CN112067809A - 靶向DNAJB6b的试剂在制备治疗肿瘤的药物中的用途 - Google Patents
靶向DNAJB6b的试剂在制备治疗肿瘤的药物中的用途 Download PDFInfo
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Abstract
本发明涉及靶向DNAJB6b的试剂在制备治疗肿瘤的药物中的用途。具体地,本发明涉及靶向DNAJB6b的试剂在制备治疗肿瘤的药物中的用途,其中所述靶向DNAJB6b的试剂降低DNAJB6b蛋白质的表达水平或拮抗DNAJB6b蛋白质的功能。另一方面,本发明提供了一种体外筛选抗肿瘤,优选抗结直肠癌的试剂的方法。
Description
技术领域
本发明涉及肿瘤治疗的技术领域。具体地,本发明涉及靶向DNAJB6b的试剂在制备治疗肿瘤的药物中的用途,其中所述靶向DNAJB6b的试剂降低DNAJB6b蛋白质的表达水平或拮抗DNAJB6b蛋白质的功能。
背景技术
结直肠癌(Colorectal cancer,CRC)是常见的消化道恶性肿瘤,其发病率和死亡率在世界范围内分别位居恶性肿瘤的第三位和第二位(参见,Bray,F.,Ferlay,J.,Soerjomataram,I.,Siegel,R.L.,Torre,L.A.,and Jemal,A.(2018)Global cancerstatistics 2018:GLOBOCAN estimates of incidence and mortality worldwide for36 cancers in 185 countries.CA Cancer J Clin 68,394-424;Ferlay,J.,Colombet,M.,Soerjomataram,I.,Mathers,C.,Parkin,D.M.,Pineros,M.,Znaor,A.,and Bray,F.(2019)Estimating the global cancer incidence and mortality in 2018:GLOBOCANsources and methods.Int J Cancer 144,1941-1953)。目前,结直肠癌造成的疾病负担在全球范围内仍在持续加剧,并且发病呈现年轻化的趋势。预计到2030年,全球结直肠癌负担将增加60%,新发病例将超过220万例、死亡病例可达10万例。最新统计数据显示,我国CRC的发病率和死亡率分别位居恶性肿瘤的第3位和第5位,且呈逐年上升趋势(参见,Arnold,M.,Sierra,M.S.,Laversanne,M.,Soerjomataram,I.,Jemal,A.,and Bray,F.(2017)Global patterns and trends in colorectal cancer incidence and mortality.Gut66,683-691;Zheng,R.S.,Sun,K.X.,Zhang,S.W.,Zeng,H.M.,Zou,X.N.,Chen,R.,Gu,X.Y.,Wei,W.W.,and He,J.(2019)[Report of cancer epidemiology in China,2015].Zhonghua Zhong Liu Za Zhi 41,19-28)。
我国CRC患者就诊时多为中晚期,尽管治疗方法和手段不断改善,但晚期患者的5年生存率一直徘徊在10%左右。化疗是晚期及转移性CRC治疗的重要手段,但是肿瘤细胞的耐药性导致多数患者疗效不佳。近年来,靶向治疗和免疫治疗取得了一些进展,但只有不到1/3的患者可从这些治疗中获益(参见,Miller,K.D.,Nogueira,L.,Mariotto,A.B.,Rowland,J.H.,Yabroff,K.R.,Alfano,C.M.,Jemal,A.,Kramer,J.L.,and Siegel,R.L.(2019)Cancer treatment and survivorship statistics,2019.CA Cancer J Clin 69,363-385;Allemani,C.,Weir,H.K.,Carreira,H.,Harewood,R.,Spika,D.,Wang,X.S.,Bannon,F.,Ahn,J.V.,Johnson,C.J.,Bonaventure,A.,Marcos-Gragera,R.,Stiller,C.,Azevedo e Silva,G.,Chen,W.Q.,Ogunbiyi,O.J.,Rachet,B.,Soeberg,M.J.,You,H.,Matsuda,T.,Bielska-Lasota,M.,Storm,H.,Tucker,T.C.,Coleman,M.P.,and Group,C.W.(2015)Global surveillance of cancer survival1995-2009:analysis of individualdata for 25,676,887patients from 279 population-based registries in67countries(CONCORD-2).Lancet 385,977-1010;参见Brenner,H.,Kloor,M.,and Pox,C.P.(2014)Colorectal cancer.Lancet 383,1490-1502;和于永扬,陈海宁,周总光(2019)我国结直肠癌的现状、制约瓶颈与反思.中国普外基础与临床杂志.26,897-902)。目前,由于对CRC的发病机理认识不足,大部分晚期及转移性CRC患者仍缺乏有效的治疗药物。因此,迫切需要寻找在结直肠癌发生发展中发挥重要作用的关键分子,为临床提供有效的药物治疗靶点。
DNAJB6属于J蛋白/HSP40家族,DNAJB亚家族(参见,Meng,E.,Shevde,L.A.,andSamant,R.S.(2016)Emerging roles and underlying molecular mechanisms of DNAJB6in cancer.Oncotarget 7,53984-53996)。DNAJB6为辅分子伴侣,在机体内广泛表达,可参与底物蛋白(或称客户蛋白)的正确折叠及其在细胞内的转运,阻止蛋白质异常聚集、调控蛋白降解及重塑等过程。此外,DNAJB6在胚胎发育、神经干细胞的自我更新、基因转录调控、激活信号分子等方面也发挥重要作用(参见,Meng,E.,Shevde,L.A.,and Samant,R.S.(2016)Emerging roles and underlying molecular mechanisms of DNAJB6 incancer.Oncotarget 7,53984-53996;Hageman,J.,Rujano,M.A.,van Waarde,M.A.,Kakkar,V.,Dirks,R.P.,Govorukhina,N.,Oosterveld-Hut,H.M.,Lubsen,N.H.,andKampinga,H.H.(2010)A DNAJB chaperone subfamily with HDAC-dependent activitiessuppresses toxic protein aggregation.Mol Cell 37,355-369;和Kampinga,H.H.,andCraig,E.A.(2010)The HSP70 chaperone machinery:J proteins as drivers offunctional specificity.Nat Rev Mol Cell Biol 11,579-592)。
