异常表观遗传沉默miRNAs对恶性血液病细胞Ras通路的激活作用

Some research showed that miR-203,miR-34a and miR-663 were silenced or down-regulated due to aberrant epigenetic regulation in the hematological malignant cells.In our previous study, the miR-663 could down-regulate H-ras expression, meanwhile the SRC that was the signal molecule in upstream region of the Ras signaling pathway,which was regulated by miR-203 and miR-34a potentially. But it is not clear if the signaling pathway of Ras will be activated because of the down-regulation of the three miRNAs. In the research, the aim was to discover the molecular mechanism of activating Ras signaling pathway and inducing malignant transformation due to the changes in epigenetic regulation in the cell lines , in which the three miRNAs expression were silenced or down-regulation because the CpG islands were methylation in the promoter regions. After the miR-203,miR-34a and miR-663 were over-expressed with the means of lentivirus vector, the expression of downstream molecules in the Ras signaling pathway were detected, then to ascertain the effects on cell proliferation, cell cycle and cell phenotype. The significance of this study is to help to enrich the understanding of the molecular mechanism of the excessive activation of the Ras signal transduction pathway and provide a potential theoretical basis for targeted therapy for the hematologic malignancies next.

已有的研究表明在恶性血液病细胞中,miR-203、miR-34a和miR-663是受异常的表观遗传学沉默或下调的miRNAs。我们既往的研究表明，miR-663可负性调控H-ras基因表达，而Ras信号传导通路上游分子SRC表达水平可能受miR-203及miR-34a调节。那么这三个微小RNA表达的下调，是否会引起Ras信号传导通路过度的激活目前尚不十分清楚。本研究中，拟在由启动子区CpG岛异常甲基化导致三种微小RNA表达下调的细胞系中，探讨这种改变对Ras信号传导通路激活作用的分子机理和对细胞恶性转化的作用。通过慢病毒介导miR-203、miR-34a及miR-663高表达，检测Ras信号通路中的下游分子的变化和由此带来的对细胞的增殖、周期、表型的影响。本研究的意义在于，有助于丰富对Ras信号传导通路过度激活的分子机制的认识，为此类恶性血液病下一步的靶向治疗提供潜在的理论依据。

研究背景：已有的研究表明，在一些恶性血液病来源的细胞中miR-34a的启动子区存在异常的高甲基化，而使miR-34a的表达下调，从而促进细胞的恶性转化和增殖。在我们前期的研究中初步表明miR-34a可能调控着Ras信号通路的上游信号分子c-SRC，本研究项目主要在miR-34a启动子区有异常高甲基化的恶性血液病细胞株中，探讨其对Ras信号通路的激活作用。.研究内容：筛选miR-34a启动子区存在异常高甲基化的恶性血液病细胞株，检测miR-34a在相关细胞系的表达水平，去甲基化后在部分细胞系检测miR-34a的表达水平。确定miR-34a对靶基因c-SRC的调控作用及作用位点。在miR-34a启动子区异常高甲基化的细胞系K-562恢复miR-34a的表达，探讨其对Ras信号通路激活的抑制作用及对细胞增殖的影响。对伊马替尼耐药的细胞株TF-1（T315I）恢复miR-34a的表达后，使用伊马替尼处理TF-1（T315I）细胞，探讨其对伊马替尼敏感性的影响。.结果及关键数据：与HEK293T细胞、间充质干细胞（MSC）、健康供者骨髓白细胞相比，恶性血液病来源的细胞株Kasumi-1、K-562、TF-1（T315I）细胞的miR-34a启动子区存在着高度甲基化，慢性髓系白血病(CML)患者的骨髓白细胞存在着半甲基化；与HEK293T 、MSC相比，miR-34a在Kasumi-1、K-562、TF-1（T315I）细胞的表达水平降低，c-SRC蛋白的表达水平上调；去甲基化处理后，能在K-562、TF-1（T315I）细胞提高miR-34a的表达而降低c-SRC蛋白的表达；miR-34a对c-SRC的两个潜在作用位点都有调控作用；miR-34a能通过下调c-SRC蛋白及其磷酸化水平，在K-562细胞抑制RAF/MEK/ERK信号通路的活性；在K-562细胞恢复miR-34a的表达，能部分通过诱导细胞G1/S期阻滞、促进细胞凋亡，而抑制细胞增殖；在伊马替尼抵抗的细胞株TF-1（T315I）恢复miR-34a的表达，能通过加强对c-SRC、ERK1/2活化的抑制，部分的提高对伊马替尼的敏感性。.科学意义：本研究表明miR-34a启动子区异常甲基化可使miR-34a表达水平下调，从而导致Ras信号通路在恶性血液病细胞系K-562、TF-1（T315I）的过度激活。