非编码RNA与肿瘤发生发展课件.pptx

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1、,非编码RNA与肿瘤发生发展,癌生物学,An artists conception shows an RNA molecule, which may have served as an early form of life on Earth.,Nicolle Rager Fuller,2,LUCA： Last Universal Common Ancestor,4,The RNA World, 4th edition,Cold Spring Harbor Laboratory (CSHL) Press,2006,2010,1999,1993,Raymond F. Gesteland,Thoma

2、s R. Cech,John F. Atkins,Edited By,5,Regulatory RNAs,Small RNA molecules regulate gene expression by post-transcriptional gene silencing.Xist: the first long non-coding RNA regulating gene expression.Non-coding RNAs control epigenetic phenomena.,http:/www.mfpl.ac.at/rna-biology/rna-history/,Non-codi

3、ng RNAs in Human Genome,6, dkfz.de,组成型非编码RNA rRNA，tRNA，snRNA，snoRNA,调控型非编码RNA 微小非编码RNA microRNA, siRNA, piRNA, etc,长非编码RNA Long non-coding RNAs (LncRNAs),人类基因组（30亿碱基对） 70% 被转录 2%转录后表达蛋白 (mRNA),环状RNA Circular RNA (circRNAs),人类基因组蕴藏着数目巨大的非编码RNA,Non-coding RNAs and Cancer,7,Douglas Hanahan and Robert A

4、. Weinberg, Cell 144, 2011: 646-674.,Oncogenic and Tumor suppressive ncRNAs,Contents,8,Small RNAs (microRNAs); Long non-coding RNAs (lncRNAs); Circular RNAs (circRNAs).,MicroRNA discovery,1993年，第一个miRNA基因lin-4 (秀丽线虫) (Lee, et al. Cell, 1993, 75:843-854),2000年，第二个miRNA基因let-7 (秀丽线虫) (Reinhart, et al.

5、 Nature, 2000, 403: 901-6),2001年，德、美等三个实验室发现约100个miRNA基因 (果蝇、线虫以及人类等) (Lagos-Quintana, et al; Lau, et al; Lee, et al. Science),http:/www.mirbase.org/index.shtml (miRBase Sanger Center),9,Release 21: June, 2014miRNA count: 28645 entries 35828 mature miRNAs in 223 speciesHomo sapiens ：1,881 precursor

6、(2,588),DNA,Cap,AAAAAAAAAA,pri-miRNA,microRNA,Exon,Intron,DNA,Cap,AAAAAAAAAA,ATG,TGA,mRNA,Protein,转录,转录,翻译,加工,microRNA:由19-25个核苷酸组成的非编码小分子RNA,10,Biogenesis and Action of microRNAs,11,MicroRNA-mRNA complex interplay,One miRNA targets multiple mRNAs,One mRNA by multiple miRNAs,12,MicroRNAs can regulat

7、e about 60% of all mRNAs!,MicroRNA Regulation of Cancer Biology,13,miR-124GastroentolgoymiR-199Hepatology,MicroRNA领域值得关注的几个问题,14,1. microRNA生物合成及表达调控机制；2. microRNA在疾病进程中的作用及调控网络；（疾病模型）3. microRNA在生理、病理中的调节作用；（KO，TG）4. microRNA转化医学研究：血清诊断、预后判断、分子分型、治疗探索等,Contents,15,Small RNAs (microRNAs); Long non-c

8、oding RNAs (lncRNAs); Circular RNAs (circRNAs).,LncRNA Discovery,16,LncRNA是一类转录本长度超过200nt的RNA分子,1980s-1990s: Individual lncRNAs (eg, Xist and H19) are discovered through traditional gene mapping approaches; Early 2000s: Large scale cDNA sequencing leads to the discovery of a surprising number of lnc

