CN101802227B - 非衰竭与衰竭的人心脏中微小rna的差异表达 - Google Patents
非衰竭与衰竭的人心脏中微小rna的差异表达 Download PDFInfo
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Abstract
本发明公开了作为治疗心力衰竭的新的生物标志物和治疗性靶的特殊miRNA。本发明公开了通过施用这些特定的miRNA的模拟物和抑制剂来治疗或预防受试者中心力衰竭的方法。本发明也公开了通过测定一种或多种miRNA的表达水平来诊断或预后受试者中心力衰竭的方法。
Description
相关申请的交叉参考
本申请要求在2007年7月18日提交的美国临时申请第60/950,565号的权益,将其通过题述整体并入本文。
背景技术
心力衰竭是一种病理生理状态,在此状态下心脏无法泵送充足的血液以达到进行代谢的组织或细胞的营养和氧需求。其为许多心脏病的主要的并发症。40岁以上的成年人具有预计21%的患上心力衰竭的终身风险(Lloyd-Jones等人,2002,Circulation 106,3068-72),因该状况住院的人数多于所有形式的癌症的总和(American Heart Association.Heart Disease and StrokeStatistics--2003 Update)。
心力衰竭为描述多种疾病过程的最终共同结局的通用术语。引起心力衰竭的最常见的原因是能够导致心肌梗死(心脏病发作)的冠状动脉疾病,通常导致心脏细胞死亡。为了进行补偿,心脏则必须用余下的有活力的组织来执行同样的工作。慢性梗阻性冠状动脉疾病(chronic obstructive coronary arterydisease)也可以在无心肌梗死的情况下引起心力衰竭。瓣膜疾病或高血压能通过增加心脏的工作负荷而导致心力衰竭。心力衰竭较少见的原因(其主要涉及心肌)被归为心肌病(尽管这一术语有时被更广泛用作涵盖心力衰竭的任何原因)。这些病症中得到最好表征的是一组肌节的单基因病症,其经常导致“肥厚性心肌病”表型(事实上是一种误称,因为许多患者并没有肥大,即存在可变的表型外显率)。相反,具有“扩张性心肌病(dilated cardiomyopathy)”的所有患者都具有扩张的薄壁的心室。这种状况的遗传学尚有待表征并很可能是多因素的,然而在许多情况中非遗传原因可以显著地起作用(例如感染、酒精、化学治疗剂)。在没有找到易于确定的原因时,采用的诊断是原发性扩张性心肌病(通常为排除诊断)。
随着心力衰竭的发展,心脏会发生多种病理生理学改变。响应于体内增加的工作负荷,直接的结果是心脏往往在大小上增加(心脏肥大)然而继发于心脏细胞的肥大(即,在不存在细胞分裂的情况下细胞大小增大)。在细胞水平和分子水平,心脏肥大的特征在于收缩蛋白质表达的增加和各种各样的信号传导途径的激活,这些特征在心力衰竭的病理生理学中所起的作用仍然没有完全理解。
目前心力衰竭的治疗包括药理学方法,仪器如心室辅助装置(VDA)、心脏再同步治疗(CRT)和心脏移植。药理学方法包括但不限于运用肌力药(inotropic agent)(即增强心肌收缩性的化合物)、神经介质阻断剂(neurohumoralblocker)(例如β-阻断剂、血管紧张肽转变酶抑制剂(angiotensin convertingenzyme inhibitor))、醛固酮拮抗剂(aldosterone antagonist)、利尿剂(diuretic)和血管扩张剂(vasodilator)。然而,这些药物单独或组合使用都不是完全有效的。移植的供应度高度受限,并且由于许多患有心力衰竭的个体健康不佳,他们往往不是外科手术的好对象。由于这些原因,心力衰竭仍然是发病和死亡的主要原因,特别是在发达国家。另外,如上所指出的,可能难以确定心力衰竭的明确的病因,这是一个妨碍开发出更特异性的疗法的因素。此外,全面缺乏在分子水平的诊断技术。因而,在本领域需要发现另外的诊断标志物和药理学靶以开发治疗心力衰竭的新的治疗方法。此外,本领域需要改良评估心力衰竭的严重性及心力衰竭对治疗的响应的技术,如在此和即将描述的。本发明解决了上述等需要。
心脏肥大以及它发展为心力衰竭的可能性是对许多心血管疾病(包括高血压、缺血性心脏病、心瓣膜病和内分泌紊乱)的普通的病理学响应。心脏肥大涉及到心脏肌肉(心肌)的增厚,这可能导致血流阻塞和/或心脏功能减退(舒张性舒张(diastolic relaxation))。当心脏对作为正常生理过程(剧烈运动(rigorousexcise))或病理生理学过程如高血压、瓣膜狭窄或关闭不全、缺血或代谢病症的结果应激时,就会发生心脏肥大;其次,心脏肥大常常伴随程序性细胞死亡(凋亡)和/或组织坏死。考虑到大部分心脏细胞不能分裂,细胞增大以试图代偿;最初,这种代偿是有用的,但是过度肥大能够进行性活化许多其他的病理生理学级联反应,其导致进行性心脏机能障碍并且最终至失代偿性心力衰竭。因为心脏肥大可以是一个独立的风险因素,在其它心脏病风险因素之外,如高血压,当高血压得到治疗后,肥大可能会或不会复原。因此,直接治疗心脏肥大是有益的。
微小RNA(miRNAs)是一类近期发现的调节性RNA,其在转录后调节基因表达。miRNAs在进化上是保守(conserved)的,长度为大约18到25个核苷酸的非编码小RNA。miRNA与特殊“靶”mRNA碱基配对,并以此来抑制翻译或促进mRNA降解(Bartel,2004 Cell,116,281-297)。
据报道miRNAs涉及有机体的发育(Ambros,Cell 113:673-676)。miRNAs在很多组织中差异表达(Xu等,2003 Curr.Biol.13:790-795;Landgraf等,2007Cell 129:1401-14)。另据报道miRNAs也涉及病毒感染过程(Pfeffer等,2004Science 304:734-736),并与肿瘤生成相关(Calin,等,2004Proc.Natl.Acad.Sci.USA 101:2999-3004;Calin等,2002,Proc.Natl.Acad.Sci.USA 99(24):15524-15529)。虽然某些miRNAs在心脏中以高丰度表达,但是这些miRNAs在各种心脏病中的作用仍然需要进一步确定。
