长非编码RNA lnc-MC及RBP-ZFP36L1在单核／巨噬细胞分化中的功能及其机制研究

发布时间：2018-06-02 03:24

  本文选题：单核/巨噬细胞分化 + PU.1　； 参考：《北京协和医学院》2016年博士论文

【摘要】：造血是由造血干细胞经过一系列增殖、分化最后形成各种成熟血细胞的过程。这个过程涉及由转录因子、细胞因子和非编码RNA等组成的复杂调控网络。单核/巨噬细胞属于白细胞,也是一类非常重要的固有免疫细胞,同时也在启动适应性免疫应答方面发挥着重要作用。单核/巨噬细胞分化是整个造血链系发育的一部分,也受到一个复杂调控网络的精细调控。它的分化发育异常与急性髓系白血病的发生发展以及机体系统紊乱等众多生理病理过程密切相关。长非编码RNA (lncRNAs)是一类长度大于200nt,不编码蛋白的RNA分子。越来越多的研究表明lncRNAs可以在染色质重塑、转录以及转录后等多个层面调控基因的表达,参与调节众多生物学过程。然而,lncRNAs在造血领域的研究还处于起步阶段,有关lncRNAs在单核/巨噬细胞分化中的功能和机制还鲜有报道。RNA结合蛋白(RBPs)是一类可以在体内结合单链或双链RNA的蛋白分子,通过特定的RNA结构域识别并结合RNA,参与RNA的剪切成熟、运输、定位、翻译、降解等众多RNA代谢过程。研究表明,RBPs介导的转录后调控在哺乳动物发育以及疾病的发生发展过程中发挥着重要作用。然而,RBPs在单核/巨噬细胞分化中的功能和机制也还鲜有报道。在本课题中,我们通过生物信息学分析以及实验验证,鉴定了一条参与单核/巨噬细胞分化的长非编码RNA (lnc-MC),也筛选到了一个介导单核/巨噬细胞分化的RNA结合蛋白ZFP36L1。通过分析白细胞里的RNA-Seq数据以及ChlPBase中的PU.1-ChIP-Seq数据,我们筛选到了390个可能受到PU.1调控又在白细胞中表达的lncRNAs。结合它们在白细胞中的表达丰度以及物种保守性和编码潜能预测分析,我们把研究集中在了lnc-MC上。Lnc-MC,又名lnc-TRIP10/TCONS 00026873/XLOC 012931,在基因组上的位置为Chr19：6656385-6662832,有两个外显子；缺乏长而有效的开放阅读框,更倾向于是一条非编码RNA；只在灵长类动物中比较保守,其它物种不保守。Lnc-MC在佛波酯(PMA)诱导THP-1和HL-60以及体外诱导脐带血来源的CD34+造血干/祖细胞(HSPCs)向单核/巨噬细胞分化过程中表达逐渐上调。敲低和过表达实验证明lnc-MC可以促进人单核／巨噬细胞分化。染色质免疫沉淀以及单核分化关键转录因子PU.1敲低和过表达实验证明在单核／巨噬细胞分化过程中,PU.1可以结合于lnc-MC基因上游1826bp位点,转录激活lnc-MC的表达。我们也曾证明PU.1抑制ri-miR-199-2转录,导致miR-199a-5p减少,而miR-199a-5p可以通过靶向ACVR1B介导的TGF-β信号通路来抑制单核／巨噬细胞分化。通过RNA荧光原位杂交以及胞质胞核RNA分别抽提并PCR实验发现lnc-MC主要定位于细胞质中,提示其可能主要作为一类转录后调控因子参与单核／巨噬细胞分化。我们通过RNAHybrid预测分析以及实验证明lnc-MC可以和miR-199a-5p相互作用,相互抑制表达并降低可利用性。然而,在单核/巨噬细胞分化过程中,PU.1调节的lnc-MC上调和miR-199a-5p下调使lnc-MC在二者相互竞争中处于优势,从而减弱了miR-199a-5p对于ACVR1B的抑制作用,促进分化。因此,我们的研究揭示了一个新的由lncRNA参与的造血调控机制,即PU.1转录调控的两个非编码RNA, lnc-MC和microRNA-199a-5p,在转录后相互拮抗,通过调节ACVR1B介导的TGF-p信号通路参与单核/巨噬细胞分化。通过分析108例急性髓系白血病(AML)病例芯片以及体外诱导髓系分化芯片资料,我们筛选到了23个既在AML病例中又在体外诱导髓系分化中差异表达的RBPs,最后我们把研究集中在ZFP36L1上。芯片分析结果表明,ZFP36L1在绝大多数AML病例芯片中表达下调,而在体外诱导HSCs向单核系分化的芯片中表达逐渐上调。实验验证发现,ZFP36L1在初诊AML病例中的表达与正常对照相比明显下调,在PMA诱导THP-1和HL-60以及体外诱导脐带血来源的CD34+ HSPCs向单核／巨噬细胞分化过程中表达逐渐上调,与芯片结果一致。敲低和过表达实验证明ZFP36L1可以促进单核／巨噬细胞分化。据文献报道,ZFP36L1属于锌指蛋白家族成员,可以结合于mRNA 3'UTR的富含AU的元件(ARE),在转录后水平负调控基因的表达。因此,为了寻找ZFP36L1下游的靶基因,我们参考AREsite数据库结合芯片分析,最终筛选到了CDK6作为一个潜在的靶点。报告基因实验以及RNA免疫沉淀实验表明,ZFP36L1可以结合于CDK6 mRNA 3'UTR的ARE元件,导致CDK6mRNA的降解以及蛋白表达水平降低。在CD34+ HSPCs中通过慢病毒介导的过表达实验证明了CDK6可以抑制单核／巨噬细胞分化。最后我们又通过ZFP36L1—CDK6的rescue实验,进一步证明了ZFP36L1是通过抑制CDK6的表达来促进单核／巨噬细胞分化的。总之,我们的研究结果揭示了一个由ZFP36L1—CDK6介导的单核／巨噬细胞分化调节通路,同时也为AML的治疗提供了一个潜在的靶点。
[Abstract]:Hematopoiesis is a process in which hematopoietic stem cells undergo a series of proliferation, differentiation and final formation of a variety of mature blood cells. This process involves complex regulatory networks composed of transcription factors, cytokines and non coded RNA. Mononuclear / macrophages belong to white cells, and are also a very important type of innate immune cells, and are also initiating adaptability. The immune response plays an important role. Mononuclear / macrophage differentiation is a part of the development of the whole hematopoietic system, and is also well regulated by a complex regulatory network. Its differentiation and development are closely related to the development of acute myeloid leukemia and the disorder of