组学技术在识别肿瘤标志物中的应用.pdf

上海伯豪公司生物标志物筛选与研究研讨会组学技术在识别肿瘤标志物中的应用 在多种层面寻找正 常和癌细胞中差异肿瘤是遗传物质受损而致的疾病(1)GenomeSomatic substitutionInsertions and deletionsCodingNon-codingIntronicIntergenicGenomic leari angeiiientsIntrachromosomal lute rchro niosoni alFusion genesCopy number segments(2)rranscripome(3)A.coding RNAsB.non-coding RNAsC.sense and antisense RNAD.allele-specitic expressionE.fusion genesF.RNA edit(RDD)epigeneticA.DNA methvlationB.Histone modification(?.iniRNAs(21-24bp)D.small RNAs(30-100bp)E.Chromtain remodelingF.others各类组学技术多种方式的验证在多种层面寻找正 常和癌细胞中差异侯选基因目录在较大规模的临床 样本中进行验证基因功能研究他求人悌的疾病预测 高危人群的疾病监测 疾病的早期诊断 临床疾病的诊断传移与复发?年存期的预测?治疗方式的选择（个体化治疗）术/化疗/放疗/牛.物治疗等细胞学水平Aer o bic gl yco l ysis inhibit o r s(Pr o aBH3 mimet ics基因功能研究动物水平C EGFR Xcycl in-dependent I inhibit o r s J I kinase inhibit o r s J七口3亘“抗生长信号的不敏感自给自足生长Jq亏 Susta i ni ngEva di ng growth suppressorsproli f era ti ve si gna li ngD eregula ti ng 细胞能量异常叱,energeti cs基因组不稳定和突PARP inhibit o r sActi va ti ng i nva si on&meta sta si sInduci ng a ngi ogenesi s 持续的血管生成Genome i nsta bi li ty&ta ti onEna bli ng f Tel o mer ase 辟咤嚷-inhibit o r s)i mmorta li ty潜力无限的复制能力Immune act ivat ing ant i-CTLA4 mAbAvoi di ngd；靠滥n避免免疫摧毁Tumorpromoti ng nf la mma ti on促进肿瘤的炎症Sel ect ive ant iinfl ammat o r y dr ugs组织浸润和转移Inhibit o r s o f VEGF signal ingInhibit o r s o f HGF/c-Met细胞学水平动物水平、我国最全的组学平台包括基因芯片，组织芯片，蛋白芯 片，Hi seq2000,Soli d 5500,454以及基因功能研究平台；肿瘤是遗传物质受损而致的疾病标志物体现在以下几个方面(1)GenomeSomatic substitutionInsertions and deletionsCodingNon-codingIntronicIntergenicGenomic leari angeiiientsIntrachromosomal lute rchro niosoni alFusion genesCopy number segments(2)rranscripome(3)A.coding RNAsB.non-coding RNAsC.sense and antisense RNAD.allele-specitic expressionE.fusion genesF.RNA edit(RDD)epigeneticA.DNA methvlationB.Histone modification(?.iniRNAs(21-24bp)D.small RNAs(30-100bp)E.Chromtain remodelingF.others-i s-:-=.-*sr_2-三三IT B8-一艮-一ra-BLr-hD)&:二3*.士；二-Llt二一 联.a-,-rL=;=d:K 一：:,Ir:3r.二总 二W 1123 4 567。12 34 56790利用基因芯片平台完成肝癌D NA copy number va ri a nt(CNV)数据分析，并发现多 个CNV变异区段及相关的高扩 增高表达基因，为开发肝癌治 疗性药物奠定了良好的基础；FEBS Letters 5S0(2006)3571 3581Correla tion between genomic DNA copy number a ltera tions a nd tra nscriptiona l expression in hepa titis B virus-a ssocia ted hepa tocellula r ca rcinomaJi a n Hua ng,.Ha i-Hui Sheng)Ti ng Shena.Yua n-Ji e Hub.Hua-Sheng Xi a ob.Q i n Zha ng.Q i ng-Hua Zha ngb.Zc-Gua