非编码基因与肿瘤课件

单击此处编辑母版标题样式,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,*,microRNA,与 肿 瘤,LITTLEJIAN1982,FUDAN UNIV,microRNA 与 肿 瘤 LITTLEJIAN19,1,1. microRNA的研究历史,2. microRNA分布和作用机制,3. microRNA与癌症,4. microRNA研究的一般步骤和方法,5 以一篇文章为例介绍一下研究的过程,1. microRNA的研究历史,2,1993,2000,2001,2002,2003,2004,2005,2006,2007,1. microRNA的研究历史,199320002001200220032004200520,3,1993,2000,2001,2002,2003,2004,2005,2006,2007,199320002001200220032004200520,4,1993,2000,2001,2002,2003,2004,2005,2006,2007,199320002001200220032004200520,5,1993,2000,2001,2002,2003,2004,2005,2006,2007,2001年 命名为microrna,199320002001200220032004200520,6,1993,2000,2001,2002,2003,2004,2005,2006,2007,The first demonstration of a link between miRNA genes and cancer.,199320002001200220032004200520,7,1993,2000,2001,2002,2003,2004,2005,2006,2007,Overall, 98 of 186 (52.5%) of miR genes are in cancer-associated genomic regions or in fragile sites,199320002001200220032004200520,8,1993,2000,2001,2002,2003,2004,2005,2006,2007,The first,quantitative real-time PCR,developed for miRNA profiling, designed for the amplification of precursor molecules.,199320002001200220032004200520,9,1993,2000,2001,2002,2003,2004,2005,2006,2007,The first study to report on miRNA profiling using an oligonucleotide microchip.,The first paper to explore, by using genome-wide miRNA profiling by microarray,the potential importance of miRNAs in the diagnosis and prognosis of a human malignancy,199320002001200220032004200520,10,1993,2000,2001,2002,2003,2004,2005,2006,2007,An elegant study that shows a pathogenetic link between miRNAs and target oncogenes.,199320002001200220032004200520,11,1993,2000,2001,2002,2003,2004,2005,2006,2007,The first paper to show that the deregulation of a single miRNA gene can lead to cancer.,199320002001200220032004200520,12,1993,2000,2001,2002,2003,2004,2005,2006,2007,通过生物信息的手段分析了microRNA在蛋白相互作用网络和转录因子-microRNA相互调控网络中的作用,199320002001200220032004200520,13,1993,2000,2001,2002,2003,2004,2005,2006,2007,21-nt dsRNAs targeting selected promoter regions of human genes caused long-lastingand sequence-specific induction of targeted genes,199320002001200220032004200520,14,1993,2000,2001,2002,2003,2004,2005,2006,2007,199320002001200220032004200520,15,1993,2000,2001,2002,2003,2004,2005,2006,2007,Nature,449, 682-688,(11 October 2007),|,199320002001200220032004200520,16,非编码基因与肿瘤课件,17,Nature Biotechnology,25, 961 (2007),Trends in biotech literature 2006,Unsurprisingly, microRNAs dominate the list of highest cited papers, and the area is witnessing rapid growth,Nature Biotechnology 25, 961 (,18,非编码基因与肿瘤课件,19,2.,microRNA分布和作用机制,2. microRNA分布和作用机制,20,Overall, 98 of 186 (52.5%) of miR genes are in cancer-associated genomic regions or in fragile sites,Overall, 98 of 186 (52.5%) of,21,Dicer,microRNA成熟过程,Garzon, R，MicroRNA expression and function in cancer.,Trends in Molecular Medicine,12, 580-587 (2006).,DicermicroRNA成熟过程Garzon, R，Mic,22,5 end of the small RNA,28, known as the seed region,do not have a complex secondary structure and are located in accessible regions of the RNA,microRNA识别靶位点,5 end of the small RNA,28, kn,23,microRNA作用机制,microRNA作用机制,24,3 Microrna,与,癌症,3 Microrna,25,microrna与癌症,microrna与癌症,26,The let-7 family negatively regulates Ras,The miRNAs that are encoded by the,let-7,family were the first group of oncomirs,shown to regulate the expression of an oncogene, specifically the Ras genes.,RAS原癌基因参与细胞的增殖和分化， 约有15-30%的肿瘤中含有RAS基因的突变,The let-7 family negatively re,27,Ras家族有3个成员，即Nras、Kras、Hras分别含有9个、8个和3个let-7的靶位点，在HepG2细胞系中过表达let-7显著降低RAS蛋白的水平，在HeLa细胞系中转染let-7的反义抑制分子，则会提高RAS蛋白的水平,Ras家族有3个成员，即Nras、Kras、Hras分别含有,28,E2F1,E2F1 Tsp1 CTGF,E2F1E2F1 Tsp1 CTGF,29,miR-1792簇,miR-1792簇位于13q31，其中包含了7个microRNA: miR-17-5p, miR-17-3p, miR-18, miR-19a, miR-20, miR-19b-1 和miR-92-1,ODonnell 66等发现MYC可以结合到miR-17-92簇所在基因C13orf25的第一个内含子上，并诱导miR-17-92簇的表达,miR-1792簇miR-1792簇位于13q31，其中,30,He将miR-1792簇中脊椎动物特异的部分miR-1719b 和c-myc在B细胞淋巴癌小鼠模型的造血干细胞中共表达，发现miR-1719b加快恶性淋巴瘤形成的速度，,共表达miR-1719b和c-myc的细胞比仅表达c-myc的细胞有更高的增殖速度和更低的死亡率；,Dews等人发现miR-17-92簇可以抑制抗血管新生的因子Tsp1 