人DNAJB6基因定位于7q36.3,可产生两个转录本,分别编码两个不同的蛋白异构体,即长异构体DNAJB6a(326aa)和短异构体DNAJB6b(242aa)。DNAJB6b缺少DNAJB6a C端的95个氨基酸,但含有另外10个独有的氨基酸(DNAJB6a的氨基酸序列请参见GenBank登录号NP_490647.1;DNAJB6b的氨基酸序列请参见GenBank登录号NP_005485.1)。除C端不同外,这两个异构体都含有一个位于N端的保守的J结构域,一个富含甘氨酸和苯丙氨酸残基的G/F结构域和富含丝氨酸(SSF-SST区)的C端结构域(参见,Meng,E.,Shevde,L.A.,and Samant,R.S.(2016)Emerging roles and underlying molecular mechanisms of DNAJB6 incancer.Oncotarget 7,53984-53996;Hageman,J.,Rujano,M.A.,van Waarde,M.A.,Kakkar,V.,Dirks,R.P.,Govorukhina,N.,Oosterveld-Hut,H.M.,Lubsen,N.H.,andKampinga,H.H.(2010)A DNAJB chaperone subfamily with HDAC-dependent activitiessuppresses toxic protein aggregation.Mol Cell 37,355-369;Kampinga,H.H.,andCraig,E.A.(2010)The HSP70 chaperone machinery:J proteins as drivers offunctional specificity.Nat Rev Mol Cell Biol 11,579-592;和Mitra,A.,Fillmore,R.A.,Metge,B.J.,Rajesh,M.,Xi,Y.,King,J.,Ju,J.,Pannell,L.,Shevde,L.A.,andSamant,R.S.(2008)Large isoform of MRJ(DNAJB6)reduces malignant activity ofbreast cancer.Breast Cancer Res 10,R22)。
既往研究显示,DNAJB6a和DNAJB6b在肿瘤细胞中的功能存在明显差异,甚至相反。而且,它们的功能和表达变化均存在明显的组织特异性(参见,Yu,V.Z.,Wong,V.C.,Dai,W.,Ko,J.M.,Lam,A.K.,Chan,K.W.,Samant,R.S.,Lung,H.L.,Shuen,W.H.,Law,S.,Chan,Y.P.,Lee,N.P.,Tong,D.K.,Law,T.T.,Lee,V.H.,and Lung,M.L.(2015)NuclearLocalization of DNAJB6 Is Associated With Survival of Patients WithEsophageal Cancer and Reduces AKT Signaling and Proliferation of CancerCells.Gastroenterology 149,1825-1836 e1825;Taguwa,S.,Maringer,K.,Li,X.,Bernal-Rubio,D.,Rauch,J.N.,Gestwicki,J.E.,Andino,R.,Fernandez-Sesma,A.,andFrydman,J.(2015)Defining Hsp70 Subnetworks in Dengue Virus ReplicationReveals Key Vulnerability in Flavivirus Infection.Cell 163,1108-1123;Sarparanta,J.,Jonson,P.H.,Golzio,C.,Sandell,S.,Luque,H.,Screen,M.,McDonald,K.,Stajich,J.M.,Mahjneh,I.,Vihola,A.,Raheem,O.,Penttila,S.,Lehtinen,S.,Huovinen,S.,Palmio,J.,Tasca,G.,Ricci,E.,Hackman,P.,Hauser,M.,Katsanis,N.,andUdd,B.(2012)Mutations affecting the cytoplasmic functions of the co-chaperoneDNAJB6cause limb-girdle muscular dystrophy.Nat Genet 44,450-455,S451-452;和Andrews,J.F.,Sykora,L.J.,Letostak,T.B.,Menezes,M.E.,Mitra,A.,Barik,S.,Shevde,L.A.,and Samant,R.S.(2012)Cellular stress stimulates nuclear localizationsignal(NLS)independent nuclear transport of MRJ.Exp Cell Res 318,1086-1093)。首先,肿瘤组织中DNAJB6异构体的表达变化存在组织特异性。例如,DNAJB6a的表达在食管癌、进展期黑色素瘤、浸润性乳腺导管腺癌组织中显著下调,但在肝癌、肺癌、宫颈癌和CRC中并无显著变化(参见,Mitra,A.,Fillmore,R.A.,Metge,B.J.,Rajesh,M.,Xi,Y.,King,J.,Ju,J.,Pannell,L.,Shevde,L.A.,and Samant,R.S.(2008)Large isoform of MRJ(DNAJB6)reduces malignant activity of breast cancer.Breast Cancer Res 10,R22;Yu,V.Z.,Wong,V.C.,Dai,W.,Ko,J.M.,Lam,A.K.,Chan,K.W.,Samant,R.S.,Lung,H.L.,Shuen,W.H.,Law,S.,Chan,Y.P.,Lee,N.P.,Tong,D.K.,Law,T.T.,Lee,V.H.,and Lung,M.L.(2015)Nuclear Localization of DNAJB6 Is Associated With Survival ofPatients With Esophageal Cancer and Reduces AKT Signaling and Proliferationof Cancer Cells.Gastroenterology 149,1825-1836e1825;和Abba,M.C.,Drake,J.A.,Hawkins,K.A.,Hu,Y.,Sun,H.,Notcovich,C.,Gaddis,S.,Sahin,A.,Baggerly,K.,andAldaz,C.M.(2004)Transcriptomic changes in human breast cancer progression asdetermined by serial analysis of gene expression.Breast Cancer Res 6,R499-513)。其次,DNAJB6的两个异构体在功能上也存在明显差异。以往的研究显示,DNAJB6a在乳腺癌、黑色素瘤和食管鳞癌中可通过下调Wnt/β-catenin和AKT等信号通路的活性,降低细胞的增殖和侵袭转移能力,从而发挥抑癌基因功能(参见,Mitra,A.,Fillmore,R.A.,Metge,B.J.,Rajesh,M.,Xi,Y.,King,J.,Ju,J.,Pannell,L.,Shevde,L.A.,and Samant,R.S.(2008)Large isoform of MRJ(DNAJB6)reduces malignant activity of breastcancer.Breast Cancer Res 10,R22;Yu,V.Z.,Wong,V.C.,Dai,W.,Ko,J.M.,Lam,A.K.,Chan,K.W.,Samant,R.S.,Lung,H.L.,Shuen,W.H.,Law,S.,Chan,Y.P.,Lee,N.P.,Tong,D.K.,Law,T.T.,Lee,V.H.,and Lung,M.L.(2015)Nuclear Localization of DNAJB6 IsAssociated With Survival of Patients With Esophageal Cancer and Reduces AKTSignaling and Proliferation of Cancer Cells.Gastroenterology 149,1825-1836e182523,24;Mitra,A.,Rostas,J.W.,Dyess,D.L.,Shevde,L.A.,and Samant,R.S.(2012)Micro-RNA-632downregulates DNAJB6 in breast cancer.Lab Invest 92,1310-1317;和Mitra,A.,Menezes,M.E.,Shevde,L.A.,and Samant,R.S.(2010)DNAJB6 inducesdegradation of beta-catenin and causes partial reversal of mesenchymalphenotype.J Biol Chem 285,24686-24694),但其在CRC细胞SW480中对AKT的活性并无调控作用,提示DNAJB6a在不同类型的肿瘤组织中可能发挥不同的作用(参见,Yu,V.Z.,Wong,V.C.,Dai,W.,Ko,J.M.,Lam,A.K.,Chan,K.W.,Samant,R.S.,Lung,H.L.,Shuen,W.H.,Law,S.,Chan,Y.P.,Lee,N.P.,Tong,D.K.,Law,T.T.,Lee,V.H.,and Lung,M.L.(2015)NuclearLocalization of DNAJB6 Is Associated With Survival of Patients WithEsophageal Cancer and Reduces AKT Signaling and Proliferation of CancerCells.Gastroenterology 149,1825-1836 e1825)。