发明内容
本发明提供了一种诊断或预后受试者中心力衰竭的方法。在一个实施方案中,所述方法包括测定来自所述受试者的生物样品中一种或多种miRNAs的表达水平;并比较在上述生物样品中一种或多种miRNAs的表达水平与在标准样品中一种或多种miRNAs的水平;其中上述生物样品中至少一种或多种miRNAs的表达水平与在所述标准样品中一种或多种miRNAs的水平相比的差异对心力衰竭是诊断性或预测性的。
在另一个实施方案中,用于诊断或预后心力衰竭的方法的一种或多种miRNAs选自hsa-miR-542-5p(SEQ ID NO:1)、hsa-miR-125b(SEQ ID NO:2)、hsa-miR-197(SEQ ID NO:3)、hsa-miR-195(SEQ ID NO:4)、hsa-miR-92a(SEQID NO:5)、hsa-miR-139(SEQ ID NO:6)、hsa-miR-100(SEQ ID NO:7)、hsa-miR-483(SEQ ID NO:8)、hsa-miR-22(SEQ ID NO:9)、hsa-miR-23a(SEQID NO:10)、hsa-miR-486(SEQ ID NO:11)、hsa-miR-150(SEQ ID NO:12)、hsa-miR-30c(SEQ ID NO:13)、hsa-miR-342(SEQ ID NO:14)、hsa-miR-133a(SEQ ID NO:15)、hsa-miR-422b(也称作miR-378;SEQ ID NO:16)、hsa-miR-221(SEQ ID NO:17)、hsa-let-7f(SEQ ID NO:18)、hsa-miR-133b(SEQID NO:19)、hsa-miR-222(SEQ ID NO:20)、hsa-miR-224(SEQ ID NO:21)、hsa-let-7a(SEQ ID NO:22)、hsa-miR-1(SEQ ID NO:23)、hsa-miR-28(SEQ IDNO:24)、hsa-miR-199a(SEQ ID NO:25)、hsa-miR-181b(SEQ ID NO:26)、hsa-miR-20a(SEQ ID NO:27)、hsa-let-7c(SEQ ID NO:28)、hsa-miR-484(SEQID NO:29)、hsa-miR-26b(SEQ ID NO:30)、hsa-let-7d(SEQ ID NO:31)、hsa-miR-10b(SEQ ID NO:32)、hsa-miR-382(SEQ ID NO:33)、hsa-miR-92b(SEQ ID NO:48)、hsa-let-7b(SEQ ID NO:49)、hsa-let-7e(SEQ ID NO:50)和hsa-let-7g(SEQ ID NO:51)。
在另一实施方案中,用于诊断或预后心力衰竭的方法的一种或多种miRNAs选自:hsa-miR-125b(SEQ ID NO:2)、hsa-miR-195(SEQ ID NO:4)、hsa-miR-92a(SEQ ID NO:5)、hsa-miR-100(SEQ ID NO:7)、hsa-miR-22(SEQID NO:9)、hsa-miR-486(SEQ ID NO:11)、hsa-miR-150(SEQ ID NO:12)、hsa-miR-30c(SEQ ID NO:13)、hsa-miR-342(SEQ ID NO:14)、hsa-miR-133a(SEQ ID NO:15)、hsa-miR-221(SEQ ID NO:17)、hsa-let-7f(SEQ ID NO:18)、hsa-miR-133b(SEQ ID NO:19)、hsa-miR-222(SEQ ID NO:20)、hsa-let-7a(SEQID NO:22)、hsa-miR-1(SEQ ID NO:23)、hsa-miR-28(SEQ ID NO:24)和hsa-miR-199a(SEQ ID NO:25)。
在另一优选实施方案中,用于诊断或预后心力衰竭的方法的一种或多种miRNAs选自hsa-miR-195(SEQ ID NO:4)、hsa-miR-92a(SEQ ID NO:5)、hsa-miR-100(SEQ ID NO:7)、hsa-miR-486(SEQ ID NO:11)、hsa-miR-150(SEQ ID NO:12)和hsa-miR-422b(也称为miR-378;SEQ ID NO:16)。
在本发明中的又一个实施方案中,心力衰竭的类型是根据比较上述生物样品中一种或多种miRNAs的水平与上述标准样品中一种或多种miRNAs的水平来诊断的,其中所述一种或多种miRNAs的水平对于缺血性心肌病(ischemic cardiomyopathy)或特发性扩张性心肌病(idiopathic dilatedcardiomyopathy)是诊断性的。优选地,对于缺血性心肌病或特发性扩张性心肌病是诊断性的一种或多种miRNAs选自:hsa-miR-125b(SEQ ID NO:2)、hsa-miR-22(SEQ ID NO:9)、hsa-miR-150(SEQ ID NO:12)、hsa-miR-30c(SEQID NO:13)、hsa-miR-342(SEQ ID NO:14)、hsa-miR-133a(SEQ ID NO:15)、hsa-miR-422b(也称为miR-378;SEQ ID NO:16)、hsa-let-7f(SEQ ID NO:18)、hsa-miR-133b(SEQ ID NO:19)、hsa-miR-222(SEQ ID NO:20)、hsa-let-7a(SEQID NO:22)、hsa-miR-1(SEQ ID NO:23)、hsa-miR-28(SEQ ID NO:24)和hsa-miR-199a(SEQ ID NO:25)。
