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1、重大疾病中的非编码RNA调控,主讲人：徐娟 副教授,Single(SNP/Gene/Tf/miRNA)multi-(SNP/Gene/Tf/miRNA)system,Cancer information flow：Two lines,Life line：,Mechanism line：,Analysis ways：,+ways,大纲,非编码RNA简介重大疾病中的miRNA调控重大疾病中的lncRNA调控非编码RNA表达谱在重大疾病中的应用基于miRNA、mRNA双重表达谱分析复杂疾病中的miRNA调控,大纲,非编码RNA简介重大疾病中的miRNA调控重大疾病中的lncRNA调控非编码RNA表达
2、谱在重大疾病中的应用基于miRNA、mRNA双重表达谱分析复杂疾病中的miRNA调控,Junk DNA may not be“junk”93%Transcribed(ENCODE,Nature,2007)95%non-coding RNAs(Science 2010),RNA World,1-2%,non-coding RNA,ncRNA,不能翻译成蛋白的功能性RNA分子Housekeeping non-coding RNAtRNAs、rRNAs、snRNAs etc.Regulatory non-coding RNAsmall non-coding RNAsiRNA、miRNA、piRNA
3、etc.Long non-coding RNA（lncRNA，200 nt）,microRNA的概念,microRNA简称miRNA，一类进化上保守的内源性的非编码RNA,长度约22个核苷酸，在转录后层面调控基因的表达。,非编码RNA,约22,调控基因的表达,内源性,转录后,转录后调控,转录调控,hsa-miR-181a-2*hsa人，mus小鼠，rat大鼠let，lin,mir，miR,181：编号，按注册顺序a：与已注册的miRNA序列高度同源2:由不同染色体上的DNA序列转录加工而成的具有相同成熟体序列的 miRNA,则在后面加上阿拉伯数字以区分*：如果一个前体的2个臂分别产生miRNA
4、,则根据克隆实验,在表达水平较低的miRNA 后加“*”;或进行如下命名 hsa-miR-188-5p(或hsa-miR-188-3p）5p：表示从 5 端的臂加工而来；3p：表示从 3 端的臂加工而来,microRNA命名规则,miRNA的生物合成过程,70-90碱基,22碱基,几百几千碱基,通过和靶基因3UTR（3非翻译区）结合导致RNA诱导的沉默复合体（RNA-induced silencing complex,简称RISC）降解其靶mRNA或阻碍其靶的翻译。,miRNA的作用机制,负向调控因子,miRNA-靶基因靶向关系的识别,实验方法转染实验、荧光素酶报告实验等pSILAC、Argo
5、naute CLIP-Seq和Degradome-Seq靶基因预测方法miRanda、DIANA-microT、TargetScan、RNAhybrid PicTar、miTarget、RNA22、PITAmiRNA靶基因预测常用的原则miRNA与其靶位点的互补性、靶位点的保守性miRNA-mRNA双链之间的热稳定性靶位点处不应有复杂二级结构miRNA 5端与靶基因的结合能力强于3(种子序列),Argonaute CLIP-Seq,Ago binds in a ternary 三元的 complex to both miRNA and mRNA,with sufficiently close
6、contacts to allow UV-crosslinking to either RNA;mRNA tags will be in the immediate vicinity of miRNA binding sites.,miRNA靶基因的高通量实验鉴定方法,Argonaute CLIP-Seq,又称为HITS-CLIP(ultraviolet cross-linking and immune-precipitationand and high-throughput sequencing)，即紫外交联免疫共沉淀与高通量测序偶联技术。CLIP 技术是研究RNA结合蛋白(或者RNA)体内
7、结合靶标的新技术。通过紫外交联将RNA结合蛋白与体内结合的RNA分子进行固定，用Ago蛋白的抗体免疫共沉淀之后酶解未受蛋白保护的RNA，可以获得Ago蛋白直接结合的RNA序列。针对AGO蛋白的CLIP-seq技术能够在全基因组范围内鉴定与AGO蛋白结合的小RNA及其mRNA靶标。Chi,SW,Zang,JB,Mele,A,Darnell,RB.2009.Argonaute HITS-CLIP decodes microRNA-mRNA interaction maps.Nature.460:479-86。,要想获得检测区域被哪个miRNA调控，还需结合预测算法,HITS-CLIP reads
8、do not precisely pinpoint the position of crosslinking between the RNA and protein,and thus can only identify a targeted region(100-nt)as opposed to a specific target site.,2010年，Gene W.Yeo采用AGO-CLIPseq技术在线虫中鉴定了Argonaute的结合位点，发现其不仅结合mRNA的3UTR区域，也会结合编码外显子区域，还发现Argonaute大量结合的区域对于miRNA 的功能非常重要，揭示了其新的自我
9、调控的功能。,Estimated:more than 60%human genes are potentially regulated by miRNAs,预测miRNA的功能,Target genes,miRNA,功能1,功能n,功能2,功能注释/功能富集,miRNA调控信息,Definition of lncRNA,Long non-coding RNAs(long ncRNAs,lncRNAs)are non-protein coding transcripts longer than 200 nucleotides(Perkel 2013).lncRNAs are transcript
