚未见报道。我们设想，在结肠癌细胞中有可能存在着一条始自miR及LKB1控制有氧酵解和凋亡的信号通路。本课题拟阐明LKB1调控有氧酵解的机制，为利用肿瘤的代谢特点干预治疗奠定理论基础。主要研究内容与重要结果：寻找并筛选与结肠癌细胞有氧酵解关系密切的候选miR分子：利用microarray技术筛选结肠癌细胞在加入二氯乙酸处理后显著变化的MicroRNA分子，结合生物信息学预测和Q-PCR技术筛选与AMPK上游激活因子（LKB1/CAB39/STRAD）相关的miR。发现miR-107与miR-543可能与结肠癌细胞有氧酵解密切相关；通过mimic/inhibitor转染技术研究所筛选的miR分子与LKB1/CAB39/STRAD分子之间的靶向关系，以探讨控制结肠癌肿瘤细胞糖酵解的上游信号途径及机制。发现miR-107和miR-543能够直接调控CAB39分子从而改变AMPK信号通路的活化情况；通过siRNA沉默，免疫印迹技术等研究miR-CAB39-AMPK磷酸化激活后如何调控下游mTOR信号分子并最终诱导了结肠癌细胞的凋亡。科学意义：本课题有望发现调控结肠癌细胞有氧酵解代谢的miR分子，有助于阐明肿瘤细胞有氧酵解代谢的机制；本课题的实施有可能发现二氯乙酸这一小分子药物作为化疗辅助用药诱导结肠癌细胞凋亡的可能，为临床结肠癌的化疗提供新的治疗方案。

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