本发明还提供了治疗或预防受试者中心力衰竭或与心力衰竭有关的疾病或状况的方法。在一个实施方案中,所述方法包括向受试者的心脏细胞施用治疗有效量的miRNA模拟物或miRNA抑制剂。
在另一实施方案中,在所述治疗或预防心力衰竭或与心力衰竭有关的疾病或状况的方法中使用的miRNA模拟物或miRNA抑制剂为选自如下组的miRNAs中的一种或多种的模拟物或抑制剂:hsa-miR-542-5p(SEQ ID NO:1)、hsa-miR-125b(SEQ ID NO:2)、hsa-miR-197(SEQ ID NO:3)、hsa-miR-195(SEQ ID NO:4)、hsa-miR-92a(SEQ ID NO:5)、hsa-miR-139(SEQ ID NO:6)、hsa-miR-100(SEQ ID NO:7)、hsa-miR-483(SEQ ID NO:8)、hsa-miR-22(SEQID NO:9)、hsa-miR-23a(SEQ ID NO:10)、hsa-miR-486(SEQ ID NO:11)、hsa-miR150(SEQ ID NO:12)、hsa-miR-30c(SEQ ID NO:13)、hsa-miR-342(SEQ ID NO:14)、hsa-miR-133a(SEQ ID NO:15)、hsa-miR-422b(也称为miR-378;SEQ ID NO:16)、hsa-miR-221(SEQ ID NO:17)、hsa-let-7f(SEQ IDNO:18)、hsa-miR-133b(SEQ ID NO:19)、hsa-miR-222(SEQ ID NO:20)、hsa-miR-224(SEQ ID NO:21)、hsa-let-7a(SEQ ID NO:22)、hsa-miR-1(SEQ IDNO:23)、hsa-miR-28(SEQ ID NO:24)、hsa-miR-199a(SEQ ID NO:25)、hsa-miR-181b(SEQ ID NO:26)、hsa-miR-20a(SEQ ID NO:27)、hsa-let-7c(SEQID NO:28)、hsa-miR-484(SEQ ID NO:29)、hsa-miR-26b(SEQ ID NO:30)、hsa-let-7d(SEQ ID NO:31)、hsa-miR-10b(SEQ ID NO:32)、hsa-miR-382(SEQID NO:33)、hsa-miR-92b(SEQ ID NO:48)、hsa-let-7b(SEQ ID NO:49)、hsa-let-7e(SEQ ID NO:50)和hsa-let-7g(SEQ ID NO:51)。在另一实施方案中,这种方法进一步包括向受试者施用第二心脏治疗。该第二心脏治疗可以包括肌力药、神经介质阻断剂、醛固酮拮抗剂、利尿剂或血管扩张药。
在本发明的又一实施方案中,治疗或预防受试者中心力衰竭或与心力衰竭有关的疾病或状况的方法包括向受试者施用药物组合物,其中所述药物组合物包含至少一种miRNA模拟物或至少一种miRNA抑制剂和药理学上可接受的载体。所述miRNA模拟物或miRNA抑制剂可以是hsa-miR-542-5p(SEQID NO:1)、hsa-miR-125b(SEQ ID NO:2)、hsa-miR-197(SEQ ID NO:3)、hsa-miR-195(SEQ ID NO:4)、hsa-miR-92a(SEQ ID NO:5)、hsa-miR-139(SEQID NO:6)、hsa-miR-100(SEQ ID NO:7)、hsa-miR-483(SEQ ID NO:8)、hsa-miR-22(SEQ ID NO:9)、hsa-miR-23a(SEQ ID NO:10)、hsa-miR-486(SEQID NO:11)、hsa-miR150(SEQ ID NO:12)、hsa-miR-30c(SEQ ID NO:13)、hsa-miR-342(SEQ ID NO:14)、hsa-miR-133a(SEQ ID NO:15)、hsa-miR-422b(也称为miR-378;SEQ ID NO:16)、hsa-miR-221(SEQ ID NO:17)、hsa-let-7f(SEQ ID NO:18)、hsa-miR-133b(SEQ ID NO:19)、hsa-miR-222(SEQ ID NO:20)、hsa-miR-224(SEQ ID NO:21)、hsa-let-7a(SEQ ID NO:22)、hsa-miR-1(SEQ ID NO:23)、hsa-miR-28(SEQ ID NO:24)、hsa-miR-199a(SEQ ID NO:25)、hsa-miR-181b(SEQ ID NO:26)、hsa-miR-20a(SEQ ID NO:27)、hsa-let-7c(SEQ ID NO:28)、hsa-miR-484(SEQ ID NO:29)、hsa-miR-26b(SEQ ID NO:30)、hsa-let-7d(SEQ ID NO:31)、hsa-miR-10b(SEQ ID NO:32)、hsa-miR-382(SEQ ID NO:33)、hsa-miR-92b(SEQ ID NO:48)、hsa-let-7b(SEQ ID NO:49)、hsa-let-7e(SEQ ID NO:50)或hsa-let-7g(SEQ ID NO:51)的模拟物或抑制剂。在另一实施方案中,药物组合物进一步包含一种或多种肌力药、神经介质阻断剂、醛固酮拮抗剂、利尿药和血管扩张药。
