HOTAIRM1通过ceRNA模式调控自噬在NPM1突变白血病中的作用探讨

Acute myeloid leukemia (AML) carrying nucleophosmin (NPM1) mutations has been defined as a distinct entity in the 2016 revision to the World Health Organization (WHO) classification of myeloid neoplasms and acute leukemia. Under the support of National Natural Science Foundation of China projects (No：30872418；81271913), we have confirmed that AKT activation and miRNA-10b upregulation are involved in cell proliferation and differentiation block in NPM1-mutated leukemia cells. Additionally, aberrant autophagy activation was observed in NPM1-mutated leukemia cells in our preliminary study. However, the potential molecular mechanisms are unclarified. In this study, to identify the functional role of long non-coding RNA (lncRNA) HOTAIRM1 in the regulation of autophagy in NPM1-mutated leukemia cells, we firstly detected high expression levels of HOTAIRM1 in NPM1-mutated OCI-AML3 cell line and primary blasts. Based on a miRNA-dependent regulation mode between lncRNA and autophagy related genes, the bioinformatics analysis are used to reveal the potential miRNAs. Thus, we try to elucidate the latent molecular mechanism how HOTAIRM1 regulates autophagy related genes (i.e. Beclin1) by acting as a ceRNA. Finally, we explore the functional involvement of HOTAIRM1 on cell proliferation and drug sensitivity. These results will provide novel insights into lncRNA in NPM1-mutated leukemia and serve as a potential biomarker and a latent strategy in AML therapy.

急性髓系白血病伴核仁磷酸蛋白（NPM1）基因突变是新近独立的白血病亚型。在国家自然科学基金支持下我们前期研究发现AKT和miRNA-10b参与调控NPM1突变白血病细胞的体外增殖和分化阻滞（No：30872418；81271913），同时发现白血病细胞具有高自噬活性表型，然而，调控自噬的分子机制并未阐明。本项目拟探讨长链非编码RNA（lncRNA）HOTAIRM1在NPM1突变白血病细胞自噬调控中的作用。预实验结果发现NPM1突变细胞系和临床样本中HOTAIRM1高表达，基于生物信息学数据库挖掘HOTAIRM1与自噬关键分子共享的miRNAs，探讨HOTAIRM1借ceRNA模式调控白血病细胞自噬关键分子的分子机制，最后观察靶向HOTAIRM1对白血病细胞增殖和药物敏感性的影响。本研究有助于阐明lncRNA在NPM1突变白血病中的重要作用，同时为临床诊疗寻找新的检验指标和潜在的治疗策略。

急性髓系白血病(acute myeloid leukemia, AML )是成人急性白血病中最常见的类型，其临床和分子生物学特征上具有高度异质性。临床数据显示近三分之一的AML患者可发生核仁磷酸蛋白（nucleophosmin 1, NPM1）基因突变。因其独特的临床特征和基因表达谱，NPM1突变AML作为单独的一类疾病亚型被纳入2016年WHO白血病分型指南中。在前一个项目中我们发现NPM1突变白血病细胞具有高自噬活性表型，然而，调控自噬的分子机制并未阐明。在本项目中，首先，我们探讨长链非编码RNA（lncRNA）HOTAIRM1对白血病细胞自噬活性的影响，通过过表达和RNA干扰手段证实了HOTAIRM1对自噬关键分子ULK3的调控作用；其次，通过双荧光素酶报告实验证实白血病细胞中存在HOTAIRM1与miR-152-3p、miR-152-3p与ULK3的相互作用，揭示了HOTAIRM1通过ceRNA模式上调ULK3 mRNA表达的分子机制，进一步证实了HOTAIRM1 通过上调ULK3促进白血病细胞自噬性存活；然后，通过Co-IP实验和CHIP实验证实转录因子KLF5与HOTAIRM1的互作及其对HOTAIRM1的调控作用，且NPM1突变蛋白可通过调控KLF5进而影响HOTAIRM1的转录活性，阐释了HOTAIRM1高表达的分子调控机制；此外，发现HOTAIRM1能够特异性结合EGR1蛋白，通过MDM2介导的泛素化修饰促进EGR1蛋白降解，同时证实HOTAIRM1通过下调EGR1促进白血病细胞自噬和增殖；最后，我们通过小鼠体内实验观察HOTAIRM1对白血病发生发展的影响，发现敲低HOTAIRM1表达可以阻碍白血病的发生。本项目不仅有助于阐明lncRNA HOTAIRM1在NPM1突变白血病中的重要作用，而且为临床个体化诊疗寻找新的检验指标和潜在的治疗策略。