医学]miRNAsandstemcell课件

1、医学miRNAsandstemcellTopic 5microRNAs and Cell FatemicroRNAs 和细胞命运和细胞命运医学miRNAsandstemcellmicroRNAs (miRNAs)1993年发现于Caenorhabditis elegans, 近年才被重视2002年Science年度评选10大科学成就高居榜首21-24nt 的小分子非编码RNA通过对RNA的裂解或翻译抑制起到基因调控的作用参与细胞生长与分化、生长发育、肿瘤形成和抑制医学miRNAsandstemcellmiRNAs 作用机制作用机制1.降解 mRNA：植物中多见2.抑制mRNA的翻译：动物众多见

2、miRNAs 通过不完全的碱基配对通过不完全的碱基配对结合结合mRNA 的的3非翻译区（非翻译区（UTRs）转录后水平上抑制基因翻译转录后水平上抑制基因翻译仅降低其靶基因的蛋白质表达仅降低其靶基因的蛋白质表达不影响不影响 mRNA 水平水平医学miRNAsandstemcellmiRNAs 的生物合成过程的生物合成过程 RNA 聚合酶聚合酶II (Pol II) 转录合成转录合成Pri-miRNA RNase III Drosha 和双链和双链RNA 结合蛋白结合蛋白Pasha 处理成约小片段的的处理成约小片段的的pre-miRNAexportin 5 将这种前体分子输送到细胞质中将这种前体分

3、子输送到细胞质中RNase III Dicer 剪切产生约剪切产生约22 nt的的miRNA双链双链被整合到被整合到miRISC 复合体中复合体中 医学miRNAsandstemcellmiRNAs 调节多个靶基因调节多个靶基因 miRNAs 和其结合位点非完全互补的，存在短的错配和GU 配对 同一家族的成熟miRNAs 在5末端具有高度的同源性 miRNA 的5末端：对于miRNA 进入miRISC及其其生物学功能相当 关键“miRNA seed”搜索：利用一个包含了成熟miRNA 的2-8 位的碱基 序列的miRNA seed”来搜索所有基因的3 非编码区域的互补序列 一个单独的miRNA

4、 可以结合多达200个靶基因 单个基因的3非翻译区域具有几个miRNAs 的结合位点 医学miRNAsandstemcellmiRNA biogenesis and its regulation医学miRNAsandstemcellTechnology to study function of miRNAsmiRNAs overexpressionDicer/DGCR8 knock down + rescue医学miRNAsandstemcellCoupling transcriptional and post transcriptional miRNA regulation in the c

5、ontrol of cell fate 医学miRNAsandstemcellIn cancer cells, inactivation of the miRNA-mediated silencing pathway and the avoidance of miRNA regulation contribute to transformation.医学miRNAsandstemcell (A)miRNAs and TFs in FFLs tend to mutually target genes from the same pathway. (B) Additionally, co regu

6、lated miRNAs and miRNAfamilies co target many genes in the same pathway, thus resulting in a significant total output, having a major effect on cell fate. Different ways by which FFLs can account for the enhanced phenotypic effect of miRNAs on cell fate医学miRNAsandstemcellmiRNA target spatiotemporal

7、avoidance医学miRNAsandstemcellMicroRNAs in Embryonic Stem Cells医学miRNAsandstemcellA total of 678 and 472 miRNA genes have been discovered in the human and mouse genomes, respectively, but only a subset of these miRNAs is expressed in ESCs. Fewer than 100 miRNAs were initially identified in human or mu

8、rine ES cells by cloning and sequencing from small RNA libraries. In independent studies, different ES cell lines show distinct miRNA profiles.医学miRNAsandstemcellhsa-let-7a, hsa-miR-103, hsa-miR-130a, hsa-miR-136, hsa-miR-143, hsa-miR-151, hsamiR-16, hsa-miR-17, hsa-miR-200c, hsa-miR-21, hsa-miR-221