10、s that are 200 nucleotides in length and do not have the potential to encode for proteins exceeding lengths of 30 amino acids(Mercer TR.Nat.Rev.Genet.2009,Li X Med.Res.Rev.2012).This somewhat arbitrary limit distinguishes long ncRNAs from small regulatory RNAs such as miRNAs,siRNAs,piRNAs,snoRNAs,an
11、d other short RNAs.,Large scale RNA-seqindicate that lncRNA number in the order of tens of thousands in mammals.9277 manually annotated genes producing 14880 transcripts(GENCODE v7).The number of protein coding genes in our genome has been revised downward multiple timeswhereas the number of known n
12、on protein coding transcripts has increased exponentially over the past decade.However,unambiguously identifying ncRNAs within the cDNA libraries is challenging,since it can be difficult to distinguish protein-coding transcripts from non-coding transcripts.,Features of lncRNAs,As of December 2012,12
13、7 LncRNAs have been functionally annotated inLncRNAdb(a database of literature described LncRNAs)(Amral 2011).,21,lncRNA分析,coding potential exonic structureconservation in mammalshistone modifications post-processiontissue-specific expression Correlations of expression between lncRNAs/mRNAs,lncRNAs
14、have a similar coding potential and contain ORFs similar in length to known lncRNAs and decoy lncRNA,but very different from protein coding genes,most GENCODE lncRNAs lack any protein-coding potential.,LncRNAs have unusual exonic structure,but exhibit standard canonical splice site signals,and alter
15、native splicing,149bp,132bp,2280bp,1602bp,dd,592bp2453bp,Human lncRNAs are under weaker selective constraints than protein-coding genes,and many are primate specific,(Red)No reliable homolog was detected,Expressed lncRNAs have typical histone modifications,Some lncRNAs may be post-processed into sma
16、ller RNAs,particularly snoRNAs,compared lncRNA genomic position with small RNAs on the same strand.,LncRNAs show lower and more tissue-specific expression than protein-coding genes,lncRNAs-mRNA/mRNA-mRNA are more positively than negatively correlated,the higher frequency of lncRNAs-lncRNAs with extr
17、eme positive correlations(6.8%)compared with lncRNAs-mRNAs,lncRNA和mRNA比较,lncRNA,lncRNA的特点类型多、数量多、作用模式多大都是pol II 转录的，具有类似于编码基因的组蛋白标记谱表达具有强的时空和组织特异性与编码基因相比，lncRNA表达水平低、保守性低生化鉴定和功能研究尚处于起步阶段,目前仅有大约100多种已知功能的lncRNAs。lncRNA通过表观遗传学调控、转录调控、转录后调控、蛋白活性调控等多种方式调控相关基因的作用,HOTAIR and PRC2,37,LncRNA as Scaffold for
18、 Transcription Repression,lncRNA研究方法,定性和定量高通量技术：芯片、RNA-seq、RNA captureSeq低通量技术：Northern blot、qRT-PCR、FISH功能分析RNAi、RIP（RIP-chip、RIP-seq）catRAPID(fast prediction of RNA and protein interactions and domains)CHIRP（CHRIP-seq）C-KLAN(combined knockdown and localization analysis of ncRNA)Spinach,Map lncRNA
19、Occupancy,ChIRP:Chromatin Isolation by RNA Purification(ChIRP)Chu et al,Mol Cell 2011,39,Chromatin is crosslinked to lncRNA:protein adducts in vivo.Biotinylated tiling probes are hybridized to target lncRNA,and chromatin complexes are purified using magnetic streptavidin beads,followed by stringent