在专利申请CN110865186A中公开了DNAJB6以及p-mTOR和MAP4的蛋白表达水平与结直肠癌患者术后总生存期之间的关系,然而,由于该项研究中使用免疫组织化学染色技术检测DNAJB6蛋白的表达水平,所用抗体不能区分DNAJB6两个异构体之间的差别,因此该研究未能明确结直肠癌组织中DNAJB6a和DNAJB6b两种异构体的表达变化及其临床意义。而且,目前现有的研究中,并没有关于DNAJB6的两种蛋白异构体DNAJB6a和DNAJB6b在结直肠癌中功能的报道。
我们前期研究发现,DNAJB6的长异构体DNAJB6a(其氨基酸序列参见GenBank登录号NP_490647.1,其编码核酸序列参见NM_058246.3)在手术切缘正常结肠上皮和癌组织中的表达水平均很低,而短异构体DNAJB6b(其氨基酸序列请参见GenBank登录号NP_005485.1,其编码核酸序列请参见NM_005494.3)的表达丰度远高于DNAJB6a。而且,与配对的正常结直肠上皮组织相比,DNAJB6b的表达水平在部分结直肠癌组织中明显上调,提示其表达变化在结直肠癌的发生发展中可能发挥重要的功能作用(参见,Zhang,T.T.,Jiang,Y.Y.,Shang,L.,Shi,Z.Z.,Liang,J.W.,Wang,Z.,Zhang,Y.,Hao,J.J.,Jia,X.M.,Xu,X.,Cai,Y.,Zhan,Q.M.,and Wang,M.R.(2015)Overexpression of DNAJB6 promotescolorectal cancer cell invasion through an IQGAP1/ERK-dependent signalingpathway.Mol Carcinog 54,1205-1213)。
基于目前对寻找结直肠癌有效治疗的药物的迫切需要,进一步研究DNAJB6b与结直肠癌肿瘤细胞恶性增殖之间的相关性,能够为结直肠癌治疗提供潜在的分子靶点。
发明内容
在第一方面,本发明涉及靶向DNAJB6b的试剂在制备治疗肿瘤的药物中的用途,其中所述靶向DNAJB6b的试剂降低DNAJB6b蛋白质的表达水平或拮抗DNAJB6b蛋白质的功能。
在一些具体的实施方案中,通过用干扰RNA敲降DNAJB6b蛋白质的表达水平。当在体内引入时,干扰RNA与其他蛋白质形成RNA诱导沉默复合物(“RISC”)并启动称为RNA干扰(RNAi)的过程。在RNAi过程中,RISC合并单链干扰RNA或双链干扰RNA的一条链。并入的链充当RISC识别互补的mRNA转录物的模板。一旦确定互补mRNA,RISC中的蛋白质组分激活并切割mRNA,导致靶基因表达的敲降。用于敲降靶基因表达的干扰RNA分子的非限制性实例包括siRNA、短发夹RNA(shRNA)、单链干扰RNA和微RNA(miRNA)。使用这些干扰RNA的方法是本领域技术人员公知的。
在本发明具体的实施方案中,通过例如Western Blot、免疫组化的方法检测蛋白质表达水平的降低。
在本发明具体的实施方案中,通过使用DNAJB6b蛋白质的拮抗剂拮抗DNAJB6b蛋白质的功能,所述DNAJB6b蛋白质的拮抗剂为特异性结合并阻断DNAJB6b蛋白质功能的拮抗剂抗体。
在另一方面,本发明提供了一种治疗受试者中DNAJB6b相关的肿瘤的方法,所述方法包括向有需要的受试者施用治疗有效量的降低DNAJB6b蛋白质的表达水平的试剂和/或拮抗DNAJB6b蛋白质功能的试剂,所述DNAJB6b相关的肿瘤选自食管癌、黑色素瘤、乳腺癌、肝癌、肺癌、宫颈癌和结直肠癌。
另一方面,本发明提供了体外筛选抗肿瘤试剂的方法,所述方法包括以下步骤:
1)体外培养肿瘤细胞株;
2)使待检测试剂与1)中培养的肿瘤细胞接触,
3)检测肿瘤细胞株中DNAJB6b蛋白质的表达水平,和
4)检测肿瘤细胞的增殖能力,
如果与接触待检测试剂之前相比,DNAJB6b蛋白质的表达水平显著降低,且肿瘤增殖能力显著降低,则所述待检测试剂可用于治疗肿瘤。
优选地,通过细胞增殖活力检测、集落形成实验等检测肿瘤细胞的增殖能力。
在进一步优选的实施方案中,根据本发明的体外筛选抗肿瘤试剂的方法,所述肿瘤为结直肠癌;所述肿瘤细胞选自SW480、HCT116和Colo205;优选地,所述待检测试剂选自敲降DNAJB6b蛋白质的表达水平的干扰RNA或拮抗DNAJB6b蛋白质功能的抗体。本领域技术人员可以根据本领域的公知常识选择这些干扰RNA和拮抗抗体。
术语
如非另外指明,本发明所使用的术语与本领域普通技术人员通常理解的具有相同的含义。
如本文所用,“DNAJB6”是指J蛋白/HSP40家族,DNAJB亚家族。DNAJB6为辅分子伴侣,在机体内广泛表达,可参与底物蛋白(或称客户蛋白)的正确折叠及其在细胞内的转运,阻止蛋白质异常聚集、调控蛋白降解及重塑等过程。此外,DNAJB6在胚胎发育、神经干细胞的自我更新、基因转录调控、激活信号分子等方面也发挥重要作用。
人DNAJB6基因定位于7q36.3,可产生两个转录本,分别编码两个不同的蛋白异构体,即长异构体DNAJB6a(326aa)和短异构体DNAJB6b(242aa)。DNAJB6a的氨基酸序列参见GenBank登录号NP_490647.1,其编码核酸序列参见NM_058246.3。DNAJB6b的氨基酸序列请参见GenBank登录号NP_005485.1,其编码核酸序列请参见NM_005494.3。
DNAJB6b缺少DNAJB6a C端的95个氨基酸,但含有另外10个独有的氨基酸。除C端不同外,这两个异构体的结构高度相似,都含有一个位于N端的保守的J结构域,一个富含甘氨酸和苯丙氨酸残基的G/F结构域和富含丝氨酸(SSF-SST区)的C端结构域。
术语“包含”旨在表示组合物和方法包括所列举的要素,但不排除其他要素。当用于定义组合物和方法时,“基本上由......组成”是指指定的材料或步骤以及不会实质上影响要求保护的本发明的基本和新颖特征的那些材料或步骤。“由......组成”是指排除多于痕量的其他成分和所述的实质方法步骤。由这些过渡术语中的每一个定义的实施方案都在本发明的范围内。
术语“任选的”或“任选地”是指随后描述的事件或情况可以发生或不发生,并且该描述包括其中事件或情况发生的事例和其中不发生的事例。
术语“多核苷酸”、“核酸”和“寡核苷酸”可互换使用,并且是指任何长度的核苷酸的聚合形式,不管是脱氧核糖核苷酸或是核糖核苷酸或其类似物。多核苷酸可以具有任何三维结构并且可以执行已知或未知的任何功能。以下是多核苷酸的非限制性实例:基因或基因片段(例如,探针、引物、EST或SAGE标签)、外显子、内含子、信使RNA(mRNA)、转移RNA、核糖体RNA、核酶、cDNA、重组多核苷酸、支链多核苷酸、质粒、载体、任何序列的分离的DNA、任何序列的分离的RNA、核酸探针和引物。
如本文所用,“降低”、“减少”、“减小”和“缩减”DNAJB6b蛋白质的表达,指与参考水平相比,DNAJB6b蛋白质的表达水平降低至少10%,例如降低至少约20%,或至少约30%,或至少约40%,或至少约50%,或至少约60%,或至少约70%,或至少约80%,或至少约90%或多至且包括100%的降低(即与参考样品相比不存在的水平),或与参考水平相比在10-100%之间的任何降低。所述参考水平可以是与施用降低DNAJB6b表达的试剂之前的同一个体相比较的水平。
术语“患者”、“受试者”、“个体”等在本文中可互换使用,并且无论是体外还是原位,是指可适用于本文所述的方法的任何动物或其细胞。在某些非限制性实施方案中,患者、受试者或个体是人。
术语“治疗”或“处理”包括在受试者如人中治疗本文所述的疾病或病症,并且包括:(i)抑制疾病或病症,即阻止其发生;(ii)缓解疾病或病症,即引起病症消退;(iii)减缓疾病的进展;和/或(iv)抑制、缓解或减缓疾病或病症的一种或多种症状的进展。
术语“施用”或“给予”治疗剂如降低DNAJB6b表达的试剂包括引入或递送治疗剂以执行预期功能的任何途径。可以通过适合于递送药剂的任何途径进行施用。因此,递送途径可包括静脉内、肌肉内、腹膜内或皮下递送。在一些实施方案中降低DNAJB6b表达的试剂直接施用于肿瘤,例如,通过注射到肿瘤中。