ng Ha nJ Chinese Natitmal fhumni Gitunne(enter af Sliunnhai.351 Guo ShaimJing Roatl,Shanghai 21)12(13.Chinah Nuiional EngBieerbig Center for Biochip at Sltiiiigltai,151 Lihin Road.Zlkingjiaiig Hi-Tech Park.Pudong.Sltaiighui 201203.Chiiid Depariinem of Chetninry of Futlan Lniversiiv,220 Hanikm Road.Sltatiglui 2()1)433.ChinaRc c vivc d 13 Marc h 2()06;raised 4 May 2(X)6;uuc vpic d 12 Muy 2()06Available online 22 May 2(X)6Ed ited by Takashi(iojobonStsI 二老-I an I月耳一.3r*r笈 旦旦总 I affft-aRn-一.CZ，r.iaC JlC-j.C c.CC.c.v-.CC.C.-、c c c-方一.C.一 8.aV;.9NW加 W.3：-uw.u SI123 4 567。12 34 56790利用高通量测序平台完成肝 脏转录的数据分析，我国首 篇利用第一代solexa技术(MPSS)研究成果;BMC GenomicsResear ch ar t icl e0 BioMed CentraOpen AccessDiscovering multiple transcripts of human hepatocytes using massively parallel signature sequencing(MPSS)lia n Hua ng11,Pei Ha o1-3,Yun-Li Zha ng11,F u-Xing Deng1,Qing Deng1,Yi Hong5,Xia oAVo Wa ng6,Yun Wa ng1,Ting-Ting Li6,Xue-Gong Zha ng6,Yi-Xue Li-4,Peng-Yua n Ya ng*1,Hong-Ya ng Wa ng*a nd Ze-Gua ng Ha n*1Ad d ress:Departmeni of Chemistry oflud an University Shanghai-Minis(r)r Key labontoiy of Disease and Health Genomic s,Chinese National Human Genome Center at Shanghai,351 Guo Shou-Jing Road.Shanghai 201203.China.;SCBrr intorsens Joint Lab,Shanghai Center tor Bioinfornuuon Tec hnology,100 Qinzhou Road,Shanghai 200235.China.Sc hool ofljfe Sc ienc e.I ud an University,2201 hnhn Road,Shanghai 200-133,China,ShanRhai Instiiutes foi Riologital Sc ienc es,Chinese Ac ad emy of Sc ienc es,320 Yue Yang Road,Shanghai 200031.China.International(2o-operaiion 1 jboraion-on Signal Transd uc tion,liaslern llepalobilian Surgery Institute,the Sec ond Military Med ic al University,Shanghai 200438,China and MOI：Key I jbor.Uan1 of lliointoiinatic s,Deparlmenl ol Aulom.uion.Tsinghua lluiversiiy.Reijing 100064,ChinaI mail:lian lluang-huangjc hgc.sh.c n;Pei I lao-happersc bit.org:Yun-Li/hang-zhangy lc hgc.sh.c n;lu-Xinp Deng-d trnglxc hgc sh c n;，【、介nc 一 ahrtv i I Ic nc .c c itync vi ic tvc An麻tc ic c hiii Qa4utv02o莹与工At MX)-一2*2 Pr c，ax”-tmZc Q-C 5-iMe J”仆 gfG 52oA&qE一工AcovetagrISO160140120100 aoV)40h200 400 6CO 800Medan coverage5 C Q U B A S P 0 O Ho射50 100 150 200 250To t al r eadsBIOC 3092K)Q18)QK心1000800600400008004 芯玉 uoyeulEQ 号s/12345 S7393 12 3 4 5利用Hi seq2000平台，建立分析 100种我国常见单基因遗传病的 检测分析技术，并发现多个新 的单基因遗传病致病基因。