IRAIN-DNMT3A正反馈回路影响急性髓细胞白血病发生发展的分子机制及临床意义

The discovery of long non-coding RNAs sheds a new light on further exploration of molecular mechanism for carcinogenesis, however, their role in the leukemogenesis for acute myeloid leukemia (AML) remains nearly unknown. A significant decreased expression of long non-coding RNA IRAIN was found in AML, but its resultant pathophysiological effects and its underlying mechanism need to be further explored. Our previous findings showed that IRAIN inhibited the growth of AML cells, and its inhibitory effects were supposed to be triggered by inhibition of IGF-1R. And then, increased expression of DNA methyltransferase DNMT3A was positively correlated with IRAIN promoter hypermethylation, suggesting that DNMT3A may be the underlying basis for the inactivation of IRAIN in AML. Furthermore, the inactivation of IRAIN may promote a higher expression of DNMT3A by enhancement of MDM2 expression. Based on the above data, we propose that a positive feedback circuitry exists between IRAIN and DNMT3A, which plays an important role in the leukemogenesis of AML. In the present study, the above hypothesis will be further investigated through multiple analysis like bisulfite sequencing and RNA interference assay, and then animal models and patients’ samples will be utilized to testify the impact of the dysregulation of IRAIN-DNMT3A circuitry on AML development. In summary, the results of the research will provide potential molecular targets for the diagnosis and therapy of AML.

长链非编码RNA的发现为深入揭示肿瘤发生的分子机制提供了全新的维度，但其影响急性髓系白血病（AML）发生发展的效应仍有待深入。长链非编码RNA IRAIN在AML中表达明显下降，但其失活的原因及其效应仍不清楚。前期研究我们发现IRAIN抑制AML细胞生长，与其抑制IGF-1R有关；而且DNA甲基转移酶DNMT3A在AML中表达升高且与IRAIN甲基化明显正相关，这提示DNMT3A可能是IRAIN失活的重要因子之一；同样重要的是，IRAIN失活后又可能通过促进MDM2表达进而上调DNMT3A的表达。由此我们推测在IRAIN与DNMT3A之间存在一条正反馈回路，该回路对AML的发生发展发挥了重要调控作用。本课题将进一步应用DNA甲基化测序及RNA干扰等手段验证该假设，并应用动物模型和临床标本验证该回路在体内对AML发生的影响，最终有望丰富AML发生的机制，并为AML诊治提供潜在的分子新靶标。

急性髓细胞白血病（AML）是成年人最常见的急性白血病，预后差，长期存活率仅5-16%。应用高剂量阿糖胞苷巩固化疗或者干细胞移植可延长患者生存期；然而，尽管有后续的巩固治疗，AML的预后依然很差。目前为止AML发病的分子机制仍然不清楚，因此迫切的需要对AML的分子机制进行深入研究，以期获得它更为细分和特异的生物标志或治疗新靶标。DNA异常甲基化是促进AML发生发展重要机制，但造成其异常的原因并不清楚。DNA甲基转移酶3A（DNMT3A）对正常发育、细胞分化（如造血干细胞的分化）、基因组印记、x染色体失活、基因调控至关重要。AML的发生常与抑癌基因的启动子区高甲基化有关，研究发现在多数恶性骨髓细胞和AML细胞中DNMT3A的表达明显增高，而长链非编码RNA IRAIN的启动子区富含CpG岛，且在AML中存在着不同程度的失活，这提示DNMT3A可能通过影响IRAIN的甲基化而调控IRAIN的表达。本项目主要研究了：1.在AML临床样本及AML细胞系中检测IRAIN及DNMT3A的表达量。2.DNMT3A对IRAIN表达的调控及AML疾病发生的影响。3.IRAIN对AML细胞生物学行为的影响。4.AML中IRAIN发挥生物学功能的分子机制。利用高通量测序分析、RT-qPCR、western blot、流式细胞术等技术发现：DNMT3A对AML的发生发展起到重要调节作用，DNMT3A通过抑制IRAIN的表达而促进AML的发生。另外对转录组数据深入分析发现，在AML中，IRAIN通过抑制NF-κB通路可以下调CD44的表达，从而抑制AML细胞的增殖，促进细胞凋亡，使细胞周期停滞在G0/G1期。该部分数据已基本完成，这些均为治疗AML提供了新的理论基础。