本发明也提供了一种调节细胞中胎儿心脏基因程序(fetal cardiac geneprogram)(FGP)的再活化的方法。在一个实施方案中,所述方法包括提供具有一种或多种miRNA抑制剂或miRNA模拟物的心脏细胞,其中所述一种或多种miRNA抑制剂或miRNA模拟物调整胎儿心脏基因程序的至少一种标志物的表达。所述一种或多种miRNA抑制剂或miRNA模拟物可以是选自下组的一种或多种miRNAs的模拟物或抑制剂:hsa-miR-542-5p(SEQ ID NO:1)、hsa-miR-125b(SEQ ID NO:2)、hsa-miR-197(SEQ ID NO:3)、hsa-miR-195(SEQ ID NO:4)、hsa-miR-92a(SEQ ID NO:5)、hsa-miR-139(SEQ ID NO:6)、hsa-miR-100(SEQ ID NO:7)、hsa-miR-483(SEQ ID NO:8)、hsa-miR-22(SEQID NO:9)、hsa-miR-23a(SEQ ID NO:10)、hsa-miR-486(SEQ ID NO:11)、hsa-miR150(SEQ ID NO:12)、hsa-miR-30c(SEQ ID NO:13)、hsa-miR-342(SEQ ID NO:14)、hsa-miR-133a(SEQ ID NO:15)、hsa-miR-422b(也称为miR-378;SEQ ID NO:16)、hsa-miR-221(SEQ ID NO:17)、hsa-let-7f(SEQ IDNO:18)、hsa-miR-133b(SEQ ID NO:19)、hsa-miR-222(SEQ ID NO:20)、hsa-miR-224(SEQ ID NO:21)、hsa-let-7a(SEQ ID NO:22)、hsa-miR-1(SEQ IDNO:23)、hsa-miR-28(SEQ ID NO:24)、hsa-miR-199a(SEQ ID NO:25)、hsa-miR-181b(SEQ ID NO:26)、hsa-miR-20a(SEQ ID NO:27)、hsa-let-7c(SEQID NO:28)、hsa-miR-484(SEQ ID NO:29)、hsa-miR-26b(SEQ ID NO:30)、hsa-let-7d(SEQ ID NO:31)、hsa-miR-10b(SEQ ID NO:32)、hsa-miR-382(SEQID NO:33)、hsa-miR-92b(SEQ ID NO:48)、hsa-let-7b(SEQ ID NO:49)、hsa-let-7e(SEQ ID NO:50)和hsa-let-7g(SEQ ID NO:51)。在优选实施方案中,一种或多种miRNA抑制剂或miRNA模拟物为hsa-miR-92a(SEQ ID NO:5)、hsa-miR-100(SEQ ID NO:7)或hsa-miR-133b(SEQ ID NO:19)的模拟物或抑制剂。在另一实施方案中,所述胎儿心脏基因程序的至少一种标志物选自由αMyHC、βMyHC、ANF、BNP、SERCA2a和骨骼肌α-肌动蛋白质组成的组中。
本发明也包括监测对受试者进行的心力衰竭治疗的方法。在一个实施方案中,所述方法包括在第一时间点从受试者获得第一生物样品,其中所述第一时间点在心力衰竭的药物治疗方案开始之前;在第二时间点从受试者获得第二生物样品,其中所述第二时间点在心力衰竭的药物治疗方案开始之后;通过miRNA阵列分析、mRNA阵列分析和定向蛋白质组学表达分析(targetedproteomic expression analysis)处理第一生物样品和第二生物样品;并且将来自第一生物样品的miRNA、mRNA和蛋白质表达数据与来自第二生物样品的表达数据比较以确定基因表达模式(gene expression pattern)的变化,其中基因表达的变化指示所述药物治疗方案的效力。在另一实施方案中,第一时间点和第二时间大约间隔三至九个月。在另一实施方案中,mRNA分析是通过Affymetrix方案进行的。
附图简述
图1.miRNAs的相对表达。A.非衰竭(NF,n=6)对衰竭的特发性扩张性心肌病(IDC,n=5)的心脏,和B.NF对衰竭的、缺血性心肌病(ISC,n=5)的心脏。在对应于Sanger human miRBase 9.0(一个470miRNAs的数据组)的LCSciences(Houston,TX)miRNA表达阵列芯片上进行分析。以赝热温标(pseudo-thermal scale)表达数据,其中用红色表示增加的表达,以及用绿色表示降低的表达。仅表示出显著上调或下调(p<0.05)的miRNA。
图2.非衰竭(NF,n=6)与衰竭(IDC或ISCH,n=5,5)人心脏中相对miRNA表达的图示。注意某些miRNA在两种类型的衰竭心脏中都上调或下调,然而其它则基于心力衰竭类型差异性地表达。
图3.具有p<0.05的差异性表达的miRNAs亚组的相对表达。
图4.通过RT-PCR从提取自非衰竭(NF)、特发性心肌病(IDC)或缺血性心肌病(ISC)受试者的RNA评估miRNA的表达水平。将miRNA表达标准化为miR-24,其表达在非衰竭和衰竭心脏之间是恒定的。使用ANOVA进行统计分析。
图5在用miR-92模拟物或miR-92抑制剂转染的新生大鼠心室肌细胞中胎儿基因表达的变化。转染后24小时用10-7M异丙肾上腺素(ISO;黑色柱)处理细胞,并且在处理后48小时收集。通过RT-PCR测量基因表达。将结果标准化为18S rRNA表达水平,并与用对照模拟物或抑制剂转染的细胞(定义为1(在刻度1处的线))比较。该图代表3-4个单独实验的平均值。
图6.在用miR-100模拟物或miR-100抑制剂转染的新生大鼠心室肌细胞中胎儿基因表达的变化。转染后24小时用10-7M异丙肾上腺素(ISO;黑色柱)处理细胞并且在处理后48小时收集。通过RT-PCR测量基因表达。将结果标准化为18S rRNA表达水平,并与用对照模拟物或抑制剂转染的细胞(定义为1(在刻度1处的线))比较。该图代表3-4个单独实验的平均值。
图7.在用miR-133b模拟物或miR-133b抑制剂转染的新生大鼠心室肌细胞中胎儿基因表达的变化。转染后24小时用10-7M异丙肾上腺素(ISO;黑色柱)处理细胞并且在处理后48小时收集。通过RT-PCR测量基因表达。将结果标准化为18S rRNA表达水平,并与用对照模拟物或抑制剂转染的细胞(定义为1(在刻度1处的线))比较。该图代表3-4个单独实验的平均值。