20、washes.We elute lncRNA-bound DNA or proteins with a cocktail of Rnase A and H.A putative lncRNA binding sequence is schematized in orange.,注释相关数据库miRBase、LNCipedia、noncode、Gencode表达相关数据库GEO、ArrayExpress、SRANRED、YM500、microRNA.org靶基因数据库TarBase、mirTarbaseMicrocosm（miRBase）、TargetScan、microRNA.orgDIANA
21、-LncBase、疾病相关数据库miR2disease、HMDD、LncRNADisease、,常用资源,大纲,非编码RNA简介重大疾病中的miRNA调控重大疾病中的lncRNA调控非编码RNA表达谱在重大疾病中的应用基于miRNA、mRNA双重表达谱分析复杂疾病中的miRNA调控,miRNA表达水平的检测方法,低通量RT-PCR、Northern blot检测的miRNA数目少，准确性相对高高通量microarray、next generation sequencing、Ribonuclease protection assay(核糖核酸酶保护实验，RPA)、Bead-based flow
22、cytometric assay(磁珠流式检测)、原位杂交等同时检测几百个miRNA，准确性相对低,miRNA表达谱,M个miRNAN1个疾病样本、N2个正常样本,利用表达谱寻找复杂疾病相关miRNA,差异表达分析 寻找异常表达miRNAFold change、T-test、SAM、ANOVA等低通量实验证实倍数法（fold change）,实验样本中的表达值,对照样本中的表达值,t检验(t-test),MiRNA与癌症,ProliferationApoptosisInvasion metastasis Angiogenesis,OncogenicmiRNAs,Tumor Suppressor
23、miRNAs,Cancer,miRNA表达的变化认为是发生癌症的共同特征致癌基因的高表达抑癌基因的低表达oncogenic miRNAs(oncomiRs),致癌和抑癌miRNA表达和定位的差别,染色体分布-两者均是成簇出现的抑癌miRNA更多的出现在人类癌症基因组的deleted region致癌miRNA更多的出现在人类癌症基因组的amplified region。表达上，相对于致癌miRNA，抑癌miRNA的表达水平更高，表达组织更广泛,miRNA的表达和作用是组织和肿瘤特异性的,It is thus likely that these miRNAs influence cell pro
24、liferation and tumorigenesis in a cell-type specific manner,depending on the milieu of target mRNAs that are expressed.,大纲,非编码RNA简介重大疾病中的miRNA调控重大疾病中的lncRNA调控非编码RNA表达谱在重大疾病中的应用基于miRNA、mRNA双重表达谱分析复杂疾病中的miRNA调控,重大疾病中的lncRNA调控,相比较miRNA，lncRNA在复杂疾病中的研究正处于起步阶段。最近的研究发现，lncRNA同样是肿瘤生物学的一个重要组分LncRNA 的异常表达与疾病
25、的发生发展有着密切关系LncRNA表达的改变会导致其靶基因的表达改变或者通过异常调控某些重要的表观修饰蛋白进而诱导疾病的发生,lncRNA的异常表达导致剪接调控异常,MALAT-1 是一种转移肺癌相关的lncRNA，它在非小细胞肺癌的转移肿瘤中异常的高表达MALAT-1 高表达的病人常常预后效果较差MALAT1调控SR剪接因子的磷酸化和在剪接斑点（splicing speckle）的分布,MALAT-1 肺癌转移相关转录物 1（metastasis-associated in lung adenocarcinoma transcript 1）,lncRNA as miRNA Decoy,Pol
26、iseno et al,Nat 2011,51,causes of abnormal expression,大纲,非编码RNA简介重大疾病中的miRNA调控重大疾病中的lncRNA调控非编码RNA表达谱在重大疾病中的应用分类人类癌症、诊断标记预后标记基于miRNA、mRNA双重表达谱分析复杂疾病中的miRNA调控,应用一、诊断标记,miRNA作为一种新的生物标记优点miRNA片段小而表达量大，易获取和检测血清中miRNA能够作为一种稳定的生物分子用于癌症的检测表达具有特异性诊断标记、预后标记、肿瘤分期等应用一、miRNA表达谱,miRNA表达谱区分正常/肿瘤样本,大多数miRNA在肿瘤组织中呈
27、现显著的低表达（217个miRNA中有129个p0.05）,并且不依赖肿瘤类型,多个miRNA作为诊断标记,Wang S,Wang L,Bayaxi N,et al.Gut(2012).,miR-375,miR-424 and miR-92a differentiated carcinomas from high-grade intraepithelial neoplasms(accuracy=89%).,26 of 28 adenomas and 103 of 105 carcinomas were correctly classified,Glioma gene expression da
28、ta used in this study were obtained from the publicly available GEO.Affymetrix HG-U133 Plus 2.0 platform2448 lncRNA transcripts with corresponding Affymetrix probe IDsSignificance Analysis of Microarrays was used to determine the differentially expressed lncRNAs2-fold,FDR10%,Differentially expressed
