mTOR/c-Myc信号轴的O-GlcNAc糖基化修饰对儿童急性B淋巴细胞白血病耐药的调控机制研究

ALL is the most common type of malignant disease among children. About 20%-30% cases died from relapspe or drug resistance of ALL. As a hot topic in post transcriptional regulation in recent years, O-GlcNAc glycosylation modification was found in a large number of tumor associated proteins. It was known that PI3K/Akt pathway play a key role in the process of drug resistance in leukemia, and mutual regulation was found between PI3K/Akt pathway and O-GlcNAc glycosylation, however, the role of O-GlcNAcylation of PI3K/Akt pathway and mTOR/c-Myc Axis in drug resistance of B-ALL has never been reported. We found that the level of O- GlcNAcylation and the expression of OGT increased in drug-resistant B-ALL cell line nalm-6, and confirmed that O- GlcNAcylation participate in the process of drug resistance of B-ALL. We speculated that the O-GlcNAcylation of c-MYC in PI3K/Akt pathway of B-ALL cells paticipated in the regulation of resistance-associated gene. To confirm the hypothesis, we will detect O- GlcNAcylation of mTOR and c-MYC axis in clinical B-ALL B lymphocytes, observe the key molecules in O- GlcNAcylation of mTOR/c-MYC axis, and the regulation mechanism of resistance-associated gene using a variety of molecular biologic techniques. This project will clarify the mechanism of B-ALL multidrug resistance from a new perspective and provide a new target for the treatment of B-ALL.

急性淋巴细胞白血病（ALL）是儿童最常见的恶性疾病，20%-30%的患儿因耐药或复发而死亡。O-GlcNAc糖基化是近年转录后调控的研究热点，大量与肿瘤相关的蛋白具有O-GlcNAc糖基化修饰。已知PI3K/Akt通路的异常在白血病耐药过程中起关键作用，PI3K/Akt通路和O-GlcNAc糖基化之间存在相互调控关系，而PI3K/Akt通路及其下游的mTOR/c-Myc轴的O-GlcNac糖基化修饰是否参与儿童B-ALL的耐药的发生，目前尚无研究。我们已发现耐药型BALL细胞株Nalm-6的O-GlcNac水平增高，说明白血病细胞耐药与O-GlcNac糖基化相关。我们将分析儿童白血病细胞的糖代谢状况及c-Myc蛋白的O-GlcNac修饰状态，寻找耐药细胞株中调控mTOR/c-Myc信号轴O-GlcNac修饰的关键分子并研究其对耐药相关基因的调控机制，为儿童B-ALL的治疗提供新靶点。

急性淋巴细胞白血病（ALL）是儿童最常见的恶性疾病，20%-30%的患儿因耐药或复发而死亡。O-GlcNAc糖基化是近年转录后调控的研究热点，大量与肿瘤相关的蛋白具有O-GlcNAc糖基化修饰。本课题收集了232例初诊ALL患儿的骨髓标本，密度梯度离心法获得单个核细胞并冻存，详细记录了每个患儿的临床资料、治疗效果及随访信息。这为今后继续进行儿童白血病的相关研究提供了丰富的临床标本。本课题以B-ALL细胞株Nalm6细胞为对象，构建了三种化疗药物的耐药细胞株，为今后进行白血病耐药机制研究提供了细胞模型。我们检测了白血病细胞中O-GlcNAc糖基化及糖代谢相关蛋白的表达水平，观察了OGT上调或下调对白血病细胞的耐药、细胞周期、细胞增殖、细胞凋亡、耐药基因表达水平的影响，筛选了O-GlcNAc糖基化在B-ALL细胞耐药中可能的关键分子和信号通路，还分析了ALL患儿化疗和/或合并放疗引起的中枢神经系统改变和认知功能损伤以及这两者之间的关系。结果发现，在B-ALL患儿的CD19+BM-MNC中存在过度的O-GlcNAc糖基化修饰以及关键催化酶OGT表达的升高。下调OGT表达，降低Nalm6白血病细胞的O-GlcNAc糖基化水平，能够降低细胞增殖，增加细胞凋亡，在耐药细胞Nalm6/VN中下调OGT表达，可以部分地逆转其耐药性，增加其对化疗药物的敏感性，而以上作用可能是通过降低PI3K/Akt/c-Myc通路中重要信号蛋白的磷酸化水平实现的。ALL患儿在治疗后可出现多种神经认知障碍，其中智力、记忆力和注意力受到的影响最大。这些认知障碍与其中枢神经系统的结构和功能改变有关。结果具有创新性和较大的科研价值，为今后进行儿童白血病耐药机制的研究做了基础性的工作，为寻找新的治疗靶点和精准治疗提供了思路。

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