图8.用模拟物miRNA或抑制剂miRNA转染后的miRNA水平。将miR-92、miR-100和miR-133b的模拟物和抑制剂转染至新生大鼠心室肌细胞。通过RT-PCR评估每种miRNA的表达水平。
图9.在新生大鼠心室肌细胞中miRNA-133b的过表达或下调调节细胞的肥大。将细胞用miR-133b模拟物(下方的组)或miR-133b抑制剂(上方的组)转染并且用异丙肾上腺素处理(右侧的组)或者不进行进一步处理(左侧的组)。用抗-辅肌动蛋白抗体使图显示免疫荧光。
图10.在新生大鼠心室肌细胞中miRNA-133b的过表达或下调调节细胞的肥大。用miR-133b模拟物或miR-133b抑制剂转染细胞,并将转染细胞的一个亚组暴露于异丙肾上腺素(黑色柱)。使用Image J软件测量细胞的尺寸。测量来自3个不同区域的总共30个细胞。
图11.将基因表达系列分析(SAGE)作为用于发现基于miRNA的靶的方法的集成方法的示意图。心力衰竭(HF)患者心内膜心肌活检(endomyocardialbiopsy)被用于为miRNA阵列分析、mRNA(Afflymetrix)阵列分析和(定向)蛋白质组学表达分析提供生物样品。
图12.用β-阻断剂治疗的人患者的miR-133的表达。对来自用β-阻断剂治疗的患者的样品进行RT-PCR分析。在末期心力衰竭(A)、治疗后3个月(B)和治疗后12个月(C)获得系列活检标本。将miR-133b(上方的组)和miR-133a(下方的组)的表达水平标准化为miR-370(右侧的组)和小RNA RNU66(左侧的组)的表达。患者20、21、25和34对β-阻断剂治疗应答,但是患者103对所述治疗无应答。
图13.用β-阻断剂治疗的人患者的miR-1和miR-19b的表达。对来自用β-阻断剂治疗的患者的样品进行RT-PCR分析。在末期心力衰竭(A)、治疗后3个月(B)和治疗后12个月(C)获得系列活检标本。miR-1(上方的组)和miR-19b(下方的组)的表达水平被标准化为miR-370(右侧的组)和小RNA RNU66(左侧的组)的表达。患者20、21、25和34对β-阻断剂治疗应答,但是患者103对治疗无应答。
图14用β-阻断剂治疗的人患者的miR-30d和miR-92a的表达。对来自用β-阻断剂治疗的患者的样品进行RT-PCR分析。在末期心力衰竭(A)、治疗后3个月(B)和治疗后12个月(C)获得系列活检标本。miR-30d(上方的组)和miR-92a(下方的组)的表达水平被标准化为miR-370(右侧的组)和小RNARNU66(左侧的组)的表达。患者20、21、25和34对β-阻断剂治疗应答,但是患者103对治疗无应答。
图15.用β-阻断剂治疗的人患者的miR-208b和miR-499的表达。对来自用β-阻断剂治疗的患者的样品进行RT-PCR分析。在末期心力衰竭(A)、治疗后3个月(B)和治疗后12个月(C)获得系列活检标本。miR-208b(上方的组)和miR-499(下方的组)的表达水平被标准化为miR-370(右侧的组)和小RNARNU66(左侧的组)的表达。患者20、21、25和34对β-阻断剂治疗有应答,但是患者103对治疗无应答。
图16.用β-阻断剂治疗的人患者的miR-let-7g的表达。对来自用β-阻断剂治疗的患者的样品进行RT-PCR分析。在末期心力衰竭(A)、治疗后3个月(B)和治疗后12个月(C)获得系列活检标本。miR-let-7g的表达水平被标准化为miR-370(右侧的组)和小RNA RNU66(左侧的组)的表达。患者20、21、25和34对β-阻断剂治疗应答,但是患者103对治疗无应答。
图17.miR-92a靶向PKCε和PDE1A。用miR-92a模拟物、miR-92a抑制剂或乱序控制序列(scrambled control sequence)转染的新生大鼠心室肌细胞的Western印迹分析。A.用PKCε探测的印迹(n=2)(blot probed for PKCε)。B.用PDE1A探测的印迹(n=1)。钙联接蛋白表达被用作上样对照。
图18.miR-133b靶向钙调蛋白。A.对用miR-133b模拟物、miR-133b抑制剂或乱序控制序列转染的新生大鼠心室肌细胞中钙调蛋白mRNA表达的RT-PCR分析。钙调蛋白的表达水平被标准化为18S核糖体亚基的表达。B.用包含连接至钙调蛋白3’UTR和miR-133b模拟物、miR-133b抑制剂或乱序控制序列的萤光素酶编码区的构建体共转染的新生大鼠心室肌细胞中的萤光素酶活性。C.对用miR-133b模拟物、miR-133b抑制剂或乱序控制序列转染的新生大鼠心室肌细胞的钙调蛋白进行的Western印迹分析。将钙联接蛋白的表达用作上样对照。所有实验n=3。
发明详述
本发明部分基于如下发现:微小RNA(miRNAs)的特殊亚组在健康心脏和衰竭心脏之间存在差异性表达。有趣的是,一些miRNAs在特定类型的衰竭心脏(如特发性扩张性心肌病或缺血性心肌病)中是上调或下调的。这些差异性表达的miRNAs为心力衰竭提供了新的生物标志物,也为心力衰竭的有效治疗的开发提供了靶。因此,本发明提供诊断和预后受试者中心力衰竭的方法。也提供治疗或预防受试者中心力衰竭的方法。
miRNAs是源自较大的前体的、长度为大约18-25个核苷酸,特别是21-22个核苷酸的小的、非编码RNAs。当miRNAs和靶mRNA的序列完全互补时,miRNAs通过促进靶mRNAs的降解来发挥其抑制剂的作用,或者当它们的序列包含错配时,miRNAs通过抑制翻译来发挥mRNAs的抑制剂的作用。