如本文所用,术语“敲降”是指与不包含减少表达的遗传修饰的对应对照细胞中靶mRNA或相应蛋白质的表达相比,遗传修饰细胞中靶mRNA或相应蛋白质表达的可测量的降低。本领域技术人员将容易理解如何使用各种遗传方法,例如siRNA、shRNA、miRNA、反义RNA或其他RNA介导的抑制技术,以基于本文所述细节敲降靶多核苷酸序列或其部分。
术语“干扰RNA”是指RNA核酸分子,其是双链或单链的,并且能够实现针对敲降靶基因表达的RNA干扰机制的诱导。
如本文所用的术语,“siRNA”是双链RNA,其通常小于30个核苷酸长。通过siRNA的基因沉默开始于siRNA的一条链并入称为RNA诱导沉默复合物(RISC)的核糖核蛋白复合物中。并入RISC中的链识别与并入的siRNA链至少部分互补的mRNA分子,且然后RISC切割这些靶mRNA或抑制它们的翻译。
术语“miRNA”是小的非编码RNA分子,其可以与mRNA分子内的互补序列杂交,从而导致mRNA的切割,或通过缩短其聚(A)尾巴使mRNA失稳定。
术语“单链干扰RNA”可以以与双链siRNA类似的方式实现mRNA沉默,尽管效率低于双链siRNA。单链干扰RNA通常具有约19至约49个核苷酸的长度,同样对于上述双链siRNA。
术语“短发夹RNA或小发夹RNA(shRNA)”是具有紧密发夹转角的人工RNA分子,其可用于通过其在细胞中产生的siRNA沉默靶基因表达。shRNA在细胞中的表达通常通过质粒的递送或通过病毒或细菌载体来实现。合适的载体包括但不限于腺相关病毒(AAV)、腺病毒和慢病毒。shRNA是siRNA的有利介质,因为它具有相对低的降解和转换率。
本文中的术语“抗体”按最广义使用,指包含两个重链和两个轻链的任何免疫球蛋白(Ig)分子,以及其任何片段、突变体、变体或衍生物,只要该片段、突变体、变体或衍生物表现出所需要的生物学活性(例如,表位结合活性)。
术语“拮抗剂抗体”在最广泛的意义上使用,并且包括抑制或降低该抗体所结合的抗原(例如,DNAJB6b)的生物活性的抗体。因此,DNAJB6b拮抗剂抗体涵盖结合DNAJB6b并且以任何有意义的程度(包括显著地)阻断、抑制、抵消、拮抗、降低DNAJB6b激动剂活性的抗体。
如本文所述的“DNAJB6b相关的肿瘤”,是指与正常组织相比,DNAJB6b mRNA或蛋白质表达水平增加的肿瘤。DNAJB6b相关的肿瘤包括但不限于食管癌、黑色素瘤、乳腺癌、肝癌、肺癌、宫颈癌和结直肠癌。
以下将结合附图以及具体实施例进一步说明本发明的实施方案,但是,不应理解为将本发明的范围限于这些具体实施例。
附图说明
图1A和图1B:结直肠癌组织和细胞系中DNAJB6a和DNAJB6b的表达情况。图1A:细胞系中DNAJB6a和DNAJB6b蛋白的Western blot检测。GAPDH为内参照。图1B:在2组独立的、来源于GEO数据库的数据集中,DNAJB6b的mRNA表达水平在结直肠癌组织中显著上调(P<0.05),而DNAJB6a的表达无显著变化。DNAJB6a和DNAJB6b的探针分别为209015_s_at和208810_s_at。
图2显示在SW480和HCT116细胞中瞬时转染DNAJB6b特异性的siRNA和阴性对照非沉默的siRNA后,进行Western blot检测。从左至右,电泳列分别表示亲本细胞、非沉默的siRNA、siD6b-1和siDb-2。
其中siD6b-1和siDb-2的靶序列如下:
siD6b-1:5′-GCACGCACTTAACAGAAAT-3′(SEQ ID NO:1)
siD6b-2:5′-GGACGATTCTTCTACAGAA-3′(SEQ ID NO:2)。
图3A和图3B显示在SW480和HCT116细胞瞬时转染DNAJB6b特异性的siRNA(siD6b-1和siDb-2)和阴性对照非沉默siRNA,进行的CCK-8细胞增殖活力检测。
图4A和图4B显示在SW480和HCT116细胞中瞬时转染DNAJB6b特异性的siRNA(siD6b-1和siDb-2)和阴性对照非沉默siRNA,进行平板集落形成实验。
图5显示稳定克隆细胞蛋白的Western blot检测。
图6A显示移植瘤的生长曲线;图6B显示移植瘤的大体观;图6C显示移植瘤的瘤重比较。
图7A显示移植瘤组织蛋白的Western blot检测;图7B显示移植瘤组织中DNAJB6和Ki67的免疫组织化学染色结果。
图8A、图8B和图8C显示在SW480和HCT116细胞中过表达DNAJB6b-WT(D6b-WT),筛选稳定细胞株,进行Western blot检测(图8A)和平板集落形成实验(图8B-图8C)。***,P<0.001。
图9A、图9B和图9C显示在Colo205和NCM460细胞中稳定过表达DNAJB6b-WT(D6b-WT),以空载体转染细胞(EV)作为对照,进行Western blot检测(图9A)和平板集落形成实验(图9B-图9C)。***,P<0.001。
具体实施方式
实验材料和方法步骤
1.Western blot检测
在本发明的实施方案中,涉及一种检测组织、细胞内DNAJB6蛋白的两种异构体DNAJB6a和DNAJB6b表达水平的方法,称Western blot。其基本原理是通过特异性抗体对凝胶电泳处理过的细胞或生物组织样品进行着色。通过分析着色的位置和着色深度获得特定蛋白质在所分析的细胞或组织中表达情况的信息。
在本方法中,可同时检测DNAJB6a和DNAJB6b的抗体购自Proteintech公司(美国),货号为66587-1-Ig。检测内参GAPDH的抗体购自Proteintech公司(美国),货号为6004-1-1G。
Western blot检测所用试剂购自北京普利莱生物科技有限公司。
在Western blot检测中,制备样本方法、Western blot方法如下:
制备样本:
(1)组织/细胞总蛋白提取;
(2)蛋白定量,制备蛋白上样样品。
蛋白表达的Western blot检测,具体方法如下:
(1)SDS-PAGE凝胶电泳和蛋白质印迹,包括制胶、电泳、转膜;
(2)目的蛋白的检测,包括封闭、孵育一抗、孵育二抗、曝光显影
(3)结果评价:
将胶片进行扫描或拍照,用凝胶图象处理系统分析目标带的分子量和净光密度值。
2.细胞培养和操作流程:
在细胞培养和操作中,使用的体外实验模型为正常结肠上皮细胞NCM460和结肠癌细胞株:SW480、HCT116、Colo205。
所需实验材料:细胞培养基和PBS(北京细工);胎牛血清、胰蛋白酶和Opti-MEM培养基(Gibco);青霉素、链抗生素和Lipofectamine 2000(Invitrogen);T25细胞培养瓶和细胞培养板(Corning);
在细胞的实验中,涉及如下实验操作流程:
细胞复苏
1)提前将水浴锅打开调节温度设定至37℃。
2)从液氮罐中快速拿出冻存的细胞并在37℃水浴锅中水浴3~5分钟。
3)1000rpm离心5分钟得到细胞沉淀。
4)从事先在T25瓶中复温的5mL培养基中取出1mL培养基重悬细胞沉淀,将放置细胞悬液的T25瓶放置于细胞培养箱培养。
细胞传代
1)待T25瓶中细胞生长至80%~90%时,用2.5mL 1×PBS清洗细胞2遍,弃掉PBS。
2)用0.7mL 1×胰蛋白酶消化细胞3~5分钟。用1.4mL培养基终止消化。
3)再将上述2.1mL细胞悬液等体积传到3个T25瓶中,每瓶再补加4.3mL培养基继续培养细胞。
细胞冻存
1)将T25细胞培养瓶中处在对数生长期中的细胞用2.5mL 1×PBS清洗细胞2遍,弃掉PBS。
2)用0.7mL 1×胰蛋白酶消化细胞3~5分钟。用1.4mL培养基终止消化。
3)将上述细胞悬液1000rpm离心5分钟,弃掉上清得到细胞沉淀。
4)用0.7mL冻存液重悬细胞,转移至冻存管中,然后4℃冰箱静置30分钟,-20℃冰箱静置2小时,-80℃冰箱过夜冻存,然后转入液氮罐中长期保存。
Lipofectamine 2000转染质粒入哺乳动物细胞
1)将生长良好的细胞于转染前一天接种到6孔板中,经过夜培养后,转染前以细胞汇合度达到70%~80%为宜。
2)将6孔板培养基换成无血清培养基,每孔1.5mL,37℃培养细胞半小时。
3)取5μL Lipofectamine 2000转染试剂加入250μL不含血清和抗生素的Opti MEM培养基,温和混匀,室温放置5分钟;同时,将2.5μg质粒DNA加入另外250μL不含血清和抗生素的Opti-MEM培养基中,温和混匀,室温放置5分钟。
4)将转染试剂逐滴加入质粒DNA溶液中,室温放置25分钟。
5)将上述混合物均匀滴加入培养基。
6)37℃培养4~6小时,换2mL含血清RPMI 1640全培养基。24~48小时后收集细胞,检测其瞬时表达情况。