the body system. Long non coded RNA (L NcRNAs) is a class of RNA molecules with a length greater than 200nt and no encoding protein. More and more studies have shown that lncRNAs can regulate the expression of genes in many levels, such as chromatin remodeling, transcription and post transcription, and participate in the regulation of many biological processes. However, the study of lncRNAs in the field of hematopoiesis is still in its infancy and lncRNAs is in mononuclear / giant. The functions and mechanisms of phagocytic differentiation have not been reported that.RNA binding protein (RBPs) is a class of protein molecules that can bind single or double stranded RNA in the body, identify and combine RNA by specific RNA domains, and participate in many RNA metabolic processes, such as shear maturation, transport, localization, translation, and degradation of RNA. The study shows that RBPs mediated posttranscriptional modulation Control plays an important role in the development of mammalian development and the development of disease. However, the functions and mechanisms of RBPs in mononuclear / macrophage differentiation are still rarely reported. In this subject, we identified a long non coded RNA (lnc-) by bioinformatics analysis and experimental verification. MC), also screened for a RNA binding protein ZFP36L1. mediated mononuclear / macrophage differentiation by analyzing RNA-Seq data in white cells and PU.1-ChIP-Seq data in ChlPBase, we screened 390 lncRNAs. that may be regulated by PU.1 and expressed in white cells in combination with their expression abundance in white cells and species. Conservatism and coding potential prediction analysis, we focus the study on lnc-MC.Lnc-MC, also known as lnc-TRIP10/TCONS 00026873/XLOC 012931, where the location of the genome is Chr19:6656385-6662832, and there are two exons; the lack of a long and effective open reading frame is more inclined to be a non coded RNA; it is only in the primates. More conserved, other species did not conservatively.Lnc-MC in THP-1 and HL-60 induced by phorbol ester (PMA) and the expression of CD34+ hematopoietic stem / progenitor cells (HSPCs) induced by umbilical cord blood in vitro during the differentiation of mononuclear / macrophages. Knockdown and overexpression experiments showed that lnc-MC could promote human mononuclear / macrophage differentiation. Chromatin immunoprecipitation And the key transcription factor PU.1 knockdown and overexpression of the key transcription factor of mononuclear differentiation demonstrated that in the process of mononuclear / macrophage differentiation, PU.1 can be combined with the upstream 1826bp site of lnc-MC gene to activate lnc-MC expression. We have also proved that PU.1 inhibits ri-miR-199-2 transcription, resulting in miR-199a-5p reduction, and miR-199a-5p can be targeted to ACVR1B. TGF- beta signaling pathway is mediated to inhibit mononuclear / macrophage differentiation. RNA fluorescence in situ hybridization and cytoplasmic nucleus RNA were separately extracted and PCR experiments showed that lnc-MC was mainly located in the