和结缔组织生长因子CTGF，从而促进肿瘤中血管的新生，这些,研究都表明miR-1792具有原癌基因的功能,而该簇中miR-17-5p和 miR-20a可以降低E2F1的水平， E2F1通过调控与DNA复制、细胞分裂和凋亡相关的基因控制细胞从G1期到S期， miR-17-92簇通过降低E2F1的水平,又起到了抑癌基因的作用,miR-17-92簇是作为原癌基因还是抑癌基因发挥作用，要依赖于miRNA所在的组织、细胞类型以及胞内存在靶基因的种类,He将miR-1792簇中脊椎动物特异的部分miR-17,31,与p53相关的miRNA,与p53相关的miRNA,32,2007-06-06,2007-06-06,33,非编码基因与肿瘤课件,34,非编码基因与肿瘤课件,35,非编码基因与肿瘤课件,36,4.,microRNA,研究的一般步骤和方法,4. microRNA研究的一般步骤和方法,37,4.,microRNA,研究的一般步骤和方法,4. microRNA研究的一般步骤和方法,38,非编码基因与肿瘤课件,39,Real-time PCR,Real-time PCR,40,Real-time PCR,Real-time PCR,41,Microarray,Direct and sensitive miRNA profiling from low-input total RNA.,RNA,13, 151-159 (2007),MicroarrayDirect and sensitive,42,Microarray-based, high-throughput gene expression profiling of microRNAs.,Nat Methods,1, 155-161 (2004).,Microarray-based, high-through,43,非编码基因与肿瘤课件,44,非编码基因与肿瘤课件,45,非编码基因与肿瘤课件,46,非编码基因与肿瘤课件,47,miR-122 regulation of lipid metabolism revealed by in vivo antisense targeting.,Cell Metab,3, 87-98 (2006).,miR-122 regulation of lipid me,48,Nature,449, 682-688 (,11 October 2007,),5 以一篇文章介绍一下研究的过程,Nature 449, 682-688 (11 Octobe,49,非编码基因与肿瘤课件,50,Out of a total of eight selected miRNAs,three (miR-155, miR-9 and miR-10b) were found to be markedly upregulated,in breast cancer cells when compared with either primary human mammary epithelial cells (HMECs) or with the spontaneously immortalized MCF- 10A cells,Out of a total of eight select,51,the expression level of miR-10b was 50-fold higher in cells of the MDA-MB-231 line, which are capable of metastasizing,than in cells of the MCF-7 human breast cancer line, which have little if any metastatic powers .,This correlation indicated that miR-10b might well have a causal role in breast cancer metastasis,.,the expression level of miR-10,52,5.1 miR-10b,cell migration and invasion,in vitro,5.2 miR-10b,tumour invasion,in vivo,5.3 miR-10b,distant metastasis,in vivo,5.4,how miR-10b expression is regulated?,5. 5 What is a direct and functional target of miR-10b?,5.6 miR-10b expression is elevated in metastatic breast tumours,5.1 miR-10b cell migration a,53,5.1 miR-10b,cell migration and invasion,in vitro,first performed in vitro loss-of-function analyses by silencing the miRNAs with,antisense oligonucleotides,suggesting that each of the transfected antisense RNAs achieved a greater than,50% inhibition,of the actions of its cognate miRNA.,5.1 miR-10b cell migration a,54,Although neither of these two antisense RNAs affected the motility of MDA-MB-231 cells,silencing of miR-10b led to a more than tenfold reduction in the invasive properties of these cells,Transwell migration assay,Matrigel invasion assay,Although neither of these two,55,Trypan blue exclusion assay(,台盼蓝拒染法,),This reduction was not due to impairment of cell viability,Taken together, these observations suggested that miR-10b function is required,for in vitro invasiveness,but,not for viability or motility of these metastatic cells.,Trypan blue exclusion assay(台盼,56,miR-10b overexpression,cloned the genomic sequence of the human mir-10b gene into a green fluorescent protein (,GFP,)-expressing, murine stem-cell retrovirus (,MSCV,)-derived vector1,miR-10b overexpressioncloned,57,These results indicated that overexpression of miR-10b is sufficient to promote both migration and invasion in vitro.,These results indicated that o,58,5.1 miR-10b,cell migration and invasion in vitro,5.2 miR-10b,tumour invasion in vivo,5.3 miR-10b,distant metastasis,in vivo,5.4,how miR-10b expression is regulated?,5. 5 What is a direct and functional target of miR-10b?,5.6 miR-10b expression is elevated in metastatic breast tumours,5.1 miR-10b cell migration a,59,5.2 miR-10b,tumour invasion,in vivo,implanted miR-10b-transduced or mock-infected SUM149 cells into the mammary fat pads of NOD-SCID mice(该品系鼠为非肥胖糖尿病/严重联合免疫缺陷鼠，不但成熟T、B细胞联合免疫缺陷，而且单核巨噬细胞、NK细胞功能下降，补体系统不健全，人类原代癌症和肿瘤很容易在其体内成癌症和肿瘤 ).,GFP expression was maintained in the tumour cells,5.2 miR-10b tumour invasion,60,the control SUM149 tumours were strictly noninvasive, as shown by their,confinement within fibrotic capsules,(A, B).,miR-10b-overexpressing SUM149 tumours