OPEN Q ACCESS Freely available online-PLS OHGIdentification of Sequence Variants in Genetic Disease-Causing Genes Using Targeted Next-GenerationSequencingXiaoming Wei15,Xiangchun Ju15,Xin Yi15,Qian Zhu1,Ning Qu1,Tengfei Liu1,Yang Chen,Hui Jiang,Guanghui Yang1,Ruan Zhen1,Zhangzhang Lan Ming Qi Jinming Wang1,Yi Yang1,Yuxing Chu Xiaoyan Li1,Yanfang Guang1,Jian Huang1,2,1 Be币ng Geno mics inst it ut e at Sh enzh en,Sh enzh ei.China.2 Sh angh ai-Minist r y Key Labo r at o r y o f Disease and Heal t h Geno mks,Nat io nal Engineer ing Cent er(o r Bio ch ip at Sh angh ai.Sh angh ai ChinaAbstractBackground:Id entific ation of gene variants plays an important role in researc h on and d iagnosis of genetic d iseases.A c ombination of enric hment of targeted genes and next-generation sequenc ing(targetejd DNA-HiSeq)results in both high effic ienc y and low c ost for targeted sequenc ing of genes of interest.I 2 个 3：；利用Solexa GAII和Soli d 4完 成肝癌肝内转移全外显子数据 分析流程，研究成果已二次修 回(Na ture Geneti cs)，补充 相关数据；Landscape of somatic mutations in advanced hepatocellular carcinomaJi a n Hua ng*2 4 Q i ng D eng12 4.Q un Wa ng12.Kun-Yu Li2.Ni n Li1*2.Bo Zhou12.Xi a o-Ya n Li u1,2.Hui Chen2.Bi ns Ca i5.Ze-Gua ns Ha n1,2*Na ti ona l Huma n Genome Center,Rui-Ji n Hospi ta l.Sha ngha i Ji a otong U ni versi ty School of Medi ci ne.197 Rui-Ji n II Roa d.Sha ngha i 200025.Chi na:-Sha ngha i-MOST Key La bora tory f or D i sea se a nd Hea lth Genomi cs.肝癌组织基因表达谱分析34557390 CENPOI CDK4 一 口.aUR.m鬻I I 丑iniiiiiiiiiiiiiiiBUBi MH.；门 刁日日 CCNA2HH需I CSCC1 ISniS!H!n=肝细胞癌发生发展中部份差异表达基因三、一个新的肝癌抑癌基因的识别和签定一个潜在药靶发现的过程SCARA5 may be a no vel candidat e t umo r suppr esso r gene?Sca venger i eceptor cjstei ne-ri ch-doma i n(SRCR)_ Colla gen-li ke doma i nISpa cer regi on一 Tra nsi nebra ne doma i n一 Intra cellula r regi onScavenger r ecept o r cyst eine-r ich do main1.Whether is SCARA5 gene really down-regulated in HCCs?A-6-7-8-9-10-11-12i-13 I14-15-161718-19Bp0.01J岂 J E 一e SVHVJSGVNCPd GVNCPdNon-HCCHCC80%79.5%The expressi on of SCARA5 versus cli ni ca l f ea turesClinicopa thologica l pa ra metersNumber of pa tientsDown*regulta ionNo Down regulta ionX2P*Genderma le3627(75%)9(25%)fema le42(50%)2(50%)1.290.3AgeN50205(25%)15(75%)502014(70%)6(30%)8.120.004Tumor Size(cm)42319(83%)4(17%)4177(41%)10(59%)7.3760.009*D own-regula ti on of MGC wa s desi gned a s JolunhAAf ter i nocula ti on(da ys)14 21 23 28 31 34 3-41 44After