环状RNA hsa_circ_0012152作为儿童AML分子标志物及其功能验证

Recent studies reveal that circular RNAs (circRNAs) in mammalian cells are abundant and stable and play important roles in regulating different aspects of cellular processes. The circRNA profile changes in disease conditions support that circRNAs could be used as molecular biomarkers. However, circRNAs and related functions in leukemia remain largely unknown. In our preliminary study, we found abnormal circRNA profile in bone marrow samples of pediatric acute myeloid leukemia (AML) patients. In these profiles, hsa_circ_0012152 was one of the most significantly elevated (> 100 times than normal controls), and such an upregulation is consistent in the analyzed samples. Our further study demonstrated that silencing endogenous hsa_circ_0012152 by shRNA inhibits leukemia cell proliferation. We, therefore, hypothesize that hsa_circ_0012152 plays important role in leukemogenesis by promoting proliferation and inhibiting differentiation and apoptosis, and the increase of hsa_circ_0012152 is associated with clinicopathological/characteristics of pediatric AML. Objectives of the proposed study are to uncover the role of hsa_circ_0012152 in pediatric AML through two aims: 1) to determine the relationship between hsa_circ_0012152 with clinicopathological characteristics of pediatric AML and 2) to investigate the roles of hsa-circ-0012152 in the proliferation, apoptosis and leukemogenesis of leukemia cells. The results of this study will establish not only the importance of hsa_circ_0012152 in pediatric AML but also its potential as diagnosis/prognosis molecular biomarker and therapeutic target.

最新研究揭示环状RNA在细胞中广泛表达，其表达丰度高、比线性更稳定的特征有可能使它成为更理想的疾病分子标志物。而环状RNA在白血病中罕见报道。我们前期筛选出一批儿童急性髓细胞白血病（AML）异常表达的环状RNA，其中hsa_circ_0012152增高100多倍。那么hsa_circ_0012152能否作为诊断和预后分子标志物？进一步实验显示：干扰其表达可抑制白血病细胞增殖。由此提出假设：异常高表达的hsa_circ_0012152与儿童AML诊断及预后相关，导致细胞恶性增殖、凋亡和分化受阻，促进AML发生。本项目拟采用定量PCR、流式细胞术及SCID小鼠成瘤等方法以及慢病毒载体和siRNA等工具，阐明hsa_circ_0012152与儿童AML临床特征及预后的相关性及其在AML发生发展中的功能，为hsa_circ_0012152作为儿童AML分子标志物和治疗新靶点提供更充分的科学依据。

环状RNA (circRNAs)是一类新型的内源性RNA分子，可形成共价闭合的连续环，无5’帽子或3’尾巴，近年来被认为是潜在的生物标志物和癌症治疗靶点。迄今为止，circRNAs在儿童AML中的表达模式和作用尚不清楚。本研究前期筛选儿童AML相关的环状RNA（hsa_circ_0012152）。在本项目中，我们通过定量PCR技术检测306例不同血液系统疾病（包括健康对照5例，初诊AML 85例，缓解AML 82例，初诊ALL 86例，初诊ITP 26例，初诊MDS 3例，以及包括CML和JMML等其他共19例）中hsa_circ_0012152的表达，并研究了它的生物学特性。通过短发夹RNA介导的干扰小分子抑制hsa_circ_0012152后证实了其功能。RNA测序分析了靶向hsa_circ_0012152后基因表达谱的变化。结果显示：hsa_circ_0012152仅在儿童AML中特异高表达，使用hsa_circ_0012152独立诊断AML与正常对照时，AUC=0.9506,其敏感性为94.12%，特异性为100%;当它与其他血液系统疾病(包括ALL, ITP, MDS, CML及JMML等)相鉴别时候，AUC=0.9073，灵敏度90.59%，特异度85.5%。它大部分定位在核内，能耐受RNaseR，半衰期超过48h；功能实验表明hsa_circ_0012152具有多效性，靶向hsa_circ_0012152能抑制AML细胞恶性增值，促进髓细胞分化和凋亡。在AML骨髓细胞中干扰hsa_circ_0012152后的基因表达谱结果显示，差异表达的基因多富集在泛素化降解及肿瘤相关通路。本研究揭示了AML来源的hsa_circ_0012152在髓系白血病的发生发展中发挥了重要作用，它有望成为诊断儿童AML的一个可靠的生物标志物。