Lipofectamine 2000转染siRNA入哺乳动物细胞
1)生长良好的细胞于转染前一天接种到6孔板中,经过夜培养后,转染前以细胞汇合度达到40%~50%为宜。
2)将6孔板培养基换成无血清培养基,每孔1.5mL,37℃培养细胞半小时。
3)取5μL转染试剂Lipo 2000加入250μL不含血清和抗生素的OptiMEM培养基,温和混匀,室温放置5分钟;同时,将5μL siRNA(母液浓度20μM)加入另外250μL不含血清和抗生素的Opti-MEM培养基中,温和混匀,室温放置5分钟。
4)将转染试剂逐滴加入siRNA溶液中,室温放置25分钟。
5)将上述混合物均匀滴加入含培养基的6孔板中。
6)37℃培养4~6小时,换2mL含血清的RMPI 1640全培养基。24~48小时后收集细胞,收集细胞提取RNA,进行逆转录,用Real-time PCR检测敲降各分子mRNA的表达变化。提取蛋白检测各分子蛋白表达情况。
基因稳定表达或缺失表达的细胞株构建
1)转染前按照每皿1×106个细胞数将293T细胞接种于6cm培养皿。
2)待细胞汇合度约为70~80%,弃掉原培养基,每皿加入不含血清和抗生素3.8mLOpti-MEM培养基,饥饿处理30分钟。
3)将含目基因片段的6g载体质粒与1.2mL Opti-MEM培养基混合孵育,再加入3g包装质粒pMD2.G、3g包装质粒psPAX2和24μL Lipo2000到上述混合液中。注意设置实验组和对照组,要同时转染空载作为对照。
4)将上述混合液室温孵育15~20分钟,混匀之后逐滴加入293T细胞。
5)4~6小时后更换5mL含30%FBS的DMEM全培养基。
6)转染48小时后收集含有慢病毒的上清液,保存于4℃。
7)用0.45μM针头滤器过滤病毒上清液并分装,4℃保存,尽量在一周内用完。暂时不用的病毒置-80℃保存,避免反复冻融。
8)将准备用于感染病毒的癌细胞接种于6孔板,待细胞汇合度约50%时,将每孔换成1.5mL新鲜培养基和500μL慢病毒上清混合液,同时加入polybrene促进转染,polybrene的终浓度控制于6~8μg/mL之间。
9)慢病毒感染靶细胞8~12小时后更换不含polybrene的新鲜培养基。
10)24小时后重新在培养基中添加适宜浓度的相应抗生素进行筛选;筛选3~7天后可获得相应抗性基因稳定过表达或缺失表达细胞。
11)Western blot检测相应基因蛋白表达水平的改变,检测相关基因是否在细胞中稳定过表达或缺失表达。
12)配置不同浓度药物或抑制剂加入六孔板中,待处理适当时间后收集细胞提取蛋白或mRNA进行后续检测。
3.细胞增殖检测(Cell Counting Kit-8)
1)取生长处于对数期的细胞,弃去培养基,1×PBS洗两次,胰酶消化细胞,加入含10%胎牛血清的RPMI 1640培养基终止消化。
2)计数细胞,调节细胞密度2×104个/mL,于96孔培养板内每孔加入100μL细胞悬液,即每孔接种2×103个细胞。每组设4个平行孔,4个空白对照孔。共接种6块96孔板,于37℃含5%CO2的培养箱培养。
3)接种约8小时后,取一块96孔板进行检测。检查时先弃掉旧培养基,按照每孔90μL新鲜培养基加10μL的CCK-8配制检测混合液加入每个孔中,在不含CO2的37℃培养箱中培养1小时后,用酶标仪测定450nm(OD450)吸光值,定义为0天。
4)每隔24小时取一块96孔板,测定OD450值,获得1~6天六个时间点的OD450值。
5)以不同处理组细胞每个时间点各复孔OD450值的平均值为纵坐标,培养时间为横坐标,绘制细胞的生长曲线。
4.平板集落形成实验
1)将对数生长期的各组细胞,用胰蛋白酶消化制备成单细胞悬液,计数,每孔接种500个细胞于六培养孔板中,每组设三个复孔,放置于5%CO2恒温培养箱中37℃培养12~14天左右。
2)每隔5~6天换液一次,培养14天左右,待集落肉眼可见时中止培养,开始准备固定和染色。
3)PBS漂洗2次,甲醇固定30分钟,弃去固定液,待稍干燥后用0.5%结晶紫染色10分钟,用蒸馏水冲洗洗去残余染液,空气干燥。
4)光镜下观察,以含大于50个细胞的团块为一个阳性集落,照相并计数克隆数。
5.动物实验:
在本实施方案中,所使用到的体外实验模型为四周龄左右雌性SCID小鼠(购自北京华阜康生物科技有限公司):SCID小鼠外观与普通小鼠差别不大,有毛,被毛白色,体重发育正常。但胸腺、脾、淋巴结的重量不及正常的30%,组织学上表现为B细胞、T细胞显著缺陷。胸腺多位脂肪组织包围,没有皮质结构,仅残存髓质,主要有类上皮细胞合成纤维细胞构成,边缘偶见灶状淋巴细胞群。脾白髓不明显,红髓正常,脾小体无淋巴细胞聚集,主要由网状细胞构成。淋巴结无明显皮质区,麸皮质区缺失,有网状细胞占据。小肠粘膜下和支气管淋巴集结较少见,结构内无淋巴聚集。SCID小鼠极易感染,在高度洁净的SPF环境下可存活一年以上,窝产仔数为3-5只。SCID小鼠是继裸鼠出现之后,人类发现的又一种十分有价值的免疫缺陷动物,常用于肿瘤生物学研究、异体移植学研究等。
在本实验中,涉及如下实验操作流程:
1)将SPF级4周龄大小雌性SCID小鼠在实验前一天称重,按体重分组,使各种体重的小鼠在平均分布在每个组中。
2)将生长状态良好且处在对数生长期的细胞消化完全,用1×PBS洗两遍,充分混悬,过100μM细胞筛,制备成单细胞悬液。
3)细胞计数,将总数为1~3×106个/只的细胞数(0.1mL)接种于小鼠上肢腋下部位,建立小鼠皮下移植瘤模型。
4)在实验期间对小鼠体重进行称重测量,持续观察,待瘤体直径达到4mm左右时开始测量,每周两次测量并记录皮下移植瘤的长度和宽度,用公式计算瘤体积,肿瘤体积=(π/6)×(长度×宽度×宽度)。
5)待皮下瘤体体积达到1cm3左右后结束实验,采用颈椎脱臼法处死小鼠,剥取肿瘤组织拍照、称重,并取部分瘤块组织使用福尔马林固定后进行石蜡包埋切片,用于后续免疫组化染色实验。
实施例1.结直肠癌中DNAJB6b的表达分析
在我们之前的研究中,我们使用Western blot技术检测发现,DNAJB6的两个蛋白异构体在正常结直肠上皮组织和结直肠癌组织中均有表达,但短异构体DNAJB6b的蛋白表达水平明显高于长异构体DNAJB6a,而且DNAJB6b蛋白在结直肠癌组织中的表达水平明显高于正常结直肠上皮组织(参见Zhang,T.T.,Jiang,Y.Y.,Shang,L.,Shi,Z.Z.,Liang,J.W.,Wang,Z.,Zhang,Y.,Hao,J.J.,Jia,X.M.,Xu,X.,Cai,Y.,Zhan,Q.M.,and Wang,M.R.(2015)Overexpression of DNAJB6 promotes colorectal cancer cell invasion through anIQGAP1/ERK-dependent signaling pathway.Mol Carcinog 54,1205-1213)。
进一步地,我们使用Western blot技术检测了正常结肠上皮细胞NCM460,以及结肠癌细胞系SW480、HCT116、HCT-8、RKO、Colo205、SW620和LOVO中DNAJB6a和DNAJB6b蛋白的表达情况。分析结果显示,DNAJB6的两个蛋白异构体在上述细胞系中均有表达。与正常结肠上皮细胞系NCM460相比,DNAJB6的短异构体DNAJB6b的表达水平在所检测的全部7个结肠癌细胞系中均有不同程度的升高,且显著高于DNAJB6a,而长异构体DNAJB6a的表达水平在多数结肠癌细胞系中则没有明显变化。实验结果如图1A所示。
为进一步明确DNAJB6不同转录本在结直肠癌中的表达变化情况,发明人进一步分析了来自GEO数据库中的两个数据集(GSE18105和GSE32323),比较配对的结直肠癌和癌旁正常组织样本中DNAJB6a和DNAJB6b的mRNA表达情况。分析结果显示,与癌旁正常组织相比,结直肠癌组织中DNAJB6b的mRNA表达水平明显上调(探针208810_s_at;P=2.98e-11和P=0.003),而DNAJB6a的mRNA表达水平无显著变化(探针209015_s_at;P=0.277和P=0.578)。实验结果如下面的图1B所示。
上述研究结果提示,DNAJB6b表达上调可能在结直肠癌发生发展中发挥着重要的作用,因此我们将DNAJB6b作为研究的主要目标。如图1A所示,在所检测的结肠癌细胞系中,DNAJB6b在SW480和HCT116细胞中高水平表达,而在Colo205和NCM460细胞中表达水平较低,因此我们选择这4种细胞系作为后续研究的细胞模型。
DNAJB6a(NM_058246)
DNAJB6b(NM_005494)
DNAJB6a氨基酸序列