inociihilion(hiys.,，,“Huh7-A(,Number o f so ft agar co l o nies o SggSgSSgRel at ive SCARA5 mRNAS-R N A I N CW R L 6 8s iN A4 8 9S5NA1 5 1 5O O P O 7 4-0 2 4d d o b MNumber o f so ft agar co l o niesS 8 2345678 ooooooooRel at ive SCARA5 mRNA o o o o0 2 4 6 8 0 2S C A R A 5 s=e n c-n g(R N A D p r o m o t e t h e E Pumher oi soft agar coloniesPIXTPRF A RRelative SCAR.45 inRNA.宁多分 L Lo；j L&o w.RNIN-N_RN.J8gX一 RN.A1 15c o _ o n y&RNANCs iR N A4 8 9 S iR N A1 5 1 5ormaionSCARA5 sil encing(RNAi)pr o mo t e t he t umo r igenicit y in vivoJ I O U-l l-W B y l l u dYY-8103 sliRNA-NC shRNA489-2 shRNA489.111.2010 13 17 2124 2731 343741 444752 55 5861 64WRL68shRNA_J89-WRL68-shRNA_NCDavs.Days QThese dat a suppo r t t hat SCARA5 co ul d inhibit t umo r o us gr o wt h.(2)SCARA5 r el at es wit h HCC met ast asisSWAsSOJd%QIVUS 0Az;0H421AP=0.022 -ArMrMrMrMnk-irMnirATk-38HCC with PVTT 40 HCC without PVTTPVTT=po r t al vein t umo r t hr o mbo sisSCARA5 inhibit s cel l invasio n and migr at io n o f HCC cel l sMHCC-LM3YY-8103I)hruo-s e u-oco。Vect o rSCARA5Vector SCARA5SCARA5B-Acti nshRNA-NC shRNA-489 236hrSCARA5 inhibit s Tumo r Met ast asis in vivoMHCC-LM3YY-8103Ad-GEPAd-SCARXSLi vera t 4 weeksshRNA-489-2shRNA-NCLiver at 16 weeks3.By wh ich signal pat h way do es SCARA5 infl uence t h e met ast asis o f HCC cel l s?（，前:西，希SJTmsjEfevk CMj17:早J:瓯1_.0包rirn lj-ajp&E 平 JZSD 瓯 VwT rj5I M硅J率由、2吁，3,1UbLasJS圭iran r/t?.G-J.rH):,k*i*_*H叶”J tF_.H,“-喑1够赢好/.TSiEt再豆一事?7一一.3.一f r息9宙一P 生7,JSL 3,j*.ni声）住“UAf A1 mr n/fy,31y3 ZE-CBD2.回&j-0 8J Se第孱诟g 南药蚩由；L-0C4少 Q1，N-14r“.走 _VX-r质Imaqe Today岁-茶泮.金SCARA5 can co-l o cal ize wit h FAK in PLC cel l sF AK SCARA5MERGE MERGEPLC cel l sSCARA5 physical l y asso ciat es wit h FAKSCARA5-mycSCARA5SCARA5FAKiitpc-ca阻)Ol d(seD0CTl 9T-iAi-(I-IS(3.(S)9ir t L(XVJ)-6TIVd j o Aiiaipb 叫i aj e|nSaj gvHVDS s30P mohH o w d o e s S C A R A 5 r e g u-a t e t h e a c t M t y o f F A K?J 一 HC(ILI-3Y Y8 O 3Hnh7Ad-GFPuiRNA-NCbIRNA-489bIRNA-1515Ad-GFPA(I-SCAR,A5shRNA-NCshRNA-489-2shRNA-489-11L O O o i l1.00 0.8 0SCARA5P I y r 3!7 E A l EAKP1YF165。