29、 lncRNA probe sets between normal brains and gliomas,Blue-normal brain tissues yellow-glioma samples.,A total of 129 lncRNA probe sets(corresponding to 102 lncRNAs)identified as significantly different between normal brain tissues and gliomas by SAM.,The lncRNA probe sets associated with glioma hist
30、ological subtypes,Green-astrocytomas,red-oligodendrogliomas,A total of 27 lncRNA probe sets were differentially expressed between astrocytomas and oligodendrogliomas.,应用二、预后标记,CANCER CELL 9,189198,MAR 2006,单个miRNA作为预后标记,应用二、预后标记,5个miRNA作为预后标记,Published online May 3,2012,Hierarchical clustering of 18
31、 non-cancer samples(blue)and 312 nasopharyngeal carcinoma samples(yellow)41 differentially expressed microRNAsUnivariate Cox regression analysis discovered 5 out of 41 miRNAs associated with disease-free survival,应用二、预后标记,5个miRNA作为预后标记,Published online May 3,2012,应用二、预后标记,5个miRNA作为预后标记,Published onl
32、ine May 3,2012,应用二、预后标记,5个miRNA作为预后标记,Published online May 3,2012,Patients were divided into high-risk or low-risk groups with the median risk score as a cutoff point.,应用二、预后标记,5个miRNA作为预后标记,Published online May 3,2012,Patients were divided into high-risk or low-risk groups with the median risk scor
33、e as a cutoff point.,应用二、预后标记,5个miRNA作为预后标记,Published online May 3,2012,Journal of Thoracic Oncology:December 2011-Volume 6,应用二、预后标记,大纲,非编码RNA简介重大疾病中的miRNA调控重大疾病中的lncRNA调控非编码RNA表达谱在重大疾病中的应用基于miRNA、mRNA双重表达谱分析复杂疾病中的miRNA调控,剖析重大疾病相关miRNA的调控机制和功能,Target genes,miRNA,功能1,功能n,功能2,It is clear that miRNAs h
34、ave many different targets dozens in some cases,hundreds in others but depending on the cells involved,it seems likely that only a small number of them have a crucial role in cancer pathogenesis.nature reviews genetics(2009),背景信息？,HITS-CLIP,预测疾病背景下miRNA调控的靶基因,数据mRNA表达谱miRNA表达谱miRNA-mRNA靶向关系皮尔森相关系数,注
35、意：miRNA、mRNA表达谱中样本顺序要一致,Case 1,前提假设miRNAs implicated in a specific tumor phenotype will show aberrant regulation of their target genesA key difference from other methods is that we identified dysregulated network edges(regulations)instead of dysregulated nodes(miRNAs)to assemble disease-related sign
36、atures.,IF：5.225,in vitro analysis,RDH11an oncogene,is located in a high-level amplifications region in tumor cells and specifically expressed in the prostate LRP1 promote the invasion and migration ability of lung cancer cells and glioblastoma cellsis directly regulated by hsa-miR-205 in lung cance
37、r cells SMAD3,a major TGF-signaling transducer,miRNA family 的协同失调作用,包含三个miRNA家族mir-125,mir-181 和mir-145家族最小的后验概率为77.4%，表明这些miRNA和前列腺癌相关这些基因富集的功能有pathways in prostate cancer,cell adhesion and apoptosis 等,重大疾病中的miRNA协同调控作用,重大疾病中的miRNA协同调控作用,被引用43次，包括PNAS，Genome research，Frontiers in Genetics，Nucleic Acids Research，,Case 2 胶质瘤恶性进展关联的miRNA-target 调控网络,A hub miRNA signature predicts survival in glioma,Thank You!,