MVDYYEVLGVQRHASPEDIKKAYRKLALKWHPDKNPENKEEAERKFKQVAEAYEVLSDAKKRDIYDKYGKEGLNGGGGGGSHFDSPFEFGFTFRNPDDVFREFFGGRDPFSFDFFEDPFEDFFGNRRGPRGSRSRGTGSFFSAFSGFPSFGSGFSSFDTGFTSFGSLGHGGLTSFSSTSFGGSGMGNFKSISTSTKMVNGRKITTKRIVENGQERVEVEEDGQLKSLTINGVADDDALAEERMRRGQNALPAQPAGLRPPKPPRPASLLRHAPHCLSEEEGEQDRPRAPGPWDPLASAAGLKEGGKRKKQKQREESKKKKSTKGNH (SEQ ID NO:5)
DNAJB6b氨基酸序列
MVDYYEVLGVQRHASPEDIKKAYRKLALKWHPDKNPENKEEAERKFKQVAEAYEVLSDAKKRDIYDKYGKEGLNGGGGGGSHFDSPFEFGFTFRNPDDVFREFFGGRDPFSFDFFEDPFEDFFGNRRGPRGSRSRGTGSFFSAFSGFPSFGSGFSSFDTGFTSFGSLGHGGLTSFSSTSFGGSGMGNFKSISTSTKMVNGRKITTKRIVENGQERVEVEEDGQLKSLTINGKEQLLRLDNK (SEQ ID NO:6)
实施例2.敲降DNAJB6b表达可显著抑制结直肠癌细胞的恶性增殖
首先,在DNAJB6b高表达的结肠癌细胞系SW480和HCT116中瞬时转染DNAJB6b特异性的siRNA,敲降DNAJB6b的表达,进行Western blot实验检测DNAJB6b的敲降效果,结果参见图2。
在确认了DNAJB6b被敲除后,通过细胞增殖活力实验(CCK-8)和集落形成能力实验探究敲降DNAJB6b表达对SW480和HCT116细胞恶性增殖能力的影响。实验结果显示,敲降DNAJB6b表达可显著抑制结肠癌细胞系SW480和HCT116的细胞增殖活力(参见图3A和图3B),并显著抑制集落形成能力(参见图4A和图4B)。
进而,发明人使用动物模型检测了DNAJB6b高表达对结肠癌细胞致瘤性的影响。首先,用表达DNAJB6b-shRNA或Ctrl-shRNA的慢病毒分别感染SW480细胞,经嘌呤霉素(puromycin)筛选3天后,获得稳定敲降DNAJB6b表达的细胞株(shDNAJB6b)及对照细胞株(shCtrl)。Western blot检测结果证实SW480细胞中DNAJB6b的表达水平被明显抑制(图5)。
随后,将稳定克隆细胞接种于雌性SCID小鼠上肢腋下,建立小鼠皮下移植瘤模型。接种后8天左右,可在对照组小鼠皮下触摸到肿块,约12天时可形成肉眼可见的瘤块,而DNAJB6b敲降组小鼠在第12天左右才能触摸到皮下肿块。此外,对皮下瘤生长速度进行监测的结果也显示,DNAJB6b敲降组皮下移植瘤的生长速度显著低于对照组(图6A)。在实验终点(接种后28天)时,与对照组相比,稳定敲降DNAJB6b表达可显著降低SW480细胞在SCID小鼠体内形成的移植瘤的重量和体积(P<0.01)(图6B至图6C)。
取移植瘤组织进行Western blot检测,结果显示DNAJB6b稳定敲降细胞形成的移植瘤组织中DNAJB6b的表达被明显抑制(图7A)。同时,免疫组织化学染色结果表明,DNAJB6b敲降组小鼠移植瘤组织中的细胞增殖相关分子Ki67及DNAJB6b蛋白表达水平,与对照组相比均明显降低(图7B)。以上结果表明,敲降DNAJB6b表达可显著降低结肠癌细胞在SCID小鼠体内的成瘤能力,该作用可能与抑制细胞恶性增殖有关。
实施例3.DNAJB6b过表达可显著增强结直肠癌细胞的恶性增殖
另一方面,发明人进一步构建了可表达野生型(D6b-WT)DNAJB6b慢病毒的表达载体,即pLVX-DNAJB6b-WT-3×flag表达载体和pLVX-DNAJB6b-delNES-3×flag表达载体,包装病毒并分别感染DNAJB6b稳定敲降的SW480、HCT116细胞。经过G418筛选,获得了可稳定表达外源野生型DNAJB6b的细胞株(简称SW480-shD6b-D6b),以感染空载体(empty vector,EV)获得的稳定细胞株(SW480-shCtrl-EV和SW480-shD6b-EV)作为对照。使用上述细胞进行集落形成实验,探讨DNAJB6b的过表达对结肠癌细胞增殖能力的影响。实验结果显示,过表达外源DNAJB6b可显著增强结肠癌细胞的集落形成能力,提示DNAJB6b在过表达可发挥促癌功能。实验结果如下面的图8B至图8C所示。
为进一步证实DNAJB6b高表达对结直肠癌细胞恶性增殖能力的影响,发明人在DNAJB6b低表达的Colo205和NCM460细胞中过表达外源DNAJB6b,结果显示DNAJB6b过表达可以显著增强这些细胞的集落形成能力(参见图9B-图9C)。
上述研究结果进一步证实,DNAJB6b高表达可增强结直肠癌细胞的恶性增殖潜能。
上述研究结果表明,DNAJB6b过表达可增强CRC细胞的恶性增殖能力,提示其异常表达在CRC的发生发展过程中发挥重要作用,可能作为结直肠治疗的潜在分子靶点。
序列表
<110> 中国医学科学院肿瘤医院
<120> 靶向DNAJB6b的试剂在制备治疗肿瘤的药物中的用途
<130> 300177CG
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(19)
<223> siD6b-1
<400> 1
gcacgcactt aacagaaat 19
<210> 2
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(19)
<223> siD6b-2
<400> 2
ggacgattct tctacagaa 19
<210> 3
<211> 2489
<212> DNA
<213> 智人(Homo sapiens)
<400> 3
ctgtttgttg ttggagaaag gagagaaagg aaagcgcgag gagccgccgc caccaccagc 60
gcagcagtcc tggagctgtg aggagattcg ggccgtcacc ctgcctcccc tgcgtcccgc 120
caccggccgc ttctgtcctc ggacccattc caacaatctc gtaaaacatg gtggattact 180
atgaagttct aggcgtgcag agacatgcct cacccgagga tattaaaaag gcatatcgga 240
aactggcact gaagtggcat ccagataaaa atcctgagaa taaagaagaa gcagagagaa 300
aattcaagca agtagcggag gcatatgaag tgctgtcgga tgctaagaaa cgggacatct 360
atgacaaata tggcaaagaa ggattaaatg gtggaggagg aggtggaagt cattttgaca 420
gtccatttga atttggcttc acattccgta acccagatga tgtcttcagg gaattttttg 480
gtggaaggga cccattttca tttgacttct ttgaagaccc ttttgaggac ttctttggga 540
atcgaagggg tccccgagga agcagaagcc gagggacggg gtcgtttttc tctgcgttca 600
gtggatttcc gtcttttgga agtggatttt cttcttttga tacaggattt acttcatttg 660
ggtcactagg tcacgggggc ctcacttcat tctcttccac gtcatttggt ggtagtggca 720
tgggcaactt caaatcgata tcaacttcaa ctaaaatggt taatggcaga aaaatcacta 780
caaagagaat tgtcgagaac ggtcaagaaa gagtagaagt tgaagaagat ggccagttaa 840
agtccttaac aataaatggt gtggccgacg acgatgccct cgctgaggag cgcatgcgga 900
gaggccagaa cgccctgcca gcccagcctg ccggcctccg cccgccgaag ccgccccggc 960
ctgcctcgct gctgagacac gcgcctcact gtctctctga ggaggagggc gagcaggacc 1020