displayed a massive desmoplastic reaction, with islands of epithelial cancer cells that had invaded the stroma (C, D).,apparent muscular and vascular invasion (E, F),苏木素-伊红染色,the control SUM149 tumours wer,61,Ki-67 proliferation marker,MECA-32 endothelial cell marker.,To determine whether miR-10b expression in the primary tumours would also affect,cell proliferation and tumour angiogenesis,the distribution, but not the total number, of Ki-671 cells,in the miR-10b-overexpressing SUM149 tumours was distinct from that seen in the control tumours,Ki-67 proliferation marker MEC,62,Quantification of Ki-67 staining (percentage of Ki-671 carcinoma cells among total carcinoma cells; e) and vessels (using MECA-32-stained sections; f) at the centre and the edge of the,SUM149 tumours.,n53 mice at 6 weeks after implantation.,Prominent,intratumoural vessels,are associated with the invasion front of miR-10b-overexpressing tumours as,demonstrated by MECA-32 staining of primary mammary tumours formed,by SUM149 cells infected with the miR-10b-expressing or empty vector,Quantification of Ki-67 staini,63,5.1 miR-10b,cell migration and invasion in vitro,5.2 miR-10b,tumour invasion in vivo,5.3 miR-10b,distant metastasis in vivo,5.4,how miR-10b expression is regulated?,5.5 What is a direct and functional target of miR-10b?,5.6 miR-10b expression is elevated in metastatic breast tumours,5.1 miR-10b cell migration a,64,5.3 miR-10b,distant metastasis,in vivo,MECA-32- and Ki-67-stained sections of a primary mammary tumour formed by miR-10btransduced SUM149 cells, at,week 6,after orthotopic transplantation.,Red arrows in panel A indicate tumour cells within a vessel, and,black arrows in panel D indicate endothelial cells,. Magnification: panels A and C, X100; panels B and D: X400.,5.3 miR-10b distant metastas,65,Figure S2 | Detection of,single cell metastases,at early stage in,lungs,of mice bearing miR-10b-overexpressing SUM149 tumours.,At week 6 after implantation,Nuclei were demonstrated by DAPI. Magnification: 200x.,Figure S2 | Detection of singl,66,H&E- and AE1/AE3-stained sections of lungs , at,week 9 after transplantation,.,Circles indicate clusters of metastatic cells,.,The arrow indicates normal,bronchial epithelium,.,Inset, AE1/AE3 staining of a SUM149 primary tumour.,H&E- and AE1/AE3-stained secti,67,At 9 weeks after implantation ,On average, we found ,1 micrometastasis,per 5-mm section,(Fig. 3c, left panel).,At week 11, there was a further increase in the number of such micrometastatic clusters (,4 micrometastases per section, Fig. 3c, right panel).,At 9 weeks after implantation,68,SUM159+,miR-10b,Bright field, GFP imaging, and H&E staining of,lungs,isolated from mice that received orthotopic injection of miR-10b-transduced or mock-infected SUM159 cells, at week 11 after transplantation.,SUM159+ miR-10bBright field, G,69,Taken together, these observations,indicate that ectopic miR-10b expression can drive tumour invasion,and metastasis in otherwise non-metastatic breast tumours, thereby,acting,as a potent pro-metastatic agent,.,Taken together, these observat,70,5.1 miR-10b,cell migration and invasion in vitro,5.2 miR-10b,tumour invasion in vivo,5.3 miR-10b,distant metastasis in vivo,5.4,how miR-10b expression is regulated?,5.,5 What is a direct and functional target of miR-10b?,5.6 miR-10b expression is elevated in metastatic breast tumours,5.1 miR-10b cell migration a,71,5.4,how miR-10b expression is regulated?,first assessed miR-10b expression in the four lines of mouse,mammary tumour cells that had been used in the identification of,Twist as a metastasis-promoting gene,the lowest expression level in 67NR cells, which are unable to intravasate from the primary tumour,the highest expression level was seen in 4T1 cells, which are capable of generating macroscopic metastases,This closely paralleled the expression pattern of Twist in these cell lines, indicating that miR-10b expression might well be upregulated by this transcription factor.,5.4 how miR-10b expression is,72,expressed either,Twist,or, as a