1 3 0 c8 p i s e c i sPIyr416鼠 S r c7&HSCARA5 o ver expr essio n inh ibit s t h e act ivit y o f MM P-9 but no t MMP-2 in MHCC-LM3 cel l sNat 2005,510MHCC-LM3YY-8103z68m-U STT68-rNZUSMMP2/MMP9RIRIP-9(92KD)MMP-2(m2KD)U NVNHqsWh ich do mains o f SCARA5 ar e r equir ed fo r it s abil it y t o bind FAK and t o inh ibit FAK ph o sph o r yl at io n?SCARA5二9I Z8I 帛I1 59 82 314 384 4951-591-821-314 1-3841-495 i60-495 iFAK缺少胞内段（1-59）SCARA5拉不下来FAKMembrane-s(ollagenDomainSRCRDomainp-Tyr-397(EAK)1.00 1.09 0.85 1.25 0.80 1.40 0.39含有SRCR do main,FAK磷酸 化水平明显降低Cell migrationTumor metastasis4.Wh y is SCARA5 gene do wn-r egul at ed in HCCs?(1)Met hyl at io nUmD.4 06 08l曲 Met hyl at io nO Un-Met h yl at io nBisul fit e sequencing assay sh o wed t h at t h e met h yl at io n l evel s o f SCARA5 pr o mo t er(CG1)was significant l y incr eased in 50%(7/14)HCCs co mpar ed wit h no n-HCCs.Her e,we sh o wed t wo r epr esent at ive r esul t s.In addit io n,no differ ence was o bser ved o n met h yl at io n l evel s o f t h e bo t h CG2 and CG3 bet ween HCC and no n-HCC sampl es.(2)LOH at t he SCA5 l o cus in HCCsdet ect t h e al l el ic imbal ance.30%HCCs sh o wed genet ic LOH.VNHUISVHVDS aARnZBnon-HCC(C29)HCC(C29)Methyi sti onE-Adobe Acrobat ProI E文揩注释表单工具R）询凶窗口给帮助耕&协作/安全 Z签名回表单=附注国文本编辑金也 叵o/O/显示倒多媒体?注释二I桢&。+CO i蹈区勺七叵1 H出产忆.(茶 The Jo ur nal o f Cl inical Invest igat io nResear ch ar t icIeGenetic and epigenetic silencing of SCARA5 may contribute to human hepatocellular carcinoma by activating FAK signalingJian Huang,12 Da-Li Zheng,12 Feng-So ng Qin,12 Na Cheng,12 Hui Chen,2 Bing-Bing Wan,2 Yu-Ping Wang,1*2 Hua-Sheng Xiao,3 and Ze-Guang Han12Nat io nal Human Geno me Cent er,Rui-Jin Ho spit al.Shanghai Jiao t o ng Univer sit y Scho o l o f Medicine,Shanghai,Peo pl es Republ ic o f China.2Shangh ai-MOST Key Labo r at o r y fo r Disease and Heal t h Geno mics,Chinese Nat io nal Human Geno me Cent er at Shangh ai,Shanghai,Peo pl es Republ ic o f China.Nat io nal Engineer ing Cent er fo r Bio chip at Shanghai,Shangh ai,Peo pl es Republ ic o f China.The epigenetic silencing of tumor suppressor genes is a crucial event during carcinogenesis and metastasis.Here,in a human genome-wide survey,we identified scavenger receptor class A,member 5(SCARA5)as a can-1.小分子化合物2.植物提取物3.已知的中药4.其它Geneti c eventsEpi geneti c eventsNext St epCRKERK pl90RhoGAP F1Uin I PKL7 1EV 田n,oliieninon CyclinDlGrb2R.AS/ERK-PLJK-AK1E-vMtlheiiuCdE/RACRACJNKDOCK180Cell migrationCell invasionVEGFIL-8Tumor metastasis（二）1）NA甲基化与肝细胞隔的防治针对DNA甲基转移髀活性和DNA甲基化模式改变.探索附 种新的思路 以析氮杂脱氧胞背（5-Aza-CdR1为代表的竞争性核 制剂.已广泛应用于逆转肿瘤细胞中的异常甲基化.他失旺的男疾病与生命科学前沿研究丛书CdR的作用机制主要是它们可以被整合到处于分裂为 键.