建立新的细胞和动物耐药模型探讨“Myc-miR328-ABC转运蛋白”调控通路在CML耐药中的作用

Primary or secondary Imatinib (IM) resistance emerges as the big hurdle of Chronic myeloid leukemia (CML) therapy. However, how these cells survive before their mutation is largely unkown. We have established both cell and mouse models of imatinib resistance, and revealed that the existence of CML stem cell (innate IM resistance) and imatinib induced resistant CML cells (IM acquried resistance) are the main reason for IM resistance. Further studies showed that miR328 is downregulated in both CML stem cell and imatinib induced resistant CML cells. Bioinformatic study reveal that these miRNAs might regulate the many ABC transporter family members. These findings suggest that both CML stem cell and imatinib induced resistant CML cells might regulate the IM resistance through the commonmiRNA-ABC transporter pathway, which thus make the cell survive upon lethal dose of imatinib and thus become possible for the subsequent mutation. Targeting this pathway might be a therapeutic strategy for overcoming drug resistance. The proposed project here aims to clarify the role of miR328 in drug resistance based on our mouse and cell model, and clinical samples. Then we will unravel the underlying mechanims how miR328 is deregulated and how it in turn regulates ABC transporter pathway by the traditional ChIP, EMSA, RNA-IP, reporter assay for transcriptional and posttranscriptional regulation. Our project would possibly find a common pathway for both the innate and acquired IM resistance, and thus shedding light on the CML therapy.

原发或继发性伊马替尼(IM)耐药是慢性髓系白血病（CML）治疗迫切需要解决的关键问题，但细胞在基因突变前如何免于IM杀伤的机制尚不清楚。我们前期发现低剂量IM处理诱导产生的耐药细胞与CML固有干细胞 miR328表达均降低，且多个ABC转运蛋白是miR328的潜在靶分子；提示两种耐药细胞均通过miR328协同调控多个耐药相关基因。综上，本课题提出IM耐药的“量变-质变”模型（即部分细胞因miR328表达降低和ABC转运蛋白表达增加，进而逃脱原本致死剂量的IM，为后续的基因突变提供充足时间）。为阻断该途径，我们构建CML耐药的细胞和动物模型，并且结合临床样本，通过研究CML干细胞和耐药细胞中表达增加的Myc转录抑制miR328的表达和miR328转录后抑制ABC转运蛋白的作用机制，旨在阐明“Myc-miR328-ABC转运蛋白”调控通路在CML发生 IM耐药的作用机制，为CML治疗提供新策略

本研究综合运用体外细胞培养和基因干预的体外模型，系统观察了miR-328与ABCG2在慢性髓系白血病伊马替尼耐药中的作用及机制；取得以下发现：1）IM浓度梯度处理K562细胞可成功建立不同耐药浓度的K562细胞系，且耐药细胞系BCR-ABL基因突变概率显著高于内参基因；2）二代测序检测比较K562细胞及其耐药系中miRNA表达差异，结果显示两者之间存在大量差异miRNAs，耐药细胞系中miR-328的表达水平明显降低。K562细胞中干涉miR328可以降低细胞对IM的敏感性，在K562耐药细胞系中过表达miR-328增加细胞对IM的敏感性；3）耐药细胞系中，ABCG2在蛋白水平显著升高，而在mRNA水平差异不明显。干涉miR-328后耐药细胞系中ABCG2表达明显升高，过表达miR-328后ABCG2表达显著降低。进一步双荧光素酶报告基因实验显示ABCG2受miR-328调控；4）耐药细胞中的miR328在溶酶体中发生选择性降解，而溶酶体的活性和数量均未发生明显变化；通过氯喹抑制溶酶体功能可以升高耐药细胞中的miR328表达水平，并提高K562对IM的敏感性；5）递送碱化外泌体来可以治疗性阻断miR328在溶酶体中的降解，并且装载miR328的碱化外泌体可以增强耐药细胞对IM的敏感性；6）在项目实施过程中发现Clathrin蛋白在细胞摄入外泌体中起到至关重要的作用。注射Clathrin siRNA 外泌体可阻断单核吞噬系统对外泌体的吞噬，进而提高治疗性外泌体在血液循环中的驻留时间。综上所述，本研究深入探讨了CML发生IM耐药的作用机制，为CML的治疗提供了新的治疗策略。