cytoplasm, suggesting that it may be involved in mononuclear / macrophage differentiation mainly as a post transcriptional regulator. We predict and analyze by RNAHybrid. And experiments show that lnc-MC can interact with miR-199a-5p to suppress each other and reduce availability. However, in the process of mononuclear / macrophage differentiation, the up regulation of PU.1 regulated lnc-MC and the downregulation of miR-199a-5p make lnc-MC dominant in the competition between the two, thus reducing the inhibitory effect of miR-199a-5p on ACVR1B and promoting the differentiation of ACVR1B. Therefore, our study revealed a new hematopoietic regulatory mechanism involved in lncRNA, namely, two non coded RNA, lnc-MC and microRNA-199a-5p of PU.1 transcriptional regulation, interacting with each other after transcriptional transcription, and participating in mononuclear / macrophage differentiation by regulating ACVR1B mediated TGF-p signaling. By analyzing 108 cases of acute myeloid leukemia (AML) disease On the chip and in vitro induction of myeloid differentiation chip data, we screened 23 RBPs for differentially expressed myeloid differentiation in both AML cases and in vitro. Finally, we concentrated the study on ZFP36L1. The results of microchip analysis showed that ZFP36L1 was downregulated in most of the AML case chips, and HSCs was induced in vitro to the mononuclear system in vitro. The expression in the differentiated chip was gradually up-regulated. The experimental results showed that the expression of ZFP36L1 in the first diagnosed AML cases was significantly lower than that of normal. The expression of CD34+ HSPCs induced by PMA in THP-1 and HL-60 and in vitro induced umbilical cord blood in the differentiation process of mononuclear / macrophage was gradually up-regulated, which was consistent with the results of the chip. Experiments have shown that ZFP36L1 can promote mononuclear / macrophage differentiation. According to the literature, ZFP36L1 is a member of the zinc finger protein family, which can be combined with the AU rich component of mRNA 3'UTR (ARE) to express the expression of negative regulatory genes at post transcriptional level. Therefore, in order to find the target genes in the downstream of ZFP36L1, we refer to the AREsite database combined with the chip analysis. CDK6 was selected as a potential target. The reporter gene experiment and RNA immunoprecipitation experiment showed that ZFP36L1 could bind to the ARE element of CDK6 mRNA 3'UTR and lead to the degradation of CDK6mRNA and the decrease of protein expression level. In CD34+ HSPCs, through the slow virus mediated overexpression experiment, CDK6 can inhibit mononuclear / giant. In the end we further demonstrated that ZFP36L1 is mediated by inhibiting the expression of CDK6 to promote mononuclear / macrophage differentiation. In conclusion, our results reveal a single nucleus / macrophage differentiation regulatory pathway mediated by ZFP36L1 CDK6, and also for the treatment of AML. A potential target.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q343
，

本文编号：1967148