本文選題：單核/巨噬細(xì)胞分化 + PU.1　； 參考：《北京協(xié)和醫(yī)學(xué)院》2016年博士論文

【摘要】：造血是由造血干細(xì)胞經(jīng)過一系列增殖、分化最后形成各種成熟血細(xì)胞的過程。這個(gè)過程涉及由轉(zhuǎn)錄因子、細(xì)胞因子和非編碼RNA等組成的復(fù)雜調(diào)控網(wǎng)絡(luò)。單核/巨噬細(xì)胞屬于白細(xì)胞,也是一類非常重要的固有免疫細(xì)胞,同時(shí)也在啟動適應(yīng)性免疫應(yīng)答方面發(fā)揮著重要作用。單核/巨噬細(xì)胞分化是整個(gè)造血鏈系發(fā)育的一部分,也受到一個(gè)復(fù)雜調(diào)控網(wǎng)絡(luò)的精細(xì)調(diào)控。它的分化發(fā)育異常與急性髓系白血病的發(fā)生發(fā)展以及機(jī)體系統(tǒng)紊亂等眾多生理病理過程密切相關(guān)。長非編碼RNA (lncRNAs)是一類長度大于200nt,不編碼蛋白的RNA分子。越來越多的研究表明lncRNAs可以在染色質(zhì)重塑、轉(zhuǎn)錄以及轉(zhuǎn)錄后等多個(gè)層面調(diào)控基因的表達(dá),參與調(diào)節(jié)眾多生物學(xué)過程。然而,lncRNAs在造血領(lǐng)域的研究還處于起步階段,有關(guān)lncRNAs在單核/巨噬細(xì)胞分化中的功能和機(jī)制還鮮有報(bào)道。RNA結(jié)合蛋白(RBPs)是一類可以在體內(nèi)結(jié)合單鏈或雙鏈RNA的蛋白分子,通過特定的RNA結(jié)構(gòu)域識別并結(jié)合RNA,參與RNA的剪切成熟、運(yùn)輸、定位、翻譯、降解等眾多RNA代謝過程。研究表明,RBPs介導(dǎo)的轉(zhuǎn)錄后調(diào)控在哺乳動物發(fā)育以及疾病的發(fā)生發(fā)展過程中發(fā)揮著重要作用。然而,RBPs在單核/巨噬細(xì)胞分化中的功能和機(jī)制也還鮮有報(bào)道。在本課題中,我們通過生物信息學(xué)分析以及實(shí)驗(yàn)驗(yàn)證,鑒定了一條參與單核/巨噬細(xì)胞分化的長非編碼RNA (lnc-MC),也篩選到了一個(gè)介導(dǎo)單核/巨噬細(xì)胞分化的RNA結(jié)合蛋白ZFP36L1。通過分析白細(xì)胞里的RNA-Seq數(shù)據(jù)以及ChlPBase中的PU.1-ChIP-Seq數(shù)據(jù),我們篩選到了390個(gè)可能受到PU.1調(diào)控又在白細(xì)胞中表達(dá)的lncRNAs。結(jié)合它們在白細(xì)胞中的表達(dá)豐度以及物種保守性和編碼潛能預(yù)測分析,我們把研究集中在了lnc-MC上。Lnc-MC,又名lnc-TRIP10/TCONS 00026873/XLOC 012931,在基因組上的位置為Chr19：6656385-6662832,有兩個(gè)外顯子；缺乏長而有效的開放閱讀框,更傾向于是一條非編碼RNA；只在靈長類動物中比較保守,其它物種不保守。Lnc-MC在佛波酯(PMA)誘導(dǎo)THP-1和HL-60以及體外誘導(dǎo)臍帶血來源的CD34+造血干/祖細(xì)胞(HSPCs)向單核/巨噬細(xì)胞分化過程中表達(dá)逐漸上調(diào)。敲低和過表達(dá)實(shí)驗(yàn)證明lnc-MC可以促進(jìn)人單核／巨噬細(xì)胞分化。染色質(zhì)免疫沉淀以及單核分化關(guān)鍵轉(zhuǎn)錄因子PU.1敲低和過表達(dá)實(shí)驗(yàn)證明在單核／巨噬細(xì)胞分化過程中,PU.1可以結(jié)合于lnc-MC基因上游1826bp位點(diǎn),轉(zhuǎn)錄激活lnc-MC的表達(dá)。我們也曾證明PU.1抑制ri-miR-199-2轉(zhuǎn)錄,導(dǎo)致miR-199a-5p減少,而miR-199a-5p可以通過靶向ACVR1B介導(dǎo)的TGF-β信號通路來抑制單核／巨噬細(xì)胞分化。