9、RNA trancscripts; 2000s-Now: The number of predicted protein coding genes in human goes down and the number of detected lncRNA transcripts increases.,GENECODE V21,LncRNA分类和特征,17,根据基因组位置分为5类：,双重活性：可结合蛋白与核酸（RNA,DNA）,Trends Genet. 2014 Oct;30(10):439-52.,LncRNA具复杂的二级与高级结构，决定其分子调控多样性,Nat Rev Genet. 2014

10、 Jan;15(1):7-21.,Sense, Antisense, Intronic, Intergenic, Bidirectional,LncRNA功能和作用机制,18,Models of nuclear lncRNA function,Models of cytoplasmic lncRNA function,Nat Rev Genet. 2014 Jan;15(1):7-21.,mRNA稳定性 mRNA翻译 miRNA sponge蛋白稳定性/修饰,染色质重塑 表观遗传修饰 基因转录 可变剪接,LncRNA具有重要分子调节功能，而不是“转录的噪音”,19,Biochim Biophy

11、s Acta. 2014;1839(11):1097-109.,LncRNA与癌症密切相关,Oncogenic and Tumor suppressive lncRNAs,20,HOX lincRNAs are systematically dysregulated in breast cancer,The lincRNA HOTAIR is increased in expression in primary breast tumours and metastases,HOTAIR has prognostic value for metastasis and survival,HOTAIR

12、 promotes invasion of breast carcinoma cells.,HOTAIR promotes metastasis of breast carcinoma cells.,The mechanism of HOTAIR in breast cancer progression,LncRNA参考文献,27,Long non-coding RNA HOTAIR reprograms chromatin state to promote cancer metastasis. Nature. 2010 Apr 15;464(7291):1071-6. PMID: 20393

13、566；A functional genomic approach identifies FAL1 as an oncogenic long noncoding RNA that associates with BMI1 and represses p21 expression in cancer. Cancer Cell. 2014 Sep 8;26(3):344-57. PMID: 25203321The STAT3-binding long noncoding RNA lnc-DC controls human dendritic cell differentiation. Scienc

14、e. 2014 Apr 18;344(6181):310-3. PMID: 24744378 ；Pubmed关键词： 肿瘤 类型+ lncRNA,Contents,28,Small RNAs (microRNAs); Long non-coding RNAs (lncRNAs); Circular RNAs (circRNAs).,29,Circular RNA,Wilusz and Sharp, Science, 2013,Exonic Circular RNA (circRNAs,环状RNA),Circular RNA (circRNA) is a type of RNA that, un

15、like linear RNA, forms a covalently closed continuous loop.,100,000 circRNAs in human,History of circular RNA,H.L. Sanger, G. Klotz, D. Riesner, H.J. Gross, A.K. Kleinschmidt.(1976)Viroids are single-stranded covalently closed circular RNA molecules existing as highly base-paired rod-like structures

16、. Proc. Natl. Acad. Sci. USA, 73 pp. 38523856.Coca-Prados, M-THaM. (1979) Electron microscopic evidence for the circular form of RNA in the cytoplasm of eukaryotic cells. Nature 280: 339340. . . .,Nigro JM, Cho KR, Fearon ER, Kern SE, Ruppert JM, et al. (1991) Scrambled exons. Cell 64: 607613.,Capel

17、 B, Swain A, Nicolis S, Hacker A, Walter M, et al. (1993) Circular transcripts of the testis-determining gene Sry in adult mouse testis. Cell 73: 10191030. . . .,Salzman J, Gawad C, Wang PL, Lacayo N, Brown PO. (2012) Circular RNAs are the predominant transcript isoform from hundreds of human genes

18、in diverse cell types. PLoS One 7(2):e30733.,Memczak S, Jens M, Elefsinioti A, Torti F, Krueger J, et al. (2013) Circular RNAs are a large class of animal RNAs with regulatory potency. Nature 495(7441):333-8.,Hansen TB, Jensen TI, Clausen BH, Bramsen JB, Finsen B, et al. (2013) Natural RNA circles f