gacctcgggc acccgggccc tgggaccccc tcgcgtccgc agcaggattg aaagaaggtg 1080
gcaagaggaa gaagcagaag cagagagagg agtcgaagaa gaagaagtcg accaaaggca 1140
atcactagac cggacttgag gcacgcggtg cacccccaga cgctggcgct ccaccgtgct 1200
cggcatgcgg tcgtgcacac gcgctaggta gcagcgtcgg tcaggactgt ctcgaggcca 1260
cactcgctcg gcaggattat gcgatcacgg atcagtcaga gcagggtcag gagacggggc 1320
tgacggcacg ggtggcgggg acagacgttt gggacttggc cgcgactctc tgcttctctc 1380
cagctctcaa tctgctgcat tttcctctag tgcttccgga tcctcttcat tcttttcggc 1440
tactcaacca ctccgcatgc tgctggaata tttctggctt tagaagtaca ggagggcgca 1500
gatggctaac tgagtaacat tcatgaaatg aggctttctg tggcggcgta gtgtttggaa 1560
ttagaaggta attcagtaga gtgtaactta gagaatattg caagtgacac attgaatcct 1620
gcccgtcagg gcaccttttc ctcagagcaa tccggccaca cgaatagaag gctgtcgtga 1680
atcacatcag atgtaaaatc attccttctg tttactcttt taattttcat cctttgcagg 1740
tagtgcaaat tcaacttcaa atatggtgta ggttttgcta gattccatat ttttttcttg 1800
gatttttgct aattattttt agcaaaaaat ttttgctcag tggcactctc cctagtgtcc 1860
atgggttagg gccatgctgg ggaaaacggg ccggtattta cacacgcgca aaacacccag 1920
agacggcaca aggaggttga actcatgttt cagttcgcga acattgactc cttacgaaag 1980
tcacttcatt ctaactagat gcgcccactt ccggtcatta tttcgtttgc atgatgtatt 2040
gcttcttcac gttttgtttt tattgagcac ggagtagaat tccagggctg ccttgacttc 2100
ttccctgcat gctccctccc agtgactttc cttccctttc acatgaggat ctgccgttca 2160
tgttgctttc tcctttgtcc tcttggactt gagggcattg tgaaaagctt tgctgtgatt 2220
taaaaatgcc agcaatttta atctagcagt gttgaagctg ggaatttttt ggcgcaatcc 2280
atgtagcagt gacccaggct tgggagccag aaacaagtgt gacctgggat tttatttaac 2340
acaactgttg ccaaagagtt ggctttgttt atttggtttt ggcggggaga ggagtggtat 2400
ttgatgcttt ctgtggacaa tgtaacccta aacacatcat gtattttaaa tgccacctac 2460
ataaataaaa cataagcata ttgaataca 2489
<210> 4
<211> 1571
<212> DNA
<213> 智人(Homo sapiens)
<400> 4
ctgtttgttg ttggagaaag gagagaaagg aaagcgcgag gagccgccgc caccaccagc 60
gcagcagtcc tggagctgtg aggagattcg ggccgtcacc ctgcctcccc tgcgtcccgc 120
caccggccgc ttctgtcctc ggacccattc caacaatctc gtaaaacatg gtggattact 180
atgaagttct aggcgtgcag agacatgcct cacccgagga tattaaaaag gcatatcgga 240
aactggcact gaagtggcat ccagataaaa atcctgagaa taaagaagaa gcagagagaa 300
aattcaagca agtagcggag gcatatgaag tgctgtcgga tgctaagaaa cgggacatct 360
atgacaaata tggcaaagaa ggattaaatg gtggaggagg aggtggaagt cattttgaca 420
gtccatttga atttggcttc acattccgta acccagatga tgtcttcagg gaattttttg 480
gtggaaggga cccattttca tttgacttct ttgaagaccc ttttgaggac ttctttggga 540
atcgaagggg tccccgagga agcagaagcc gagggacggg gtcgtttttc tctgcgttca 600
gtggatttcc gtcttttgga agtggatttt cttcttttga tacaggattt acttcatttg 660
ggtcactagg tcacgggggc ctcacttcat tctcttccac gtcatttggt ggtagtggca 720
tgggcaactt caaatcgata tcaacttcaa ctaaaatggt taatggcaga aaaatcacta 780
caaagagaat tgtcgagaac ggtcaagaaa gagtagaagt tgaagaagat ggccagttaa 840
agtccttaac aataaatggt aaggagcagc tgctgcgctt ggataacaag taattcaacg 900
cacgcactta acagaaatgt taaactataa caagcaccat ttgaggatta acaggaacat 960
ttttttgaag atttcaaacg aactcgactt tcagtataat tgtacctaaa gtatttataa 1020
acagctcatc ggagcctcta tttgtcatag acttttgagt tgattgttgg gaccacataa 1080
taggaccatt ttttttttgt ctttaaaatt gttgtaaatc tctgtatgca ctttgctttt 1140
ttattaaacg tactccaagg tgagtcttga ctctttagtg taggacaaga ttgtacacta 1200
acaccagcat ggacctgctt ttcattgtgt ctgaaatgtg agccacgtag tgtcggcctg 1260
ctgtgaagtt aacattgcca ggacgattct tctacagaaa taatttcaat ttttttcagt 1320
atttagtagt gaaagatatt aatacattaa tggtaataca tttctggttt aatataaatt 1380
aaggatgttt tctagttgtg catgaatgct ggcaacttag taagttttga caattgttta 1440
aatatgtaat gttaagctta ggtttaaaaa agtaaagctg gtaaactggg tctttgtcat 1500
ttgctttaaa aaaaaaaaaa aagaaaataa atgcgaatgt gttggtgcat tcttcctgag 1560
tgggatctgg a 1571
<210> 5
<211> 326
<212> PRT
<213> 智人(Homo sapiens)
<400> 5
Met Val Asp Tyr Tyr Glu Val Leu Gly Val Gln Arg His Ala Ser Pro