9、, hsa-miR-222, hsa-miR-26a, hsa-miR-28, hsa-miR-29b, hsa-miR-301, hsa-miR-302a, hsa-miR-302b, hsa-miR-302c, hsa-miR-302d, hsa-miR-320, hsa-miR-367, hsa-miR-368, hsa-miR-371, hsa-miR-372, hsa-miR-373, hsa-miR-378, hsa-miR-423, hsamiR-425, hsa-miR-708, and hsa-miR-93a miRNA expression signature charac

10、teristic of human ES (hES) cellsthe human homologs of the ES-specific murine miR-290 cluster also form a tight genomic cluster (hsa-miR-371,hsa-miR-372, hsa-miR-373, and hsa-miR-373) and have also been shown to be expressed in ES cells but not in embryonal carcinoma or other cells医学miRNAsandstemcell

11、miRNAs in mouse ES (mES) cells1. 390 mouse miRNAs were detected in Balb/c embryos atstages E9.5, E10.5, and E11.5. 2. Temporal expression patterns demonstrate that miRNAs are expressed at specific stages of embryonic development.医学miRNAsandstemcell1. miR-290 cluster identified as a 2.2-kb region on

12、chromosome 7 was determined to be ES-specific by Northern blotting. The single, spliced, capped and poly-adenylated primary transcript produced at this locus generates eight mature miRNAs (miR-290-5p, miR-290-3p, miR-291a, miR-291b, miR-292, miR-293, miR-294, miR-295), all of which share similar seq

13、uences and a 50-proximal AAGUGC seed motif. 2. miR-302 cluster (miR-302a, miR-302b, miR-302c, miR-302d, miR-367) Expression of the miRNA cluster increased during preimplantation development and remained high in undifferentiated ES cells but decreased after ES cell differentiation医学miRNAsandstemcell3

14、. miR-17-92 cluster of oncogenic miRNAs(mir-17-3p, mir-17-5p, mir-18, mir-19a, mir-19b-1, mir-92-1) was found to be down-regulated at ES to EB differentiation.During development of germ cells, the miR-17-92 cluster and the otherwise ES cell-specific miR-290 cluster were highly expressed in primordia

15、l germ cells (PGCs, 原始生殖细胞) and spermatogonia （精原干细胞）.医学miRNAsandstemcell4. Several miRNAs were rare in ES cells but abundant indifferentiated cells (e.g., HeLa and STO cells). These include miR-15a, 16, 19b, 92, 93, 96, 130, 130b in mouse as well as let-7a, miR-301, 374, 21, 29b, 29 in human.Murine

16、 miR-21 and -22 were expressed in undifferentiated ES cells and their expression levels increased dramatically upon differentiation, suggesting that these arise from the subpopulation of spontaneously differentiating cells医学miRNAsandstemcellRegulatory Circuits Controlling ESC Identity医学miRNAsandstem

17、cellKey ES cell transcription factors promote the ES cell miRNA expression program and integrate miRNAs into the regulatory circuitry controlling ES cell identityConnecting microRNA Genes to the Core Transcriptional Regulatory Circuitry of Embryonic Stem Cells医学miRNAsandstemcellOccupancy of miRNA Pr

18、omoters by Core ES Cell Transcription FactorsCell 134, 521533, August 8, 2008医学miRNAsandstemcellIn murine ES cells, Oct4, Sox2, Nanog, and Tcf3 co-occupied the promoters for 55 distinct miRNA transcription units, which included three clusters of miRNAs that are expressed as large polycistrons, thus

19、suggesting that these regulators have the potential to directly control thetranscription of 81 distinct mature miRNAs.Extensive conservation of the set of miRNA genes that were occupied at their promoters by Oct4.These key ES cell miRNAs were generally downregulated in response to Oct4 depletion and

20、 upregulated upon Tcf3 depletion.医学miRNAsandstemcellRegulation of Oct4/Sox2/Nanog/Tcf3-Bound miRNA Genes during DifferentiationOct4 and Nanog are silenced as ES cells begin to differentiate targetmiRNAs, the targets should be differentially expressed whenES cells are compared to a differentiated cel