从而抑制甲基化转移酶活性随着每一次细胞分 性减少 临床上已有应用5-Aza-CdR治疗白血病及卡 于其明显的骨髓抑制和可能导致基因突变的毒副作用 一个药物Zebula rine是近年来在研究中新发现的.具 Ca lvisi等通过动物实验发现.Zebula rine能激活多个 括RASSFJA,CIS和SOCS1等）.从而抑制肝癌细七 副作用小.具有广阔的临床应用前景M另一种非爰剂普鲁卡因酰股 Proca ina mide）.其作用机制互噬残基甲基化受阻.而非直接聚化到bNA链中I 布临床应用治疗肿瘤的潜力 尽管目前一些潜住DN.J 胞系中证实可恢复甲基化沉默的肿瘤相关是因的表达 的动物和临床试验证实其疗效.但是我们相信.随着 移酶抑制剂必将对肝细胞癌的综合治疗起到重要的推,C29NC29CQ Un-Met hyl at io n Met hyl at io n肿瘤表观遗传学第十八章肝癌表遗传学研究,黄健，2011年Next St epEpi geneti c events1.小分子化合物2.植物提取物3.已知的中药4.其它Geneti c eventsCRKERK pl90RhoGAP F1Uin I PKL-P13K-AK17 1EV 田n,oliieninonR.AS/ERK CyclinDlGrb2E-vMtlheiiuRACJNKCMpaiu Rho CdE/RACDOCK180Cell migrationCell invasionVEGFIL-8Tumor metastasisFAK wer e o ver expr essed in HCC1 a ble 1 CorreU ni on between FAK a nd Phospho-FAK Tyr39/expressi on a nd cli ni copa thologi c va ri a bles of pa ti ents wi th HCC4 2 8642 I I o o o o B ztfE.E3rsdNUE XVHc.EEag/YNBE Yqb.D V N N-U upm 工No nnict ast al ic HCCMet ast at ic HCCVa ri a blenFAKPPho 叩 hFAKP+Serum AFP(ng/b().6480.53 2()13863 7584 54Ca psula r i nva si on0.0430.018Absence11860 5882 36Presence8229 5343 39Tumor gra de0.2320.4(412914 1521 8II13162 6978 53i n4()13 2726 14TNM sta ee0.0380.03316132 2946 151!5623 3334 22Ill8326 5745 38Va scula r i nva si on0.0440.010Absence14069 7196 44Presence6020 4029 31Intruhepa ti c0.0380.024mei a sta si sAbsence12764 6387 40Presence7325 48平 35Ki-670.549().2X650%6S28 4039 29FAK expr essio n l evel s significant l y and r ever sel y co r r el at ed wit h po st o per at ive pat ient sur vival.A a sroAJAJns a&l a s e a s QB 亘一看ns=，法months after operationmonths after operationJ Cancer Res Cl in Onco l.2010 Oct;136(10):1489-96FAK kno ckdo wn inhibit ed cel l adhesio n,migr at io n and invasio n_SKhepl_Control Control siRNA FAKsiRNASMMC7721_Control Control siRNA FAKsiRNACmifral Control siRNA FAKiiKNA()=6oh24hDo wn-r egul at io n o f FAK inhibit ed cel l invasio n t hr o ugh r egul at ing MMP-2 and MMP-9O MMP-9SMMC7721 Ml M P-2 MMP-9cs哥 dKNS1VIN4C7721-onlro Control siRNA OKsfNNA_/-肿瘤研究产（识别差异（确定应用，研究功能）Ackno wl edgement sChinese Nat io nal Human Geno me Cent er at ShanghaiZe-Guang Han,Dal i Zheng,Yul i Zhang,Xiao mei Teng.Nat io nal Engineer ing Cent er fo r Bio chip at ShanghaiHua-Sheng Xiao,Junso ng HanCr editCo l l abo r at io nCo mmunicat io nCollaboration原则Strong desireIdeas Technology ResourceMoney有了问题，有人讨论;全面的解决方案 有了困难，有人解决;先进的技术平台 有了成果，有人分享。优质的售后服务