医学]miRNAs and stem cell.pptVIP

Topic 5 microRNAs and Cell Fate microRNAs 和细胞命运 microRNAs (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 miRNAs 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 miRNAs 通过过不完全的碱基配对对 结结合mRNA 的3非翻译译区（UTRs） 转录转录 后水平上抑制基因翻译译 仅仅降低其靶基因的蛋白质质表达 不影响 mRNA 水平 miRNAs 的生物合成过过程 RNA 聚合酶II (Pol II) 转录转录 合成Pri-miRNA RNase III Drosha 和双链链RNA 结结合蛋白 Pasha 处处理成约约小片段的的pre-miRNA exportin 5 将这这种前体分子输输送到细细胞质质中 RNase III Dicer 剪切产产生约约22 nt的miRNA双链链 被整合到miRISC 复合体中 miRNAs 调节调节 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 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 miRNA biogenesis and its regulation Technology to study function of miRNAs miRNAs overexpression Dicer/DGCR8 knock down + rescue Coupling transcriptional and posttranscriptio nal miRNA regulation in the control of cell fate In cancer cells, inactivation of the miRNA-mediated silencing pathway and the avoidance of miRNA regulation contribute to transformation. (A)miRNAs and TFs in FFLs tend to mutually target genes from th e same pathway. (B) Additionally, coregulated miRNAs and miR NAfamilies cotarget many genes in the same pathway, thus result ing 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 “miRNAtarget spatiotemporal avoidance“ MicroRNAs in Embryonic Stem Cells 1. A 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. 2. Fewer than 100 miRNAs were initially identified in human or murine ES cells by cloning and sequencing from small RNA libraries. 3. In independent studies, different ES cell lines show distinct miRNA profiles. hsa-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, 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-93 a miRNA expression signature characteristic of human ES (hES) cells the 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 miRNAs in mouse ES (mES) cells 1. 390 mouse miRNAs were detected in Balb/c embryos at stages E9.5, E10.5, and E11.5. 2. Temporal expression patterns demonstrate that miRNAs are expressed at specific stages of embryonic development. 1. miR-290 cluster identified as a 2.2-kb region on 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 sequences 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 3. 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 primordial germ cells (PGCs, 原始生殖细胞) and spermatogonia （ 精原干细胞）. 4. Several miRNAs were rare in ES cells but abundant in differentiated 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 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 Regulatory Circuits Controlling ESC Identity Key ES cell transcription factors promote the ES cell miRNA expression program and integrate miRNAs into the regulatory circuitry controlling ES cell identity Connecting microRNA Genes to the Core Transcriptional Regulatory Circuitry of Embryonic Stem Cells Occupancy of miRNA Promoters by Core ES Cell Transcription Factors Cell 134, 521533, August 8, 2008 In 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 suggesting that these regulators have the potential to directly control the transcription 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 upregulated upon Tcf3 depletion. Regulation of Oct4/Sox2/Nanog/Tcf3-Bound miRNA Genes during Differentiation Oct4 and Nanog are silenced as ES cells begin to differentiate target miRNAs, the targets should be differentially expressed when ES cells are compared to a differentiated cell type. Polycomb Group Proteins Co-Occupy Tissue- Specific miRNAs that Are Silenced in ES Cells miRNA Modulation of the Gene Regulatory Network in ES Cells Lefty1 and Lefty2: actively expressed in ES cells occupied at their promoters by Oct4/Sox2/ Nanog/Tcf3 conserved targets of the miR-290295 miRNA family TFs exerts both positive and negative effects on its target Let-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 Dnmt3a and Dnmt3b: indirectly upregulated by the miR-290295 miRNAs occupied at their promoters by Oct4/Sox2/Nanog/Tcf3 Multilevel Regulatory Network Controlling ES Cell Identity miRNAs function in embryonic stem cell differentiation miR-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 Klf4 let-7 family: Antagonizes Myc and Lin28 protein production GENES & DEVELOPMENT 24:27322741 2010 miRNAs Regulate the G1/S Transition in Mouse Embryonic Stem Cells The proliferation rate of mouse ES cells is extremely fast with a cycling time of 10 h. The length of cycling time is increased to more than 18 h in differentiated cells. 1. ES cells have an unusually short G1 phase (2 h) with most (70%) cells in S phase. 2. ES cells lack a G1 restriction point or checkpoint, therefore can proliferate even in the absence of growth factors or mitogens. 3. In mouse ES 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 dependent Cdks: cyclin-dependent kinases G1期PCC为单线状，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 Cdks: cyclin-dependent kinases 细细胞周期蛋白依赖赖性激酶 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。因此， CDK激酶和其调节因子又被称作 细细胞周期引擎。 A) 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 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 of G1 Progression in mESCs Cell 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. Screening strategy to identify cell cycle- regulating miRNAs in miRNA-deficient ES cell model ESCC miRNAs promote the G1/S transition in ES cells by repressing multiple inhibitors along the Cdk2-Cyclin E pathway Transfection of ESCC miRNAs (miR-291a-3p, miR- 291b-3p, miR-294, and miR- 295) individually fully rescues 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 Cell Cycle Regulation by miRNAs in Human Embryonic Stem Cells The Role of miRNAs in Cell Reprogramming These reprogramming cocktails have miR connections. Oct4, Sox2 and Nanog inducing the expression of the miR-290 and miR- 302 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 derivation 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 miRNAs 和ESC 的分化 Dicer-deficient ES cells fail to differentiate Size 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. Tuj1+ cells (neurons) GFAP+ cells (astrocytes) miR-9 miR-9/ miR-124 in neuronal system miR-9 was inhibited alone (2-OMe-9) or together with miR-124a miRNAs（miRNA1/miRAN133） 和心血管系统统分化 原位杂杂交技术观术观 察发发育过过程中miR1和果蝇蝇肌细细胞前体细细胞（Dmef2+）分化的关系 miRNA1通过过抑制Notch singnaling抑制心脏发脏发 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 miR-134 levels alone in mouse ES cells enhanced differentiation toward ectodermal lineages, whereas inhibition of miR-134 blocked differentiation miR-134 and ectodermal differentiation miR-134 Induces Morphological Changes microRNA (miR)-134 modulates transcript levels of lineage-specific biomarkers, downregulates protein levels o