通過RNA熒光原位雜交以及胞質(zhì)胞核RNA分別抽提并PCR實(shí)驗(yàn)發(fā)現(xiàn)lnc-MC主要定位于細(xì)胞質(zhì)中,提示其可能主要作為一類轉(zhuǎn)錄后調(diào)控因子參與單核／巨噬細(xì)胞分化。我們通過RNAHybrid預(yù)測分析以及實(shí)驗(yàn)證明lnc-MC可以和miR-199a-5p相互作用,相互抑制表達(dá)并降低可利用性。然而,在單核/巨噬細(xì)胞分化過程中,PU.1調(diào)節(jié)的lnc-MC上調(diào)和miR-199a-5p下調(diào)使lnc-MC在二者相互競爭中處于優(yōu)勢,從而減弱了miR-199a-5p對于ACVR1B的抑制作用,促進(jìn)分化。因此,我們的研究揭示了一個(gè)新的由lncRNA參與的造血調(diào)控機(jī)制,即PU.1轉(zhuǎn)錄調(diào)控的兩個(gè)非編碼RNA, lnc-MC和microRNA-199a-5p,在轉(zhuǎn)錄后相互拮抗,通過調(diào)節(jié)ACVR1B介導(dǎo)的TGF-p信號通路參與單核/巨噬細(xì)胞分化。通過分析108例急性髓系白血病(AML)病例芯片以及體外誘導(dǎo)髓系分化芯片資料,我們篩選到了23個(gè)既在AML病例中又在體外誘導(dǎo)髓系分化中差異表達(dá)的RBPs,最后我們把研究集中在ZFP36L1上。芯片分析結(jié)果表明,ZFP36L1在絕大多數(shù)AML病例芯片中表達(dá)下調(diào),而在體外誘導(dǎo)HSCs向單核系分化的芯片中表達(dá)逐漸上調(diào)。實(shí)驗(yàn)驗(yàn)證發(fā)現(xiàn),ZFP36L1在初診AML病例中的表達(dá)與正常對照相比明顯下調(diào),在PMA誘導(dǎo)THP-1和HL-60以及體外誘導(dǎo)臍帶血來源的CD34+ HSPCs向單核／巨噬細(xì)胞分化過程中表達(dá)逐漸上調(diào),與芯片結(jié)果一致。敲低和過表達(dá)實(shí)驗(yàn)證明ZFP36L1可以促進(jìn)單核／巨噬細(xì)胞分化。據(jù)文獻(xiàn)報(bào)道,ZFP36L1屬于鋅指蛋白家族成員,可以結(jié)合于mRNA 3'UTR的富含AU的元件(ARE),在轉(zhuǎn)錄后水平負(fù)調(diào)控基因的表達(dá)。因此,為了尋找ZFP36L1下游的靶基因,我們參考AREsite數(shù)據(jù)庫結(jié)合芯片分析,最終篩選到了CDK6作為一個(gè)潛在的靶點(diǎn)。報(bào)告基因?qū)嶒?yàn)以及RNA免疫沉淀實(shí)驗(yàn)表明,ZFP36L1可以結(jié)合于CDK6 mRNA 3'UTR的ARE元件,導(dǎo)致CDK6mRNA的降解以及蛋白表達(dá)水平降低。在CD34+ HSPCs中通過慢病毒介導(dǎo)的過表達(dá)實(shí)驗(yàn)證明了CDK6可以抑制單核／巨噬細(xì)胞分化。最后我們又通過ZFP36L1—CDK6的rescue實(shí)驗(yàn),進(jìn)一步證明了ZFP36L1是通過抑制CDK6的表達(dá)來促進(jìn)單核／巨噬細(xì)胞分化的�？傊�,我們的研究結(jié)果揭示了一個(gè)由ZFP36L1—CDK6介導(dǎo)的單核／巨噬細(xì)胞分化調(diào)節(jié)通路,同時(shí)也為AML的治療提供了一個(gè)潛在的靶點(diǎn)。
[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.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：Q343
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本文編號：1967148