control, a second EMT(,上皮一间质转变,)-inducing transcription factorsnail (,SNAI1,)in the non-tumourigenic, immortalized HMECs,ectopic expression of Twist1 led to a 4.5-fold increase in the level of this miRNA in these HMECs,expressed either Twist or, as,73,Twist bind to E-box sequences CANNTG,Examined the 4-kb genomic sequence upstream of the human mir-10b stemloop and identified two conserved E-boxes, at-313 bp (E-box 1) and -2,422 bp (E-box 2), respectively,The,CHIP,experiments revealed that TWIST1 bound to E-box 1, but not to E-box 2,Twist bind to E-box sequences,74,whether this miRNA is required for Twist1-induced migration and invasion?,.,Strikingly, miR-10b inhibition consistently led to a fivefold reduction in the motility and invasiveness of Twist1- overexpressing HMECs,whether this miRNA is required,75,5.1 miR-10b,cell migration and invasion in vitro,5.2 miR-10b,tumour invasion in vivo,5.3 miR-10b,distant metastasis,in vivo,5.4,how miR-10b expression is regulated?,5.5 What is a direct and functional target of miR-10b?,5.6 miR-10b expression is elevated in metastatic breast tumours,5.1 miR-10b cell migration a,76,5.5 What is a direct and functional target of miR-10b?,Among the approximately 100 targets predicted by both the,TargetScan,and,PicTar,search programs, two genes,homeobox D10 (HOXD10) and RB1CC1,were previously implicated in suppression of cell migration and/or invasion.,HOXD10 was of particular interest, because its expression has been found to be progressively,lost in breast tumours,showing increasing degrees of malignancy.,More importantly,restored expression,of HOXD10 in MDA-MB-231 cells has been found to,impair migration and invasion,in vitro as well as tumour progression in vivo,5.5 What is a direct and funct,77,miR-10b overexpression did not cause degradation of HOXD10 mRNA,it did, however, reduce the activity of a,luciferase reporter gene fused to the wild-type HOXD10 UTR (,48% reduction,), indicating that miR-10b targets HOXD10 through translational inhibition,miR-10b overexpression did not,78,a clear reduction in the level of the endogenous HOXD10 protein in miR-10b-overexpressing cells,HOXD10,represses,expression of genes that are involved in cell migration and extracellular matrix remodelling, including,RHOC, a3 integrin, matrix metalloproteinase-,14, and urokinase-type plasminogen activator receptor,RHOC has been identified as an especially important player in metastasis, and its expression correlates with metastatic spread of various types of carcinomas,a clear reduction in the level,79,whether reduction of HOXD10 levels might provide an explanation for the induction of cell motility and invasiveness observed following miR-10b overexpression?,Overexpressed miR-10b in SUM149 cells together with a construct expressing HOXD10 constitutively; this construct encodes the entire HOXD10 coding sequence but lacks the UTR of HOXD10-encoding mRNA,constitutive expression of HOXD10 completely abrogated miR-10b-induced cell motility and invasiveness,whether reduction of HOXD10 le,80,Transfection of RHOC siRNA (small-interfering RNA), which caused a.90% reduction in the level of the RHOC protein (Fig. 5f), led to a strong but not complete suppression of miR-10b-induced cell migration,Transfection of RHOC siRNA (sm,81,5.6 miR-10b expression is elevated in metastatic breast tumours,miR-10b expression level was lower in all of the breast arcinomas,from metastasis-free patients (5/5).,n contrast, 50% of the metastasis-positive patients (9/18) had elevated miR-10b levels in their primary tumours,5.6 miR-10b expression is elev,82,They observed that ectopic miR-10b expression in non-tumourigenic, immortalized EpH4 mouse mammary epithelial cells42 did not transform them to a tumourigenic state, whereas overexpression of Ras indeed enabled their tumourigenic growth (data not shown).,Collectively, our findings indicate that,miR-10b plays a part specifically in the metastatic process but not in primary tumour formation,.,They observed that ectopic miR,83,非编码基因与肿瘤课件,84,Thank you !,Thank you !,85,