miRNAs通过RNA聚合酶II(polII)或RNA聚合酶III(pol III;参见Qi等(2006)Cellular & Molecular Immunology Vol.3:411-419)进行转录,并且其可以源于单个的miRNA基因、源于蛋白质编码基因的内含子或源于经常编码多个、紧密相关的miRNAs的多顺反子的转录物。通过RNA pol II或pol III的miRNA基因的转录产生最初的转录物,称为初级miRNA转录物(初miRNA(pri-miRNAs)),其长度通常为数千个碱基。在细胞核中,初级miRNA通过RNA酶、Drosha的处理,得到70-至100-核苷酸的发夹形前体(前miRNA(pre-miRNAs))。在转运至胞质后,发夹前miRNA由Dicer进一步加工产生双链miRNA。然后将成熟的miRNA链并入RNA诱导的沉默复合物(RISC)(RNA-induced silencing complex),其通过碱基对互补性与其靶mRNAs结合。在相对罕见的情况中,miRNA与靶mRNA的碱基完美配对,其促进mRNA降解。更常见的是,miRNA与靶mRNA形成不完全异源双链,影响mRNA的翻译。miRNA跨越2-8个碱基的5’部分,称为种子区(‘seed’region),对靶识别尤其重要(Krenz and Robbins(2004)J.Am.Coll.Cardiol.,Vol.44:2390-2397;Kiriazis and Kranias(2000)Annu.Rev.Physiol.,Vol.62:321-351)。所述种子的序列,连同靶序列的系统发生学保守性,形成了许多当前靶预测模型的基础。
本发明提供了一种通过测定获得自受试者的生物样品中特殊miRNAs的表达水平而诊断和预后所述受试者中的心力衰竭的方法。如在此所用的,术语“心力衰竭”广泛涉及降低心脏泵血能力的任何状况。因此,在组织形成充血和水肿。最常见的,心力衰竭是由冠状动脉血流减少导致的心肌的收缩性降低所引起的,然而,许多其它因素也可导致心力衰竭,包括心瓣膜损伤,维生素缺乏和原发性心肌病。
在本发明的一个实施方案中,所述方法包括测定来自受试者的生物样品中一种或多种miRNAs的表达水平,并且比较在上述生物样品中一种或多种miRNAs的表达水平与在标准样品中一种或多种miRNAs的水平,其中在上述生物样品中至少一种或多种miRNAs的表达水平与在上述标准样品中一种或多种miRNAs的水平相比的差异对心力衰竭是诊断性和预测性的。生物样品可以包括,但不局限于,血液、血清、血浆和组织样品。在一个优选实施方案中,生物样品是心肌组织(如心耳、心内膜心肌活检等)。“标准样品”是指达标无病状态的样品,尤其是代表不存在无心力衰竭或其它任何相关状况的状态(即健康状态)。例如,标准样品可以是生物样品,如从健康受试者中获得的心肌组织,所述健康受试者与为其提供诊断和预后的受试者年龄相似。标准样品可以是混合样品(composite sample),其中将获得自来自若干健康受试者(即,无心力衰竭的症状的对照受试者)的生物样品的数据平均,从而产生混合样品。
测定样品中miRNAs的表达水平的方法为本领域普通的技术人员所熟知。这些方法包括聚合酶链反应(PCR)技术,如逆转录酶-PCR和实时PCR、Northern印迹和微阵列分析。在本发明的另一个实施方案中,所述方法包括测定选自下组的一种或多种miRNAs的表达水平:hsa-miR-542-5p(SEQ IDNO:1)、hsa-miR-125b(SEQ ID NO:2)、hsa-miR-197(SEQ ID NO:3)、hsa-miR-195(SEQ ID NO:4)、hsa-miR-92a(SEQ ID NO:5)、hsa-miR-139(SEQID NO:6)、hsa-miR-100(SEQ ID NO:7)、hsa-miR-483(SEQ ID NO:8)、hsa-miR-22(SEQ ID NO:9)、hsa-miR-23a(SEQ ID NO:10)、hsa-miR-486(SEQID NO:11)、hsa-miR-150(SEQ ID NO:12)、hsa-miR-30c(SEQ ID NO:13)、hsa-miR-342(SEQ ID NO:14)、hsa-miR-133a(SEQ ID NO:15)、hsa-miR-422b(也称为miR-378;SEQ ID NO:16)、hsa-miR-221(SEQ ID NO:17)、hsa-let-7f(SEQ ID NO:18)、hsa-miR-133b(SEQ ID NO:19)、hsa-miR-222(SEQ ID NO:20)、hsa-miR-224(SEQ ID NO:21)、hsa-let-7a(SEQ ID NO:22)、hsa-miR-1(SEQ ID NO:23)、hsa-miR-28(SEQ ID NO:24)、hsa-miR-199a(SEQ ID NO:25)、hsa-miR-181b(SEQ ID NO:26)、hsa-miR-20a(SEQ ID NO:27)、hsa-let-7c(SEQ ID NO:28)、hsa-miR-484(SEQ ID NO:29)、hsa-miR-26b(SEQ ID NO:30)、hsa-let-7d(SEQ ID NO:31)、hsa-miR-10b(SEQ ID NO:32)、hsa-miR-382(SEQ ID NO:33)、hsa-miR-92b(uauugcacucgucccggccucc;SEQ ID NO:48)、hsa-let-7b(ugagguaguagguugugugguu;SEQ ID NO:49)、hsa-let-7e(ugagguaggagguuguauaguu;SEQ ID NO:50)和hsa-let-7g(ugagguaguaguuuguacaguu;SEQ ID NO:51)。