长链非编码RNA ANRIL通过AdipoR1信号通路调控AML细胞有氧糖酵解 及增殖的分子机制研究

Acute myeloid leukemia (AML) presented uncontrolled leukemia cell proliferation, since its low cure rate and poor prognosis, the clinical treatment is facing severe challenges. Studies have shown that aerobic glycolysis can provide sufficient energy and biological molecules for rapid tumor cell proliferation and replication. In recent years, long non-coding RNA (lncRNA) was found to regulate tumor formation and development in many aspects. Our previous studies have found that lncRNA ANRIL was significantly overexpressed in AML patient samples, and silencing its expression with RNAi may inhibit aerobic glycolysis via AdipoR1 in AML cells. This study intends to use in vivo and in vitro methods to explore the function of ANRIL mainly on three aspects: (1) determine the role of ANRIL in aerobic glycolysis in AML cells and its interaction with AdipoR1; (2) analyze the molecular mechanism of ANRIL in regulating AML cell aerobic glycolysis and rapid replication and proliferation by using expression profiling, bioinformatics and functional studies; (3) validate the molecular mechanism of ANRIL and its regulatory mechanism in clinical AML specimens, and determine its clinical significance. Thus revealing the new pathogenesis of AML, and providing the theoretical basis for a potential therapeutic target.

急性髓细胞白血病（AML）癌细胞不可控增殖、治愈率低、预后差、治疗面临严峻挑战。研究表明，有氧糖酵解反应可为肿瘤细胞增殖提供充足的能量和快速复制原料。近年来长链非编码RNA（lncRNA）被发现在多种层面调控肿瘤的发生、发展。我们的前期研究发现lncRNA ANRIL在AML病人样本中显著高表达，且沉默其表达可能通过AdipoR1抑制AML细胞有氧糖酵解反应。本研究拟利用大量临床标本、小鼠体内实验和体外培养细胞，以ANRIL为切入点，开展三方面研究：（1）确定ANRIL在AML细胞有氧糖酵解中的作用及其与AdipoR1的相互作用方式；（2）利用表达谱分析、生物信息学分析和功能研究，分析ANRIL调控AML有氧糖酵解及快速复制、增殖的分子机制；（3）分析ARNIL及其调控基因在AML临床标本中的表达及临床意义。从而揭示AML新的致病机制，为寻找新的治疗靶点提供理论依据。

已有的研究表明，长链非编码RNA ANRIL的异常表达在不同癌症中发挥重要作用。我们的前期研究发现lncRNA ANRIL在急性髓性白血病(AML)病人样本中显著高表达，且在体外HL-60和MOLM-13细胞系中抑制其表达可显著抑制AML细胞的增殖和有氧糖酵解。本课题组进一步对ANRIL发挥功能的机制进行探索，发现在体外AML细胞水平发现抑制ANRIL的表达可以促进细胞的衰老、抑制细胞增殖、促进药物诱导的细胞凋亡。对sh-ANRIL和sh-NC转染后的MOLM-13细胞进行RNA-seq分析，GO分析表明表达异常基因在细胞糖代谢通路及细胞死亡调控通路显著富集，其中AdipoR1基因受ANRIL转录抑制。AdipoR1是调节肿瘤细胞Warburg effect，即有氧糖酵解的关键分子，为肿瘤细胞的增殖迅速提供能量。敲减ANRIL的表达可下调AdipoR1表达。而沉默AdipoR1表达则可以促进AML细胞的衰老、凋亡，同时降低细胞增殖。围绕ANRIL对AdipoR1的调控，以及其在AML细胞有氧糖酵解功能中的作用，我们发现敲减ANRIL或AdipoR1后，AML细胞中的葡萄糖摄取显著减少，同时培养细胞液中的乳酸含量减少。同时下游通路中pAMPKa和SIRT1蛋白的表达水平显著下降。同时，我们通过体内研究对ANRIL的调控机制进行验证。NOD/SCID小鼠尾静脉注射转染了sh-ANRIL的MOLM-13细胞后，与对照组相比，肿瘤细胞的增殖及浸润在敲减ANRIL的样本中显著降低，小鼠存活时间更长。本研究表明ANRIL可以通过AdipoR1信号通路促进AML细胞的存活和有氧糖酵解，从而促进AML的进展，是潜在的AML预后标志物和治疗靶点。