1、非非编码编码RNARNA与肿瘤发生发展与肿瘤发生发展癌生物学癌生物学An artists conception shows an RNA molecule, which may have served as an early form of life on Earth.Nicolle Rager Fuller2 LUCA： Last Universal Common Ancestor4The RNA World, 4th editionCold Spring Harbor Laboratory (CSHL) Press2006201019991993Raymond F. GestelandTh
2、omas R. CechJohn F. AtkinsEdited By5Regulatory 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-codin
3、g RNAs in Human Genome6 dkfz.de组成型非编码组成型非编码RNA rRNA，tRNA，snRNA，snoRNA调控型非编码调控型非编码RNA 微小非编码微小非编码RNA microRNA, siRNA, piRNA, etc 长非编码长非编码RNA Long non-coding RNAs (LncRNAs)人类基因组人类基因组（30亿碱基对） 70% 被转录 2%转录后表达蛋白 (mRNA)环状环状RNA Circular RNA (circRNAs)人类基因组蕴藏着数目巨大的非编码人类基因组蕴藏着数目巨大的非编码RNANon-coding RNAs and Ca
4、ncer7Douglas Hanahan and Robert A. Weinberg, Cell 144, 2011: 646-674.Oncogenic and Tumor suppressive ncRNAsContents8 Small RNAs (microRNAs); Long non-coding RNAs (lncRNAs); Circular RNAs (circRNAs).MicroRNA discovery1993年，第一个年，第一个miRNA基因基因lin-4 (秀丽线虫秀丽线虫) (Lee, et al. Cell, 1993, 75:843-854)2000年，第二
5、个年，第二个miRNA基因基因let-7 (秀丽线虫秀丽线虫) (Reinhart, et al. Nature, 2000, 403: 901-6)2001年，德、美等三个实验室发现约年，德、美等三个实验室发现约100个个miRNA基因基因 (果蝇、线虫以及人类等果蝇、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)9Release 21: June, 2014miRNA count: 28645
6、entries 35828 mature miRNAs in 223 speciesHomo sapiens ：1,881 precursor (2,588)DNACapAAAAAAAAAApri-miRNAmicroRNAExonIntronDNACapAAAAAAAAAAATGTGAmRNAProtein转录转录翻译加工microRNA:由由19-25个核苷酸组成的非编码小分子个核苷酸组成的非编码小分子RNA10Biogenesis and Action of microRNAs11MicroRNA-mRNA complex interplayOne miRNA targets multi
7、ple mRNAsOne mRNA by multiple miRNAs12MicroRNAs can regulate about 60% of all mRNAs!MicroRNA Regulation of Cancer Biology13miR-125b, HepatologymiR-210HepatologymiR-423CarcinogenesismiR-151Nature Cell BiologymiR-195, HepatologymiR-194, Science SignalingmiR-30d, HepatologymiR-124GastroentolgoymiR-199H
8、epatologyMicroRNA领域值得关注的几个问题领域值得关注的几个问题141. microRNA生物合成及表达调控机制；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）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；（在生理、病理中的调节作用；（KO，TG）4. microRNA转化医学研究：转化医学研究：血清诊断、预后判断、分子分型、治疗探索等血清诊断、预后判断、分子分型、治疗探索等Contents15 Small RNAs (microRNAs); Long non-coding RNAs (lncR
9、NAs); Circular RNAs (circRNAs).LncRNA Discovery16LncRNA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 lncRNA trancscripts; 2
10、000s-Now: The number of predicted protein coding genes in human goes down and the number of detected lncRNA transcripts increases.mRNA 19881sncRNA9534LncRNA 30344GENECODE V21LncRNA分类和特征分类和特征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
11、Genet. 2014 Jan;15(1):7-21.Sense, Antisense, Intronic, Intergenic, BidirectionalLncRNA功能和作用机制18Models of nuclear lncRNA function Models of cytoplasmic lncRNA function Nat Rev Genet. 2014 Jan;15(1):7-21.l mRNA稳定性l mRNA翻译l miRNA spongel蛋白稳定性/修饰l 染色质重塑l 表观遗传修饰l 基因转录l 可变剪接LncRNA具有重要分子调节功能，而不是“转录的噪音”19Bi
12、ochim Biophys Acta. 2014;1839(11):1097-109.LncRNA与癌症密切相关Oncogenic and Tumor suppressive lncRNAs20HOX lincRNAs are systematically dysregulated in breast cancerThe lincRNA HOTAIR is increased in expression in primary breast tumours and metastases HOTAIR has prognostic value for metastasis and survival
13、HOTAIR promotes invasion of breast carcinoma cells.HOTAIR promotes metastasis of breast carcinoma cells.The mechanism of HOTAIR in breast cancer progressionLncRNA参考文献参考文献271. Long non-coding RNA HOTAIR reprograms chromatin state to promote cancer metastasis. Nature. 2010 Apr 15;464(7291):1071-6. PMI
14、D: 20393566；2. 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: 252033213. The STAT3-binding long noncoding RNA lnc-DC controls human dendritic cell different