在另一个实施方案中,所述一种或多种miRNAs选自hsa-miR-125b(SEQ ID NO:2)、hsa-miR-195(SEQ ID NO:4)、hsa-miR-92a(SEQ ID NO:5)、hsa-miR-100(SEQ ID NO:7)、hsa-miR-22(SEQ ID NO:9)、hsa-miR-486(SEQ ID NO:11)、hsa-miR-150(SEQ ID NO:12)、hsa-miR-30c(SEQ ID NO:13)、hsa-miR-342(SEQ ID NO:14)、hsa-miR-133a(SEQ ID NO:15)、hsa-miR-221(SEQ ID NO:17)、hsa-let-7f(SEQ ID NO:18)、hsa-miR-133b(SEQ ID NO:19)、hsa-miR-222(SEQ ID NO:20)、hsa-let-7a(SEQID NO:22)、hsa-miR-1(SEQ ID NO:23)、hsa-miR-28(SEQ ID NO:24)和hsa-miR-199a(SEQ ID NO:25)。在又一个实施方案中,所述一种或多种miRNAs选自hsa-miR-195(SEQ ID NO:4)、hsa-miR-92a(SEQ ID NO:5)、hsa-miR-100(SEQ ID NO:7)、hsa-miR-486(SEQ ID NO:11)、hsa-miR-150(SEQ ID NO:12)和hsa-miR-422b(也称为miR-378;SEQ ID NO:16)。
在本发明的另一个实施方案中,所述方法还包括根据与上述标准样本中一种或多种miRNAs的水平相比的上述生物学样本中一种或多种miRNAs的水平来诊断心力衰竭的类型,其中所述一种或多种miRNAs的水平对缺血性心肌病或特发性扩张性心肌病是诊断性的。如在此所使用的,“特发性扩张性心肌病”(IDC)定义为左、右或双心室扩张,伴有未知原因的损伤的收缩性。IDC是产生心力衰竭症状的扩张性心肌病的一种普通形式。IDC可能出现或不出现明显的充血性心力衰竭并且常见心率不齐(arrhythmias),且预后通常不佳。如在此使用的“缺血性心肌病”(ISC)指的是由于输送至心肌的氧不足而导致心脏中肌肉衰弱的患者,通常是由于冠状动脉疾病。ISC是充血性心力衰竭的常见原因,并且患有该状况的患者可能曾经一度经历心脏病发作、心绞痛或不稳定心绞痛。在美国ISC是心肌病的一种最常见类型;它影响大约1%的人口,最常见为中年至老年人。
可用于诊断IDC或ISC的一种或多种miRNAs包括hsa-miR-125b(SEQ IDNO:2)、hsa-miR-22(SEQ ID NO:9)、hsa-miR-150(SEQ ID NO:12);hsa-miR-30c(SEQ ID NO:13)、hsa-miR-342(SEQ ID NO:14)、hsa-miR-133a(SEQ ID NO:15)、hsa-miR-422b(也称为miR-378;SEQ ID NO:16)、hsa-let-7f(SEQ ID NO:18)、hsa-miR-133b(SEQ ID NO:19)、hsa-miR-222(SEQ ID NO:20)、hsa-let-7a(SEQ ID NO:22)、hsa-miR-1(SEQ ID NO:23)、hsa-miR-28(SEQID NO:24)和hsa-miR-199a(SEQ ID NO:25)。
本发明也提供一种治疗和预防受试者心力衰竭或与心力衰竭相关的疾病或状况的方法。在一个实施方案中,所述方法包括向受试者的心脏细胞施用治疗有效剂量的miRNA模拟物或miRNA抑制剂。“治疗有效剂量”是足以改善或预防至少一种心力衰竭症状的量。心力衰竭的症状包括,但不限于,呼吸困难(dyspnea)、端坐呼吸(orthopnea)、头晕(dizziness)、精神错乱(confusion)、出汗(diaphoresis)、外周性水肿(peripheral edema)、腹水(ascites)、肝肿大(hepatomegaly)、疲劳(fatigue)、恶心(nausea)、心率增快(increased heart rate)和肺水肿(pulmonary edema)。
在另一个实施方案中,所述方法包括施用选自下组的miRNAs中的一种或多种的miRNA模拟物或miRNA抑制剂:hsa-miR-542-5p(SEQ ID NO:1)、hsa-miR-125b(SEQ ID NO:2)、hsa-miR-197(SEQ ID NO:3)、hsa-miR-195(SEQ ID NO:4)、hsa-miR-92a(SEQ ID NO:5)、hsa-miR-139(SEQ ID NO:6)、hsa-miR-100(SEQ ID NO:7)、hsa-miR-483(SEQ ID NO:8)、hsa-miR-22(SEQID NO:9)、hsa-miR-23a(SEQ ID NO:10)、hsa-miR-486(SEQ ID NO:11)、hsa-miR-150(SEQ ID NO:12)、hsa-miR-30c(SEQ ID NO:13)、hsa-miR-342(SEQ ID NO:14)、hsa-miR-133a(SEQ ID NO:15)、hsa-miR-422b(也称为miR-378;SEQ ID NO:16)、hsa-miR-221(SEQ ID NO:17)、hsa-let-7f(SEQ IDNO:18)、hsa-miR-133b(SEQ ID NO:19)、hsa-miR-222(SEQ ID NO:20)、hsa-miR-224(SEQ ID NO:21)、hsa-let-7a(SEQ ID NO:22)、hsa-miR-1(SEQ IDNO:23)、hsa-miR-28(SEQ ID NO:24)、hsa-miR-199a(SEQ ID NO:25)、hsa-miR-181b(SEQ ID NO:26)、hsa-miR-20a(SEQ ID NO:27)、hsa-let-7c(SEQID NO:28)、hsa-miR-484(SEQ ID NO:29)、hsa-miR-26b(SEQ ID NO:30)、hsa-let-7d(SEQ ID NO:31)、hsa-miR-10b(SEQ ID NO:32)、hsa-miR-382(SEQID NO:33)、hsa-miR-92b(uauugcacucgucccggccucc;SEQ ID NO:48)、hsa-let-7b(ugagguaguagguugugugguu;SEQ ID NO:49)、hsa-let-7e(ugagguaggagguuguauaguu;SEQ ID NO:50)和hsa-let-7g(ugagguaguaguuuguacaguu;SEQ ID NO:51)。
