Lnc-RP11-611D20.2调控Notch1影响肿瘤细胞增殖在急性淋巴细胞白血病中的机制研究

The pathogenesis of Acute Lymphocytic Leukemia (ALL) is highly associated with the abnormal proliferation and differentiation of the bone marrow cells. Recently, the underlying mechanism of long non-coding RNA (LncRNA) correlated with cell proliferation and differentiation in hematological disease still remains unknown. This study aimed to investigate the function of lncRNAs involved in the pathogenesis of ALL based on an lncRNA microarray assay. Lnc-RP11-611D20.2 and the potential target gene Notch1 was selected according to the results of the microarray and bioinformatics methods. Further results confirmed that Lnc-RP11-611D20.2 was positively correlated with Notch1, and was over-expressed in the bone marrow of T-ALL patients while no difference was obtained in B-ALL patients. In addition, it was found that the increased level of Lnc-RP11-611D20.2 could up-regulate the expression of Notch1 gene and increase proliferation of T-ALL cells in vitro assay. Based on the findings above, we proposed the hypothesis that the consistently high expression level of Lnc-RP11-611D20.2 would accelerate the expression of Notch1 which was indicated as an on-off gene in the pathogenesis of T-ALL and B-ALL, cause the abnormal proliferation of cells, and finally resulte in T-ALL. We sought to explore the mechanism of how Lnc-RP11-611D20.2 mediated Notch1 expression and caused the dysfunction of proliferation of T cells, besides, we will explore whether Lnc-RP11-611D20.2 could promote the proliferation of T cells. This study will give a new pathogenesis of ALL, providing a potential target of prevention and treatment for ALL in the future.

细胞增殖和分化异常在白血病的发病中扮演重要的角色，目前关于长链非编码RNA（LncRNA）参与细胞增殖和分化异常在血液疾病中尚未有明确的机制研究。本课题通过芯片技术拟研究lncRNA在儿童急性淋巴细胞白血病（ALL）的调控机制，并依据生物信息学方法及疾病背景筛选出具有潜在触发效应的Lnc-RP11-611D20.2及其靶基因Notch1，进一步在大样本人群中验证及体外细胞实验初步发现该lncRNA具有调控Nocth1潜在触发T-ALL的效应。结合生物信息学预测研究拟阐明具有染色体特殊位置关系（FLANK10K）的lncRNA和蛋白编码基因间的具体调控方式，探索lncRNA在儿童ALL中新的调控机制。本研究从人群出发，通过体外体内试验，验证其对Notch1的cis-调控机制，激活Notch1通路，从而促进细胞增殖能力。本研究可为T-ALL发生机制提供新视点和未来靶向治疗提供理论基础。

背景：细胞增殖和分化异常在白血病的发病中扮演重要的角色，目前关于长链非编码RNA（LncRNA）参与细胞增殖和分化异常在血液系统疾病中的研究尚未有明确深入的机制，并且在儿童急性淋巴细胞白血病（ALL）的研究中，亦罕有关于lncRNA的报道。本研究通过生物信息学探索发现，在与白血病发病机制密切相关的NOTCH1上游1000bp内，存在着一个lncRNA，命名为Lnc-NALT。本研究旨在探讨NALT通过调控NOTCH1介导T-ALL细胞增殖的作用机制。.方法：运用qRT-PCR方法检测Lnc-NALT在 T-ALL患儿中的表达水平；通过CCK8、EDU荧光检测Lnc-NALT对ALL细胞增殖能力影响；采用Hochest33342直标在维拉帕米介导下鉴定Jurkat细胞群中Side-population细胞（SP细胞）分布；采用Gal4-λN/BoxB 报告基因系统鉴定Lnc-NALT对NOTCH1及其信号通路调控机制。.结果：通过高通量芯片检测及大样本人群验证Lnc- RP11-611D20.2在T-ALL中显著高表达，并呈现与NOTCH1明显的正相关关系。体外CCK8及EDU检测发现，Lnc- RP11-611D20.2可通过正向调控NOTCH1促进细胞增殖，此外Lnc- RP11-611D20.2敲除后可产生与GSI(NOTCH1抑制剂)一致的抑制细胞增殖效果且该效应可被NICD(NOTCH1胞内活性段)所扭转。进一步研究发现Lnc- RP11-611D20.2可明显提高Jurkat细胞群SP亚群细胞比例，通过将Lnc- RP11-611D20.2与Gal4-λN/BoxB 报告基因系统进行共转染发现，Lnc- RP11-611D20.2可以产生与LUNAR1类似的促进转录现象，提示Lnc- RP11-611D20.2可作为转录激动剂进行对NOTCH1产生cis-调控。.结论：Lnc- RP11-611D20.2通过对其临近的蛋白编码基因NOTCH1转录水平，产生以cis-为主的调控机制，促进NOTCH1的表达，继而激发NOTCH1胞内段ICN1的活性，激活NOTCH1下游通路，促进T细胞增殖，进而触发T-ALL的发生。