19、unction as efficient microRNA sponges. Nature 495(7441):384-8.Zhang XO, Wang HB, Zhang Y, Lu X, Chen LL, Yang L. (2014) Complementary sequence-mediated exon circularization. Cell. 25;159(1):134-47. . .,30,31,Julia Salzman,Salzman et. al., PLoS one, 2012,32,Memczak et al., Nature, 2013,Hansen et al.,

20、 Nature, 2013,Nikolaus Rajewsky,MCD, Berlin, Germany,Jrgen Kjems,Aarhus University, Denmark,33,Biogenesis of circular RNA,Direct backsplicing,Exon skipping,Jeck and Sharpless, Nature Biotechnology, 2014,34,Liang et al., Genes and Development, 2014,Starke et al., Cell Reports, 2014,Ashwal-Fluss et al

21、., Molecular Cell ,2014,Ivanov et al., Cell Reports, 2015,Conn et al., Cell, 2015,Biogenesis of circular RNA,Zhang et al., Cell, 2014,环状RNA研究现状及问题,一、鉴定（高通量测序生物信息分析）二、生物合成和调控三、功能和机制四、意义,RNA前体剪切加工的副产物？对细胞功能？机制？,相对稳定，是否作为疾病诊疗靶点？,如何生成？! 出核？去向（代谢）？,鉴定方法；组织、癌症相关环状RNA的存在情况？数量？表达？,35,RNA高通量测序鉴定环状RNA,36,人类正常

22、和肿瘤组织中存在大量的环状RNA,38,环状RNA具有组织特异性，在脑组织高表达,N,T,N,T,all cancer tissues,HCC tissues,环状RNA在肿瘤组织中存在差异表达,环状RNA在肿瘤组织中具有重要的临床意义,环状RNA具有重要的功能,环状RNA作用机制？,环状RNA作用机制,44,Summary and prospect,发现 数量 功能 机制,45,Summary and prospect,46,Questions？ Circular RNA is enriched and stable in exosomes: a promising biomarker fo

23、r cancer diagnosis,Questions or Continued？,Cell Res. 2015 Aug;25(8):981-4.,47,The metabolism of circular RNAs?,Highly stable,Predominantly cytoplasmic,Exonucleases,?,48,Exosomes: cell-cell communications,Protein,mRNA,microRNA,?,Note： exosomes nuclear exosome (RNase complex),Raposo et. al., JCB, 2013

24、,30-100 nm,49,cells,FBS-free, overnight,Ultracentrifugation300 g 10min;15000 rpm 20min;40000 rpm 70min,Exosomes,RNA-seq analysis of cell and cell-derived exosomes RNAs,Transmission electron micrograph,50,MHCC-LM3 cells,Total RNA,Exosomes RNA,Ribo (-) Stranded RNAseq,Unmapped Reads,Back-Spliced Reads

25、,Candidate Circular RNAs,TopHat2,Anchor alignment,Circular RNAs are enriched in exosomes,51,Circular RNAs are enriched in exosomes,52,Circular RNAs are enriched in exosomes,53,Circular RNAs sorting to exosomes are regulated,Cellular circRNAs,Exosomal circRNAs,Regulated？,54,Circular RNAs sorting to e

26、xosomes are regulated by associated miRNAs,circRNA,miRNA,cytosol,exosomes,circRNA+miRNA,miRNA,circRNA,circRNA+miRNA,“free” circRNAs in exosomes,?,55,Circular RNA remains biological function in exosomes,cells,Cell proliferation,Cells transfected with associated miRNA,Exosomes,Overexpression of circRN

27、A,56,Identification of circular RNAs in human serum exosomes,350 nt,57,Circular RNAs are intact and stable in serum,58,Tumor-derived circular RNA is detectable in serum,Human cancer cells injected,Harvest serum at 28 days post-injection,Serum exosomal RNA extraction and qRT-PCR for circRNA,59,Exosomal circRNAs distinguish CRC from normal person,60,Conclusion and discussion,CircRNAs are present and enriched in exosomes; Regulation and function of circRNAs in exosomes? Exosomal circRNAs serve as potential biomarkers for cancer and other disease?,Thanks for your attention!,