miR-603在人参皂苷Rg3联合三氧化二砷治疗APL中的作用及机制研究

In addition to PML-RARα fusion gene, there are other genetic and epigenetic changes involved in the occurrence and progression of acute promyelocytic leukemia (APL). Our previous data showed that the expression of microRNA-603 (miR-603) was significantly lower in relapsed /resistant APL patients. Inhibition of miR-603 resulted in the blockage of APL cell differentiation and reduced the effect of ATO treatment. Ginsenoside Rg3 combined with arsenic trioxide (ATO) increased the expression of miR-603 and inhibited the proliferation of APL cells. We propose the following hypothesis: promoter methylation leads to miR-603 expression decrease which may participate in the relapse/resistance of APL. Ginsenoside Rg3 combined with ATO can increase the expression of microRNA-603 by decreasing promoter methylation, thus inhibiting its target NTRK2 and downstream signaling pathways, and promoting APL cell differentiation and apoptosis. Based on the preliminary experiments, this study intends to explore the role of miR-603 in the development of APL and its related molecular mechanisms through cell transfection and immunoprecipitation techniques in cell and animal models, and explore the molecular mechanism and regulatory network of ginsenoside Rg3 combined with ATO in the treatment of APL, so as to provide theoretical basis for the precise targeting of APL.

除了PML-RARα等融合基因之外，仍存在其他的遗传学改变和表观遗传学修饰参与急性早幼粒细胞白血病（APL）的发生和进展。我们的前期数据发现miR-603在复发耐药患者中显著低表达，抑制miR-603导致APL细胞分化受阻，降低ATO治疗的效果，人参皂苷Rg3联合三氧化二砷（ATO）升高miR-603表达，显著抑制APL细胞增殖。我们提出如下假说：miR-603启动子甲基化使表达减低参与导致APL复发耐药，人参皂苷Rg3联合ATO通过降低启动子甲基化升高miR-603表达，从而抑制其靶点NTRK2及下游信号通路，促进APL细胞分化及凋亡。在预实验的基础上，本研究拟在细胞和动物模型中通过细胞转染、免疫沉淀技术等实验方法，深入探讨miR-603在APL发生及发展中的作用及相关分子机制，探索人参皂苷Rg3联合ATO治疗APL的分子机制和调控网络，为实现APL的精准靶向联合治疗提供理论依据。

急性早幼粒细胞复发耐药影响预后的主要原因，而发病机制及治疗尚未阐明。除PML-RARa融合基因外，其他的表观遗传学变化受到越来越多的重视。我们通过芯片分析，发现miR-603在复发耐药患者中显著低表达，其靶基因NTRK2在患者中显著高表达。我们猜测miR-603可能由于启动子甲基化导致其低表达，其靶基因高表达，参与APL发生及发展。我们首先选择初发和复发耐药APL患者中完成miR芯片，通过生物信息学分析和PCR，Western blot等实验验证，miR-603在复发的患者中显著低表达，而它的靶向基因NTRK2却显著高表达，两者的表达呈负相关，通过双荧光素酶实验和PCR等实验证实了两者的靶向关系，通过MTT，流式细胞仪检测细胞周期，凋亡和分化等实验证实，mi-603通过NTRK2参与了APL细胞系NB4细胞的细胞周期，分化，凋亡等通路。研究结果显示miR-603低表达可能与APL复发耐药及预后不良相关，miR-603主要通过下游靶点NTRK2调控其下游信号通路，导致APL细胞分化受阻，凋亡受抑。该项研究证实了miR-603在APL中预测预后的临床意义和分子机制和下游的信号通路，为复发耐药的APL的靶向治疗提供了实验依据。