CASP8AP2与ARS2相互作用参与microRNA-210生成及其在儿童急性淋巴细胞白血病复发与耐药中的作用

Relapse and drug resistance are the main obstacles to the improvement of long-term disease-free survival in childhood ALL. Our previous studies found that patients in low expression groups of CASP8AP2 or miR-210 were subject to relapse. Several studies demonstrated that interaction of CASP8AP2 with ARS2 was necessary for S phase progression; meanwhile, ARS2 was involved in miRNAs biogenesis, and depletion of ARS2 reduced the level of miR-210. We propose that interaction of CASP8AP2 with ARS2 is involved in biogenesis of miR-210, and low expression of CASP8AP2 results in decrease of miR-210 level, which leads to relapse and drug resistance in ALL. This study will verify the role of interaction of CASP8AP2 with ARS2 in biogenesis of miR-210 in ALL cells by overexpression of CASP8AP2 mutant,in which the interacting domain with ARS2 is deleted, or by silencing of ARS2. Furthermore, it will be proved that low level of miR-210 is related with relapse and drug resistance as apoptosis is reduced in above ALL cells after treatment with chemotherapy drugs. This study innovatively demonstrates that CASP8AP2 is involved in miRNA biogenesis, which plays an important role in relapse and drug resistance of ALL. It will help to clarify the mechanism of relapse and drug resistance associated with low expression of CASP8AP2 and to provide new therapeutic targets in childhood ALL.

儿童ALL复发与耐药是提高患儿长期无病生存率的主要障碍。我们的前期研究发现CASP8AP2与miR-210低表达均与复发密切相关。已知CASP8AP2与ARS2的相互作用是细胞周期S期进展所必需；ARS2参与miRNA的生成，其沉默导致miR-210水平明显下降。我们推测CASP8AP2与ARS2相互作用参与miR-210生成，CASP8AP2低表达导致miR-210生成减少，造成白血病复发及耐药。拟通过高表达CASP8AP2突变体（缺失与ARS2相互作用的功能域）或沉默ARS2表达，证明CASP8AP2与ARS2相互作用参与miR-210生成；上述细胞经化疗药物处理后凋亡减少，证明miR-210水平与复发、耐药相关。创新点是证明CASP8AP2参与miRNA的生成，从而在ALL复发与耐药中发挥重要作用，有助于阐明儿童ALL中CASP8AP2低表达导致复发与耐药的机制，并提供新的治疗靶点。

复发与耐药是提高儿童急性淋巴细胞白血病（ALL）长期无病生存率的主要障碍。本研究目的是揭示CASP8AP2与ARS2相互作用在miR-210生成中的作用，明确miR-210表达水平差异与对化疗药物敏感性的关系，阐明CASP8AP2低表达导致白血病耐药和复发的机制。首先采用qRT-PCR方法检测了204例儿童ALL初诊时骨髓样本中的ARS2表达水平，证明初诊时ARS2低表达是儿童ALL的独立预后因素，联合分析ARS2与CASP8AP2两个基因的表达水平能更准确地判断预后、预测复发。我们采用干扰慢病毒分别感染Reh、Jurkat细胞以下调ARS2的表达后，pri-miR-210、pre-miR-210和miR-210成熟体均明显下调，证明ARS2调控miR-210生成。在Reh、Jurkat细胞中诱导CASP8AP2表达下调后，miR-210成熟体和pre-miR-210的表达均明显降低，而pri-miR-210的表达轻度升高；与过表达野生型WT-CASP8AP2相比，过表达△FARB-CASP8AP2（缺失与ARS2相互作用的功能域）的293T细胞中，△FARB-CASP8AP2与ARS2的相互作用明显减少，miR-210成熟体表达水平明显降低，而pri-miR-210的表达水平明显升高，提示CASP8AP2与ARS2的相互作用通过影响pri-miR-210的加工进而调控miR-210的表达水平。下调ARS2或CASP8AP2表达后，Reh、Jurkat细胞的增殖受到明显抑制，细胞在柔红霉素（DNR）作用后的存活率明显提高，IC50值显著增高。在293T细胞中，过表达△FARB-CASP8AP2的细胞在DNR作用后的存活率明显高于过表达WT-CASP8AP者，提示CASP8AP2与ARS2相互作用是白血病细胞耐药的重要机制。应用miR-210特异性的激动剂、拮抗剂转染Reh、RS4;11细胞株，再经不同浓度的化疗药物作用，上调或下调miR-210的表达水平能显著影响细胞对化疗药物的敏感性。最后通过芯片检测下调miR-210表达Reh细胞中基因表达谱的变化，以筛选靶基因。本研究创新性地证明了CASP8AP2参与调控miRNA生物合成的功能，并在ALL复发与耐药中发挥作用，有望为白血病治疗提供新靶点，开辟新的治疗途径，最终提高难治性及复发儿童白血病的疗效。