节点文献
含微小RNA的p53网络的动力学和功能研究
Dynamics and Functions of the p53 Signaling Network Including microRNAs
【作者】 周春红;
【导师】 刘锋;
【作者基本信息】 南京大学 , 物理学, 2015, 博士
【摘要】 细胞信号转导的研究对了解细胞基本生命活动的分子机制和揭示生命的本质有重要的意义。细胞每时每刻都在监控胞内外环境的变化,并针对不同的应激信号作出恰当的反应。肿瘤抑制因子p53可调控细胞应答多种应激信号,如DNA损伤、癌基因活化、端粒受损、组织缺氧等。本文通过数学建模和数值模拟,研究以p53为中心的信号网络如何介导细胞分别应答电离辐射引起的DNA双链断裂和低氧环境,结合描述网络的动力学来阐明细胞命运抉择的分子机制,讨论microRNA(miRNA)在信号转导中的作用。全文分为四章。在第一章中,我们介绍了有关的生物学背景知识。首先介绍了基因表达的概念和主要的调控机制。miRNA是一类仅含20~24个核苷酸的单链非编码RNA,能抑制信使RNA的翻译或促进其降解,抑制蛋白质的合成。我们介绍了miRNA的生成和功能、与疾病的关联等。描述了p53蛋白的结构、主要功能及其调控,介绍了 p53如何介导细胞周期阻断和细胞凋亡。最后,我们指出p53网络中的miRNAs参与调控细胞的应激反应。在第二章中,我们选择具有代表性的p53靶miRNA(miR-605和miR-34a)来研究miRNA对细胞命运抉择的影响。实验发现,miR-605和miR-34a分别在p53信号通路的上游和下游起作用:miR-605能抑制Mdm2的生成,促进p53的稳定,而miR-34a抑制Bcl-2的生成,促进细胞凋亡。但它们如何影响电离辐射后的细胞命运抉择,依然是个未解决的问题。为此,我们构建了含4个模块的p53网络的数学模型,分别刻画DNA损伤的产生和修复,激酶ATM的活化,以p53为中心的多个反馈结构的调控,p53选择性表达下游基因进而决定细胞命运。数值模拟的结果表明,细胞命运与p53网络动力学密切相关。在低辐射剂量下,p53蛋白浓度呈现周期性振荡(脉冲),而且脉冲的个数小于3。p53诱导细胞周期阻断,促进修复DNA损伤;损伤修复后,细胞回到正常的分裂期。在高辐射剂量下,p53浓度同样先周期性振荡,但3个脉冲后,p53浓度从低水平跃迁到高水平,p53诱导细胞凋亡。虽然增加miR-605的表达对p53脉冲的幅度影响较小,但加快p53动力学从低态向高态的转变,促进细胞的凋亡。miR-34a能加快游离态的p53AIP1的累积,使细胞凋亡提前发生。因此,miR-605和miR-34a都能影响细胞的命运,调控细胞凋亡的时间;它们协作促进细胞凋亡,增强细胞对电离辐射的敏感性。通过正反馈和信号转导的级联反应,miRNA对基因表达的影响被放大,能产生显著的生理效应。这些结果提示,可基于miRNA来调控细胞的凋亡,提高肿瘤细胞对放疗和化疗的敏感性。低氧环境是实体瘤的一个显著特征。在第三章中,我们探讨了在低氧环境下,细胞实现命运抉择的动力学机制。我们构建了一个以p53和低氧诱导因子HIF-1α为中心的信号转导网络。p53和HIF-1α都是转录因子,需竞争结合有限的辅助因子p300激活转录活性,又都能被Mdm2泛素化降解。我们发现,在不同的缺氧条件下,HIF-1α和p53的相互调控极大地影响细胞的命运抉择。中度缺氧条件下,HIF-1α累积、诱导p21表达,进而阻断细胞周期,促进细胞适应低氧环境。此时,p53水平很低。严重缺氧时,p53稳定性增加,并与HIF-1α竞争结合p300,通过抑制miR-17-92的表达来诱导细胞凋亡。在极端缺氧条件下,p53的浓度和转录活性显著增加,转录抑制miR-17-92的同时转录激活PUMA,快速诱导细胞凋亡。相反,HIF-1α失去转录活性并且被Mdm2降解。由于p300介导的乙酰化阻止Mdm2介导的蛋白质降解,我们认为p53和HIF-1α竞争结合p300不仅调节它们的转录活性,还调控它们的浓度。这些结果加深了人们对细胞应答低氧环境的分子机制的了解。第四章对本论文的主要研究结果作了总结,并对以后的工作提出了展望。
【Abstract】 The study of signal transduction is important for uncovering the molecular mecha-nisms for essential cellular activities and revealing the essence of life.Cells are contin-uously monitoring the changes in their internal and external environments and respond properly upon stress signals.The tumor suppressor p53 mediates the cellular response to various stresses,including DNA damage,oncogene activation,telomere erosion and hypoxia.In this thesis,by constructing mathematical models and performing numerical simulations,we explore how the p53-centered signaling networks respond to double-strand breaks induced by ionizing radiation and hypoxia,respectively,elucidate the molecular mechanisms for cell-fate decision by characterizing the network dynamic-s,and probe the roles for microRNAs(miRNAs)in signal transduction.The thesis consists of four chapters.In Chapter 1,we introduce some biological backgrounds.The concepts of gene expression and the underlying mechanisms are first described.miRNAs are single-strand,non-coding RNAs with 20-24 nucleotides.miRNAs can inhibit the translation of message RNAs or target them for degradation,suppressing protein synthesis.A description of the synthesis of miRNAs,their functions and correlation with diseases is presented.We describe the structure and main functions of p53,modulation of its activity,and its roles in inducing cell-cycle arrest