lncRNA-ncRAN在促进急性早幼粒细胞白血病细胞增殖中的机制研究

The researches on the pathogenesis of acute promyelocytic leukemia (APL) are focus on key PML/RARα target proteins that play essential role in the leukemogenesis and epigenetic-modifying factors recruited by PML/RARα. Long non-coding RNAs (lncRNAs) are emerging as new players in the cancers demonstrating potential roles in tumorigenesis. It has been reported that many lncRNAs are frequently aberrantly expressed in a variety of human cancers, however the role of lncRNAs in APL remain largely unknown. The study on lncRNAs dysregulated in APL will open a new field for leukemia research. In our previous work, we found that ncRAN, a lncRNA appears to have oncogenic properties in solid tumors, was highly expressed in APL cell line and patient samples. And the expression of ncRAN was downregulated in the differentiation process of APL cells induced by ATRA. Furthermore, knockdown of ncRAN in APL patient-derived NB4 cells was found to decrease cell growth significantly. Based on these findings, we predict that ncRAN may play a critical role in the uncontrolled proliferation of APL cells. In this study, we will perform cell biology and xenograft experiments to validated the growth-promoting function of ncRAN in acute promyelocytic leukemia cells, and a series of molecular biology experiments and microarray-bioinformatics analysis to identify the machanisms of APL cell overgrowth promoted by ncRAN. This study will not only provide new insights into leukemogenesis, but also explore potentially therapeutic target for effective leukemia treatment.

长链非编码RNA(lncRNA)在细胞生理功能及肿瘤发生发展中都有重要作用。研究显示：除了致病蛋白PML/RARα及其造成的靶基因调变和染色质修饰异常外，还有其他潜在机制参与急性早幼粒细胞白血病(APL)的发病。申请人前期发现：ncRAN，这一具有癌基因特性的lncRNA，在NB4细胞和临床APL样本中都高表达；ncRAN在全反式维甲酸诱导APL细胞分化过程中下调。在模拟APL发病的PR9细胞中，ncRAN在诱导PML/RARα表达后上调。同时，沉默ncRAN明显减慢APL细胞增殖速度。都提示ncRAN可能通过影响细胞增殖参与APL发病。因此，本项目拟通过细胞生物学、分子生物学和动物学实验明确ncRAN促进APL细胞增殖的作用，并整合生物信息分析和功能实验诠释ncRAN促进APL细胞增殖的分子机制。本研究不仅为全面认识APL的发病机制提供新思路，也为有效治疗白血病提供潜在有意义的靶点。

急性早幼粒细胞白血病（APL）的发病机制研究主要集中在致病蛋白PML/RARα及其靶基因表达变化和染色质修饰异常，长链非编码RNA（lncRNA）的作用研究较少。在本研究中，1）我们首先发现lncRNA ncRAN在APL患者来源的NB4细胞及初发APL患者骨髓单个核细胞都高表达，而在全反式维甲酸处理后表达明显下调。2）利用慢病毒沉默NB4细胞的ncRAN，通过细胞生长曲线、CCK8 检测和细胞周期分析检测细胞增殖变化，发现沉默ncRAN后NB4细胞增殖减慢，G0/G1期细胞比例增加。3）通过生物信息学分析我们发现ncRAN启动子区的-86bp到-71bp位置存在PML/RARα蛋白结合位点DR4，随后利用ChIP-PCR和荧光素酶报告基因检测发现PML/RARα通过结合到ncRAN启动子区DR4位点起转录激活作用。4）生物信息学预测发现ncRAN上存在let-7c的结合位点（长型1398bp-1404bp），通过构建ncRAN长型和短型及let-7c结合位点突变的质粒，利用荧光素酶报告基因检测明确let-7c通过ncRAN上的1398bp-1405bp（长型）位点对ncRAN起抑制作用。我们通过RNA免疫共沉淀和RNA pull down实验发现在NB4细胞内let-7c与ncRAN结合。5）我们利用表达谱芯片技术分析ncRAN对NB4细胞转录组的影响。但沉默和过表达ncRAN后共同调变的基因数较少，不能聚出有意义的与细胞增殖相关的通路。下一步计划从差异表达基因中筛选并验证let-7c的靶基因。6）文献报道lncRNA NEAT1在APL细胞分化过程中很重要。我们研究了NEAT1在APL细胞分化过程中表达上调的机制。通过RT-qPCR验证了NEAT1在NB4细胞分化过程中表达上调，ChIP-qPCR发现C/EBPβ结合在-54和-1453位点。荧光素酶报告基因实验发现C/EBPβ通过激活NEAT1启动子区的-54和-1453位点促进NEAT1表达。最后利用RNA干扰技术将C/EBPβ沉默，发现NEAT1的表达降低。本研究确认了ncRAN促进APL细胞增殖的作用，阐明了ncRAN在APL细胞高表达的机制和ncRAN促进APL细胞增殖的机制，并且明确了lncRNA NEAT1在APL细胞分化过程中表达上调的分子机制。本研究为全面认识APL的发病机制提供了新思路。