LncRNA-AC004893通过增强JAK2/STAT信号通路促进骨髓增殖性肿瘤发生发展的机制研究

Constitutive activation of Janus kinase 2/signal transducers and activators of transcription (JAK2/STAT) signaling plays an important role in the pathogenesis of myeloproliferative neoplasm (MPN). However, the underlying mechanism by which phosphorylated STAT protein cannot be inactivated is still unknown. Although Long non-coding RNAs (LncRNAs) are frequently deregulated in various diseases including hematological malignancies and have multiple functions in a wide range of biological processes, it is still unknown whether LncRNAs take part in the pathogenesis of MPN. This project was aimed to analyze LncRNAs mediated in the pathogenesis of MPN. Using LncRNA microarray analysis, we compared LncRNA expression levels between primary MPN cells and normal controls. Among the deregulated LncRNAs, the expression of Lnc-AC004893 was significantly increased in primary MPN cells in comparison with normal controls. Further, Lnc-AC004893 facilitated the constitutive activation of STAT5 signaling and enhanced cell proliferation through binding with STAT5 protein and preventing the de-phosphorylation of p-STAT5. We then aimed to determine whether Lnc-AC004893 prevented SHP1-induced de-phosphorylation of p-STAT5 through RNA pull down, RIP, and other experiments in cell lines, primary MPN cells, and animal model. Finally, we were planning to investigate the molecular mechanism underlying the higher expression of Lnc-AC004893 in MPN cells compared with normal controls through ChIP-seq analysis. In conclusion, establishing the different expression levels of LncRNAs and explaining the functional role of LncRNAs in MPN cells will shed light on the molecular pathogenesis leading to MPN and provide the new strategy for the diagnosis and clinical treatment.

持续激活的JAK2/STAT信号通路是骨髓增殖性肿瘤（MPN）关键的致病因素之一。然而，活化的p-STAT蛋白不被去磷酸化的机制尚不清楚。长链非编码RNA（LncRNA）通过多种机制对细胞生物学功能产生重要影响，但未见报道它是否参与MPN的致病机制。我们前期通过LncRNA基因芯片发现：在MPN细胞中表达增高的Lnc-AC004893通过结合STAT5蛋白抑制其去磷酸化，从而促进STAT信号通路持续活化，最终通过增强细胞增殖参与MPN发生发展。我们将进一步通过RIP等实验，在体外细胞株、临床病例样本和体内动物模型三个层面深入研究AC004893阻止蛋白酪氨酸磷酸酶（SHP1）对p-STAT5蛋白去磷酸化作用的分子机制。在MPN细胞中建立差异表达的LncRNA图谱，对其进行表达及功能验证，将有助于从LncRNA这一新的层面深刻了解MPN的发病机制，为MPN的诊断及治疗提供有意义的分子靶点。

骨髓增殖性肿瘤（MPN）是指分化相对成熟的一系或多系骨髓细胞不断地克隆性增殖所致的一组造血干细胞来源的疾病。深入解析MPN发病过程中的分子信号通路调控网络变化，对于全面了解MPN细胞恶性转化为白血病的分子机制、筛选治疗新靶点以及改善疾病治疗效果等提供有意义的探索。长链非编码RNA( lncRNA)是一类长度超过200个碱基的非编码RNA，其通过表观遗传学调控、转录调控、以及转录后调控等多个层面调节目标基因的表达水平，影响细胞的增殖、分化和凋亡等功能。然而，目前尚不清楚lncRNA是否在MPN发生和发展中发挥一定的作用。. 项目申请人通过lncRNA基因芯片发现lnc-AC004893在MPN患者和正常对照细胞中存在差异表达。课题组进一步通过qRT-PCR分析lnc-AC004893在150例MPN患者和20例正常对照细胞中的表达情况。通过发夹状RNA(shRNA)敲低lnc-AC004893表达，检测细胞增殖、凋亡和集落等各项指标。课题组通过RNA-pull down、RIP实验和Co-IP实验验证STAT5和lnc-AC004893相互结合。最后，课题组构建KrasG12D; ERT2cre转基因小鼠模型，模拟体内MPN发生和发展过程。通过以上系列实验，课题组发现lnc-AC004893表达量在MPN细胞中明显高于正常对照细胞。Lnc-AC004893促进MPN细胞增殖和自我更新。分子机制研究表明：lnc-AC004893通过抑制SHP1和STAT5蛋白结合，阻止p-STAT5蛋白去磷酸化，最终促进JAK2/STAT5信号通路持续活化。体内实验结果表明KrasG12D; ERT2cre转基因小鼠中敲低lnc-ac004893表达，大幅降低小鼠外周血白细胞总数，减少脾脏组织重量，延长小鼠生存期。课题组的研究结果有助于在lncRNA这一新的层面深刻了解MPN的发病机制，为MPN诊疗提供新靶点。.同时课题组发现HOTAIR通过抑制p15基因表达，促进白血病干细胞自我更新； miR-193a/miR-600通过和WT1基因形成相互调控网络，抑制AML发生和发展；miR-375通过调控HOXB3-CDCA3/DNMT3B信号通路，抑制AML细胞增殖。课题组通过对以上非编码RNA进行基因表达和功能机制研究，为MPN和AML诊断和靶向治疗提供新的靶点。