3-BrPA通过自噬诱导MCT1表达异常影响MYCN扩增神经母细胞瘤代谢的分子机制研究

Malignant tumors maintain rapid cell proliferation by reprogramming of energy metabolism. The metabolism way of MYCN-amplified neuroblastoma is mainly aerobic glycolysis, accompanied by glutamine metabolism, lipid metabolism and other processes, and autophagy can affect the glycolysis process of the malignant tumors. In our preliminary experiment, we found that the autophagy process was activated in the MYCN-amplified neuroblastoma, and the glucose metabolism inhibitor 3-BrPA could inhibit autophagy in NB cells and affect the MCT1 expression. To further investigate the effect of autophagy in the MYCN-amplified neuroblastoma, we hypothesize that 3-BrPA inhibits autophagy in the MYCN-amplified neuroblastoma by inhibiting the Wnt/β-catenin pathway and inhibiting the expression of MCT1, then the metabolism of NB cells collapses. To test this hypothesis, we will collect the neuroblastoma tumor tissues and cells with different MYCN amplification levels, detect the autophagy activity in the neuroblastoma cells by immunofluorescence, electron microscopy and immunohistochemistry, and analyze the metablism of MCT1 silencing NB cells by GC-MS and gene chip. On the other hand, after the NB cells were treated with 3-BrPA, the autophagic activity in the NB cells and the protein in the Wnt/β-catenin pathway will be examined to clarify the molecular mechanism of the inhibition of MCT1 expression. Thus, this study will start from the effect of autophagy in the MYCN-amplified neuroblastoma metabolism, and provide new ideas and theoretical basis for MYCN-amplified neuroblastoma treatment by the glucose metabolism inhibitor 3-BrPA.

恶性肿瘤通过代谢重编程维持细胞快速增殖，MYCN扩增NB以有氧糖酵解为主，谷氨酰胺代谢、脂质代谢等过程交互影响，且自噬能影响恶性肿瘤糖酵解。我们预实验发现MYCN扩增NB内自噬激活，3-BrPA抑制其自噬，影响MCT1表达。为深入探讨自噬在MYCN扩增NB代谢中的影响，我们提出假说：3-BrPA抑制MYCN扩增NB自噬，影响Wnt/β-catenin抑制MCT1表达，造成NB细胞代谢崩塌。为验证该假说，我们收集MYCN扩增不同的NB肿瘤组织和细胞，通过免疫荧光、电镜、免疫组化等方法，明确细胞内的自噬活性，并采用GC-MS、基因芯片，分析MCT1沉默对NB细胞代谢的影响。另一方面，3-BrPA处理NB细胞后，检测细胞内的自噬活性及Wnt/β-catenin通路中的蛋白，明确MCT1抑制的分子机制。本研究将从自噬影响MYCN扩增NB代谢着手，为3-BrPA用于治疗MYCN扩增NB提供新依据。

研究背景:细胞代谢的改变，如增强的有氧糖酵解，已被确定为癌细胞的显著特征。3-溴丙酮酸(3-BrPA)是一种己糖激酶(HK)-II抑制剂，可抑制癌细胞能量代谢。雷帕霉素是一种新型的大环内酯，能抑制丝氨酸/苏氨酸蛋白激酶mTOR。为了了解3-BrPA对体外自噬活性的影响，我们用不同的人神经母细胞瘤(NB)细胞系进行了一系列实验。.研究内容:用3-BrPA和/或雷帕霉素处理人NB细胞株，用CCK-8法检测细胞增殖活性。采用实时定量聚合酶链反应(QPCR)分析3-BrPA和/或雷帕霉素处理后细胞mRNA的表达情况。Western Blotting (WB)法检测细胞蛋白表达。流式细胞术检测3-BrPA和/或雷帕霉素对细胞周期和细胞凋亡的影响。同时，通过相关代谢分析试剂盒分析细胞葡萄糖吸收率、乳酸分泌率和ATP含量。.结果:我们的研究结果表明，3-BrPA可诱导细胞凋亡，并以剂量依赖性的方式抑制生长。3-BrPA联合雷帕霉素对NB细胞起协同抑制作用，影响细胞凋亡、细胞周期及代谢途径。在与3-BrPA和雷帕霉素孵育的NB细胞中，上调的LC3-II积累是有意识的。雷帕霉素单独抑制mTOR信号通路，而与3-BrPA联合可增强这种现象，影响NB细胞代谢。.结论:3-BrPA联合雷帕霉素可通过抑制mTOR活性诱导NB细胞凋亡。综上所述，我们的研究提示mTOR信号通路和糖酵解活性的双重抑制作用可能为NB的化学预防提供一种有效的治疗策略。