and apoptosis.Finally,it is pointed out that miRNAs join the p53 network to modulate cellular stress responses.In Chapter 2,we explore how miR-605 and miR-34a,direct transcriptional tar-gets of p53,affect the cell-fate decision following ionizing radiation.miR-605 and miR-34a function by acting upstream and downstream of p53,respectively.miR-605 promotes p53 accumulation by repressing the expression of mdm2,while miR-34a pro-motes p53-dependent apoptosis by suppressing the expression of antiapoptotic genes such as bcl-2.What roles they play in the p53-mediated DNA damage response is less well understood.Here,we develop a four-module model of the p53 network,char-acterizing the production and repair of DNA damage,ATM activation,p53-centered feedback loops,and cell-fate decision.Results of numerical simulation indicate that the cell fate is closely associated with network dynamics.The concentration of p53 undergoes few pulses in response to repairable DNA damage,or it first oscillates and then switches to high plateau levels after irreparable damage.The amplitude of p53 pulses rises to various extents depending on miR-605 expression,and miR-605 accel-erates the switching behavior of p53 levels to induce apoptosis.In parallel,miR-34a promotes apoptosis by enhancing the accumulation of free p53AIP1,a key proapop-totic protein.Thus,both miR-605 and miR-34a can mediate cellular outcomes and the timing of apoptosis.Together,miR-605 and miR-34a collectively contribute to apop-tosis induction and enhance the sensitivity of cells to DNA damage.These results have important implications:modulating apoptosis induction via miRNAs can improve the efficacy of cancer treatments.Lack of oxygen,or hypoxia,is typical of solid tumors.In Chapter 3,we explore the dynamic mechanism for cell-fate decision under hypoxia.We develop a mathemat-ical model of the p53 and hypoxia-inducible factor-la(HIF-lα)pathways.As tran-scription factors,p53 and HIF-1α compete for binding to limiting co-activator p300;they are targeted for degradation by Mdm2.We find that the interplay between p53 and HIF-lα significantly influences cellular outcome.In mild hypoxia,HIF-la accu-mulates and induces p21 to evoke transient cell-cycle arrest,whereas p53 is kept at basal levels.Consequently,the cell adapts to mild hypoxia.Under severe hypoxia,p53 accumulates and competes with HIF-1α for binding to p300,and induces apop-tosis by repressing the expression of miR-17-92.In anoxia,p53 is fully activated,transactivating PUMA and repressing miR-17-92 simultaneously.Thus,apoptosis is quickly induced.In contrast,HIF-la loses its transcriptional activity and is degrad-ed by Mdm2.Because p300-mediated acetylation prevents Mdm2-dependent protein degradation,the competition between p53 and HIF-la for binding to p300 not only de-termines their transcriptional activity,but also regulates their abundance.These results shed new light on the molecular mechanisms for hypoxic responses.In Chapter 4,a summary of main results is presented,and some outlooks for further research are also given.
【Key words】 Signal transduction; p53 network; miRNA; cell-fate decision; mathematic models; dynamics;