15、iation. Science. 2014 Apr 18;344(6181):310-3. PMID: 24744378 ；4. Pubmed关键词：关键词： 肿瘤肿瘤 类型类型+ lncRNAContents28 Small RNAs (microRNAs); Long non-coding RNAs (lncRNAs); Circular RNAs (circRNAs).29Circular RNAWilusz and Sharp, Science, 2013Exonic Circular RNA (circRNAs,环状环状RNA)Circular RNA (circRNA) is a
16、type of RNA that, unlike linear RNA, forms a covalently closed continuous loop. 100,000 circRNAs in humanHistory of circular RNAH.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 r
17、od-like structures. 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
18、 64: 607613.Capel 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 hundred
19、s of human genes 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) Natura
20、l RNA circles function 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. . . 发 现（1976） 拓 展（1990s） 热 点（2010s）3031Julia SalzmanSalzman et. al., PLoS one, 201232Me
21、mczak et al., Nature, 2013Hansen et al., Nature, 2013Nikolaus RajewskyMCD, Berlin, GermanyJrgen KjemsAarhus University, Denmark 33Biogenesis of circular RNADirect backsplicingExon skippingJeck and Sharpless, Nature Biotechnology, 201434Liang et al., Genes and Development, 2014Starke et al., Cell Rep
22、orts, 2014Ashwal-Fluss et al., Molecular Cell ,2014Ivanov et al., Cell Reports, 2015Conn et al., Cell, 2015Biogenesis of circular RNAZhang et al., Cell, 2014环状环状RNA研究现状及问题研究现状及问题一、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？
23、35RNA高通量测序鉴定环状高通量测序鉴定环状RNA36人类正常和肿瘤组织中存在大量的环状人类正常和肿瘤组织中存在大量的环状RNA38环状环状RNA具有组织特异性，在脑组织高表达具有组织特异性，在脑组织高表达NTNTall cancer tissuesHCC tissues环状环状RNA在肿瘤组织中存在差异表达在肿瘤组织中存在差异表达环状环状RNA在肿瘤组织中具有重要的临床意义在肿瘤组织中具有重要的临床意义环状环状RNA具有重要的功能具有重要的功能环状环状RNA作用机制？作用机制？环状环状RNA作用机制作用机制44Summary and prospectmiRNA 1990-00s 2000
24、明确 单一LncRNA 1980-90s 30000 待探索 多样复杂circRNA 1970-90s 100000 待探索 未知 发现发现 数量数量 功能功能 机制机制45Summary and prospectmiRNAcircRNALncRNA46 Questions？ Circular RNA is enriched and stable in exosomes: a promising biomarker for cancer diagnosisQuestions or Continued？Cell Res. 2015 Aug;25(8):981-4.47The metabolism
25、 of circular RNAs?Highly stablePredominantly cytoplasmic Exonucleases 48Exosomes: cell-cell communications ProteinmRNAmicroRNA?Note： exosomes nuclear exosome (RNase complex)Raposo et. al., JCB, 201330-100 nm49cellsFBS-free, overnightUltracentrifugation300 g 10min;15000 rpm 20min;40000 rpm 70minExoso
26、mesRNA-seq analysis of cell and cell-derived exosomes RNAsTransmission electron micrograph50MHCC-LM3 cellsTotal RNAExosomes RNARibo (-) Stranded RNAseqUnmapped ReadsBack-Spliced ReadsCandidate Circular RNAsTopHat2Anchor alignmentCircular RNAs are enriched in exosomes51Circular RNAs are enriched in e
27、xosomes52Circular RNAs are enriched in exosomesCircular exonLinear-Spliced Reads (L1)Linear-Spliced Reads (L2)Back-Spliced Reads (B)Back-Spliced Ratio= B / (0.5 X L1+0.5 X L2)53Circular RNAs sorting to exosomes are regulatedCellular circRNAsExosomal circRNAsRegulated？54Circular RNAs sorting to exoso
28、mes are regulated by associated miRNAscircRNAmiRNAcytosolexosomescircRNA+miRNAmiRNAcircRNAcircRNA+miRNA“free” circRNAs in exosomes55Circular RNA remains biological function in exosomescellsCell proliferationCells transfected with associated miRNAExosomesOverexpression of circRNA56Identification of c
29、ircular RNAs in human serum exosomes350 nt57Circular RNAs are intact and stable in serum58Tumor-derived circular RNA is detectable in serumHuman cancer cells injectedHarvest serum at 28 days post-injection Serum exosomal RNA extraction and qRT-PCR for circRNA59Exosomal circRNAs distinguish CRC from normal person60Conclusion 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!