1 5 10 15
Glu Asp Ile Lys Lys Ala Tyr Arg Lys Leu Ala Leu Lys Trp His Pro
20 25 30
Asp Lys Asn Pro Glu Asn Lys Glu Glu Ala Glu Arg Lys Phe Lys Gln
35 40 45
Val Ala Glu Ala Tyr Glu Val Leu Ser Asp Ala Lys Lys Arg Asp Ile
50 55 60
Tyr Asp Lys Tyr Gly Lys Glu Gly Leu Asn Gly Gly Gly Gly Gly Gly
65 70 75 80
Ser His Phe Asp Ser Pro Phe Glu Phe Gly Phe Thr Phe Arg Asn Pro
85 90 95
Asp Asp Val Phe Arg Glu Phe Phe Gly Gly Arg Asp Pro Phe Ser Phe
100 105 110
Asp Phe Phe Glu Asp Pro Phe Glu Asp Phe Phe Gly Asn Arg Arg Gly
115 120 125
Pro Arg Gly Ser Arg Ser Arg Gly Thr Gly Ser Phe Phe Ser Ala Phe
130 135 140
Ser Gly Phe Pro Ser Phe Gly Ser Gly Phe Ser Ser Phe Asp Thr Gly
145 150 155 160
Phe Thr Ser Phe Gly Ser Leu Gly His Gly Gly Leu Thr Ser Phe Ser
165 170 175
Ser Thr Ser Phe Gly Gly Ser Gly Met Gly Asn Phe Lys Ser Ile Ser
180 185 190
Thr Ser Thr Lys Met Val Asn Gly Arg Lys Ile Thr Thr Lys Arg Ile
195 200 205
Val Glu Asn Gly Gln Glu Arg Val Glu Val Glu Glu Asp Gly Gln Leu
210 215 220
Lys Ser Leu Thr Ile Asn Gly Val Ala Asp Asp Asp Ala Leu Ala Glu
225 230 235 240
Glu Arg Met Arg Arg Gly Gln Asn Ala Leu Pro Ala Gln Pro Ala Gly
245 250 255
Leu Arg Pro Pro Lys Pro Pro Arg Pro Ala Ser Leu Leu Arg His Ala
260 265 270
Pro His Cys Leu Ser Glu Glu Glu Gly Glu Gln Asp Arg Pro Arg Ala
275 280 285
Pro Gly Pro Trp Asp Pro Leu Ala Ser Ala Ala Gly Leu Lys Glu Gly
290 295 300
Gly Lys Arg Lys Lys Gln Lys Gln Arg Glu Glu Ser Lys Lys Lys Lys
305 310 315 320
Ser Thr Lys Gly Asn His
325
<210> 6
<211> 241
<212> PRT
<213> 智人(Homo sapiens)
<400> 6
Met Val Asp Tyr Tyr Glu Val Leu Gly Val Gln Arg His Ala Ser Pro
1 5 10 15
Glu Asp Ile Lys Lys Ala Tyr Arg Lys Leu Ala Leu Lys Trp His Pro
20 25 30
Asp Lys Asn Pro Glu Asn Lys Glu Glu Ala Glu Arg Lys Phe Lys Gln
35 40 45
Val Ala Glu Ala Tyr Glu Val Leu Ser Asp Ala Lys Lys Arg Asp Ile
50 55 60
Tyr Asp Lys Tyr Gly Lys Glu Gly Leu Asn Gly Gly Gly Gly Gly Gly
65 70 75 80
Ser His Phe Asp Ser Pro Phe Glu Phe Gly Phe Thr Phe Arg Asn Pro
85 90 95
Asp Asp Val Phe Arg Glu Phe Phe Gly Gly Arg Asp Pro Phe Ser Phe
100 105 110
Asp Phe Phe Glu Asp Pro Phe Glu Asp Phe Phe Gly Asn Arg Arg Gly
115 120 125
Pro Arg Gly Ser Arg Ser Arg Gly Thr Gly Ser Phe Phe Ser Ala Phe
130 135 140
Ser Gly Phe Pro Ser Phe Gly Ser Gly Phe Ser Ser Phe Asp Thr Gly
145 150 155 160
Phe Thr Ser Phe Gly Ser Leu Gly His Gly Gly Leu Thr Ser Phe Ser
165 170 175
Ser Thr Ser Phe Gly Gly Ser Gly Met Gly Asn Phe Lys Ser Ile Ser
180 185 190
Thr Ser Thr Lys Met Val Asn Gly Arg Lys Ile Thr Thr Lys Arg Ile
195 200 205
Val Glu Asn Gly Gln Glu Arg Val Glu Val Glu Glu Asp Gly Gln Leu
210 215 220
Lys Ser Leu Thr Ile Asn Gly Lys Glu Gln Leu Leu Arg Leu Asp Asn
225 230 235 240
Lys
Claims (9)
1.靶向DNAJB6b的试剂在制备治疗肿瘤的药物中的用途,其中所述靶向DNAJB6b的试剂降低DNAJB6b蛋白质的表达水平或拮抗DNAJB6b蛋白质的功能。
2.根据权利要求1所述的用途,其中所述降低DNAJB6b蛋白质的表达水平的靶向DNAJB6b的试剂选自敲降DNAJB6b蛋白质的表达水平的干扰RNA。
3.根据权利要求2所述的用途,其中所述敲降DNAJB6b蛋白质的表达水平的干扰RNA选自siRNA、短发夹RNA、单链干扰RNA和微RNA。
4.根据权利要求1所述的用途,其中所述拮抗DNAJB6b蛋白质的功能的靶向DNAJB6b的试剂选自特异性结合DNAJB6b的拮抗剂抗体。
5.根据权利要求1至4中任一项所述的用途,其中所述的肿瘤是高表达DNAJB6b蛋白质的肿瘤。
6.根据权利要求5所述的用途,其中所述高表达DNAJB6b蛋白质的肿瘤选自结直肠癌、食管癌、黑色素瘤、乳腺癌、肝癌、肺癌和宫颈癌。
7.一种体外筛选抗肿瘤试剂的方法,所述方法包括以下步骤:
1)体外培养肿瘤细胞株;
2)使待检测试剂与1)中培养的肿瘤细胞接触,
3)检测肿瘤细胞株中DNAJB6b蛋白质的表达水平,和
4)检测肿瘤细胞的增殖能力,
如果与接触待检测试剂之前相比,所述肿瘤细胞株中DNAJB6b蛋白质的表达水平显著降低,且肿瘤增殖能力显著降低,则所述待检测试剂可用于治疗肿瘤。
8.根据权利要求7所述的方法,其中所述肿瘤为结直肠癌,并且所述肿瘤细胞选自SW480、HCT116和Colo205。
9.根据权利要求7或8所述的方法,其中所述待检测试剂选自敲降DNAJB6b蛋白质的表达水平的干扰RNA和拮抗DNAJB6b蛋白质功能的抗体。
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CN107907685A (zh) * | 2017-11-07 | 2018-04-13 | 中国医学科学院肿瘤医院 | DNAJB6、Hsp70及Hsp90α的组合在Ⅱ期结肠癌预后判断中的应用 |
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CN110865186A (zh) * | 2018-08-28 | 2020-03-06 | 中国医学科学院肿瘤医院 | 蛋白标志物或其组合在结直肠癌预后判断中的应用 |
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