21、l type.医学miRNAsandstemcell医学miRNAsandstemcellPolycomb Group Proteins Co-Occupy Tissue-Specific miRNAs that Are Silenced in ES Cells医学miRNAsandstemcellmiRNA Modulation of the Gene Regulatory Network in ES CellsLefty1 and Lefty2: actively expressed in ES cells occupied at their promoters by Oct4/Sox2/

22、 Nanog/Tcf3 conserved targets of the miR-290295 miRNA familyTFs exerts both positive and negative effects on its target医学miRNAsandstemcellLet-7g and Lin28: Promoters occupied by Oct4/Sox2/Nanog/Tcf3. Lin28 blocks the maturation of Let-7g Proper expression of pri-Let-7g is dependent on Oct4医学miRNAsan

23、dstemcellDnmt3a and Dnmt3b: indirectly upregulated by the miR-290295 miRNAs occupied at their promoters by Oct4/Sox2/Nanog/Tcf3医学miRNAsandstemcellMultilevel Regulatory Network Controlling ES Cell Identity医学miRNAsandstemcellmiRNAs function in embryonic stem cell differentiation 医学miRNAsandstemcellmiR

24、-145: Expressed at low levels in selfrenewing human ESCs, but substantially upregulated during differentiation Acting as a key antagonist of ESC maintenance Directly targets and suppresses the mRNAs encoding the transcription factors Oct4, Sox2 and Klf4let-7 family: Antagonizes Myc and Lin28 protein

25、 production医学miRNAsandstemcell医学miRNAsandstemcell医学miRNAsandstemcellGENES & DEVELOPMENT 24:27322741 2010医学miRNAsandstemcellmiRNAs Regulate the G1/S Transitionin Mouse Embryonic Stem Cells医学miRNAsandstemcellThe proliferation rate of mouse ES cells is extremely fast with a cycling time of 10 h. The le

26、ngth of cycling time is increased to more than 18 h in differentiated cells. ES cells have an unusually short G1 phase (2 h) with most (70%) cells in S phase. ES cells lack a G1 restriction point or checkpoint, therefore can proliferate even in the absence of growth factors or mitogens. In mouse ES

27、cells, the Cdk4/Cdk6Cyclin D complex is not present or active, while the Cdk+2Cyclin E complex is constitutively active throughout the cell cycle. During differentiation, the Cdk2Cyclin E activity is decreased and becomes cell cycle dependentCdks: cyclin-dependent kinases 医学miRNAsandstemcellG1期PCC为单

28、线状，因DNA未复制。S期PCC为粉末状，因DNA由多个部位开始复制。G2期PCC为双线染色体，说明DNA复制已完成。细胞生长周期细胞生长周期医学miRNAsandstemcell医学miRNAsandstemcellCdks: cyclin-dependent kinases 细胞周期蛋白依赖性激酶细胞周期蛋白依赖性激酶激活的CDK1可将靶蛋白磷酸化而产生相应的生理效应，如将核纤层蛋白磷酸化导致核纤层解体、核膜消失，将H1磷酸化导致染色体的凝缩等等。这些效应的最终结果是细细胞周期的不断运行胞周期的不断运行。因此，CDK激酶和其调节因子又被称作细胞周期细胞周期引擎引擎。医学miRNAsands

29、temcell医学miRNAsandstemcellA) mESCs exhibit high mir-290 miRNA levels and elevated Cdk2-cyclin E activity, leading to accelerated G1-S progression and a corresponding short G1 phase. mir-290 inhibits the accumulation of p21Cip1, allowing unrestrained Cdk2-cyclin E activity. (B) As mir-290 miRNAs are

30、down- regulated during differentiation, or in cells deficient for Dcgr3, p21Cip1 accumulates and assembles into complexes that inactivate Cdk2-cyclin E. The diminished cdk activity is thought to delay rogression from G1 into S phase, thus lengthening overall generation times.mir-290 miRNA Regulation

