白血病细胞系TKI耐药的miRNA机制及与PI3K/Akt/FOXO/Bcl6信号通路的关系

It is important for us to explore the mechanisms of resistance to tyrosine kinase inhibitor (TKI) in the treatment of Ph chromosome positive leukemia. Moreover, resistance-associated epigenetic research and signaling pathways are currently hotspot fields. Combined the understanding of PI3K/Akt/FOXO/Bcl6 signaling pathway in the leukemia and drug resistance with our previous work which was Bcl6 expression and common chemotherapy treatment negatively correlated, this suggest us that Bcl6-related signaling pathway is involved in the occurrence of drug resistance. This study focus on TKI resistance theme in CML and Ph chromosome-positive ALL, explore the differences in miRNA expression profiling in sensitive and resistant cell lines, identify miRNA which are involved in PI3K/Akt/FOXO/Bcl6 signaling pathway and miRNA regulated target genes. Fianlly, further identification of target genes and the effect of miRNA in the regulation of signaling pathway and invovling the drug resistance. This project contribute to TKI resistance mechanisms exploration and provide a new strategy in the treatment of drug resistance, so as to achieve the ideal treatment for CML and Ph chromosome-positive ALL.

酪氨酸激酶抑制剂（TKI）在Ph染色体阳性的白血病治疗中的耐药机制的认识有重要意义，并且耐药相关的表观遗传学及信号通路的研究是目前的热点。结合目前国际上对PI3K/Akt/FOXO/Bcl6信号通路在白血病发生耐药中的最新认识，我们在前期工作中发现Bcl6高表达与常规化疗的治疗效果负相关，提示我们Bcl6相关信号通路是否参与耐药的发生。本研究围绕TKI耐药的主题，探讨CML和Ph染色体阳性ALL细胞中敏感株和耐药株中miRNA表达谱的差别，筛选出耐药株共表达的参与PI3K/Akt/FOXO/Bcl6信号通路的miRNA及miRNA调控靶基因，并进一步鉴定靶基因及miRNA通过调控信号通路在参与耐药的作用。本课题有助于深入TKI耐药发病机制的研究，并为其治疗提供新的策略，从而为实现干预达到对CML和Ph染色体阳性ALL 的理想治疗。

为探讨酪氨酸激酶抑制剂（TKI）在慢性粒细胞白血病（CML）的表观遗传学及分子学耐药机制，本实验通过分析CML细胞系中敏感株K562和耐药株K562/G中miRNA表达谱的差别，及相关靶基因分析提示PI3K/Akt/FOXO/Bcl6信号通路参与耐药发生。结果显示：miRNA10a3p,30a5p,1185-2-3p,186-5p为人Bcl6基因相关且为芯片上表达差异大的miRNA，并已行qPCR验证。Western Blot 法分析耐药株和敏感株比较FOXO和Bcl6表达上调，用定量Realtime-PCR检测显示FOXO从mRNA 水平存在表达差异。建立FOXO的慢病毒过表达载体，验证转染后蛋白水平检测候选信号通路靶基因及其编码蛋白在转染前后表达差异。用MTT 法检测细胞转染前后的IC50影响。FOXO/Bcl6信号通路与白血病TKI耐药有相关性，其蛋白及miRNA水平检测有望成为预测和治疗耐药的指标。