31、 of G1 Progression in mESCs医学miRNAsandstemcellCell cycle profiles of ES cells, differentiated ES cells, and miRNA-deficient ES cells.More cells accumulate in G1 phase in differentiated cells and miRNA deficient ES cells than undifferentiated ES cells.医学miRNAsandstemcellScreening strategy to identify

32、 cell cycle-regulating miRNAs in miRNA-deficient ES cell model医学miRNAsandstemcellESCC miRNAs promote the G1/S transition in ES cellsby repressing multiple inhibitors along the Cdk2-Cyclin E pathwayTransfection of ESCC miRNAs (miR-291a-3p, miR-291b-3p, miR-294, and miR-295) individually fully rescues

33、 the G1 accumulation phenotype along with enhanced proliferation.ESCC miRNAs promote the G1/S transition by suppressing inhibitors (p21, Rbl2, and Lats2) along the Cdk2Cyclin E pathway as this is the key G1/S regulating pathway in ES cellsas targets of ESCC miRNAs医学miRNAsandstemcell医学miRNAsandstemce

34、ll医学miRNAsandstemcellCell Cycle Regulation by miRNAs in HumanEmbryonic Stem Cells医学miRNAsandstemcell医学miRNAsandstemcellThe Role of miRNAs in Cell Reprogramming医学miRNAsandstemcellThese reprogramming cocktails have miR connections. Oct4, Sox2 and Nanog inducing the expression of the miR-290 and miR-30

35、2 clusters and c-Myc inducing the miR-1792 cluster. c-Myc can also repress let7 family members indirectly through up-regulation of Lin28B.Transient overexpression of ESC-specific miRs could replace c-Myc when reprogramming fibroblasts with Oct4, Sox2, and Klf4, with miR-294 increasing iPSC derivatio

36、n efficiencies by 20 fold.Adding both c-Myc and miR-294 at the same time had no effect, suggesting that one reason behind the enhancing effect of c-Myc on reprogramming is the induction of ESC-specific miRs医学miRNAsandstemcell医学miRNAsandstemcellmiRNAs 和和ESC 的分化的分化医学miRNAsandstemcell医学miRNAsandstemcel

37、lDicer-deficient ES cells fail to differentiateSize of teratomas following injection of the indicated ES cells into nude mice is plotted as a function of time (weeks post-implantation).Semiquantitative RTPCR analyses of mesoderm- and ectodermspecific differentiation markers.医学miRNAsandstemcellTuj1+

38、cells (neurons)GFAP+ cells (astrocytes)miR-9miR-9/ miR-124 in neuronal system医学miRNAsandstemcellmiR-9 was inhibited alone (2-OMe-9) or together with miR-124a医学miRNAsandstemcellmiRNAs（miRNA1/miRAN133）和心血管系统分化和心血管系统分化原位杂交技术观察发育过程中原位杂交技术观察发育过程中miR1和果蝇肌细胞前体细胞（和果蝇肌细胞前体细胞（Dmef2+）分化的关系）分化的关系医学miRNAsandstem

39、cellmiRNA1通过抑制通过抑制Notch singnaling抑制心脏发育抑制心脏发育miRNA overexpression 使cardioblast 从6个减少到4个miRNA overexpression 使肌前体细胞（Dmef+）数目减少使果蝇翅膀静脉数目增加，但四肢变短医学miRNAsandstemcellmiR-134 levels alone in mouse ES cells enhanced differentiation toward ectodermal lineages, whereas inhibition of miR-134 blocked differentiationmiR-134 and ectodermal differentiationmiR-134 Induces Morphological Changes医学miRNAsandstemcellmicroRNA (miR)-134 modulates transcript levels of lineage-specific biomarkers, downregulates protein levels of pluripotency markers医学miRNAsandstemcelltransfection of PmiR-134 into E14 and D3 mESCs