“miRNA模拟物”是一种用于增加miRNA表达和/或功能的作用物。miRNA模拟物还能增加、补充或取代天然miRNA的功能。在一个实施方案中,miRNA模拟物可以是包含成熟miRNA序列的多核苷酸。在另一个实施方案中,miRNA模拟物可以是包含初miRNA或前miRNA序列的多核苷酸。所述miRNA模拟物可以包含化学修饰,如锁核酸、肽核酸,糖修饰,如2’-O-烷基(如2’-O-甲基、2’-O-甲氧基乙基),2’-氟和4’硫修饰,和主链修饰,如一种或多种硫代磷酸酯、吗啉代或膦酰基羧酸酯连接(phosphonocarboxylatelinkage)。某些miRNA模拟物从公司购得,如Dharmacon(Lafayette,CO)和Ambion,Inc。
在一些实施方案中,miRNA模拟物可以在体内从载体表达。“载体”是可以用于将目标核酸递送至细胞的内部的物质组合物。本领域已知多种载体,包括,但不局限于,线性多核苷酸、与离子的或两亲性化合物联合的多核苷酸、质粒和病毒。因此,术语“载体”包括自主复制质粒或病毒。病毒载体的实例包括,但不限于,腺病毒载体、腺伴随病毒载体、逆转录病毒载体等。表达构建体可以在活细胞中复制,或者其能够以合成方法制备。就本申请而言,术语“表达构建体”、“表达载体”和“载体”可以互换地使用以总体的、说明的方式阐述本发明的应用,而并非意欲限制本发明。
在一个实施方案中,表达miRNA模拟物的表达载体包含与编码特定miRNA的多核苷酸“可操作地连接”的启动子。如在此所用的短语“可操作地连接”或“在转录控制下”意思是所述启动子在与多核苷酸相关的正确位置和方向以控制通过RNA聚合酶进行的转录的起始和所述多核苷酸的表达。编码所述miRNA的多核苷酸可以编码初级-微小RNA序列(初miRNA)、前体-微小RNA序列(前miRNA)或成熟miRNA序列。在一个优选实施方案中,多核苷酸包含hsa-miR-133b(SEQ ID NO:19)的序列。编码特定的miRNA的多核苷酸长度可以是大约18至大约2000个核苷酸、大约70至大约200个核苷酸、大约20至大约50个核苷酸、或大约18至大约25个核苷酸。在另外的实施方案中,编码特定的miRNA的多核苷酸位于编码内含子的核酸中或位于编码mRNA非翻译区的核酸中或位于非编码RNA中。
在一些实施方案中,所述miRNA模拟物包含hsa-miR-197(SEQ ID NO:3)、hsa-miR-92a(SEQ ID NO:5)、hsa-miR-139(SEQ ID NO:6)、hsa-miR-483(SEQ ID NO:8)、hsa-miR-22(SEQ ID NO:9)、hsa-miR-486(SEQ ID NO:11)、hsa-miR-150(SEQ ID NO:12)、hsa-miR-30c(SEQ ID NO:13)、hsa-miR-133a(SEQ ID NO:15)、hsa-miR-422b(也称为miR-378;SEQ ID NO:16)、hsa-miR-221(SEQ ID NO:17)、hsa-let-7f(SEQ ID NO:18)、hsa-miR-133b(SEQID NO:19)、hsa-miR-222(SEQ ID NO:20)、hsa-miR-224(SEQ ID NO:21)、hsa-let-7a(SEQ ID NO:22)、hsa-miR-1(SEQ ID NO:23)、hsa-miR-20a(SEQ IDNO:27)、hsa-let-7c(SEQ ID NO:28)、hsa-miR-484(SEQ ID NO:29)、hsa-let-7d(SEQ ID NO:31)或hsa-miR-10b(SEQ ID NO:32)的序列。在一个优选实施方案中,所述miRNA模拟物包含hsa-miR-133b(SEQ ID NO:19)的序列。
“miRNA抑制剂”是以序列特异性方式抑制miRNA功能的作用物。在一个实施方案中,miRNA抑制剂是一种微小RNA拮抗剂(antagomir)。由Krützfeldt和同事(Krützfeldt等(2005)Nature,Vol.438:685-689)首先描述,“微小RNA拮抗剂”是单链的,化学修饰的核糖核苷酸,其至少与miRNA序列部分互补。微小RNA拮抗剂可以包含一种或多种修饰的核苷酸,如2’-O-甲基-糖修饰。在一些实施方案中,微小RNA拮抗剂仅包含修饰的核苷酸。微小RNA拮抗剂也可以包含一种或多种硫代磷酸酯连接,其导致部分或完全硫代磷酸酯主链。为了促进体内递送和稳定性,可以将微小RNA拮抗剂在其3’末端与胆固醇部分连接。适合抑制miRNAs的微小RNA拮抗剂的长度可以是大约15至大约50个核苷酸,更优选是大约18至大约30个核苷酸,并且最优选是大约20至25个核苷酸。“部分互补”指的是与靶多核苷酸序列至少大约75%、80%、85%、90%、95%、96%、97%、98%或99%互补的序列。所述微小RNA拮抗剂可以与成熟miRNA序列至少大约75%、80%、85%、90%、95%、96%、97%、98%或99%互补。在一些实施方案中,所述微小RNA拮抗剂与成熟miRNA序列100%互补。
在另一个实施方案中,miRNA抑制剂是靶向成熟miRNA序列的反义寡核苷酸。所述反义寡核苷酸可以包含核糖核苷酸或脱氧核糖核苷酸。在一些实施方案中,所述反义寡核苷酸具有至少一种化学修饰。反义寡核苷酸可以包含一种或多种“锁苷酸(locked nucleic acid)”。“锁核酸”(LNAs)是修饰的核糖核苷酸,其包含位于核酸糖部分的2’和4’碳之间的额外的桥键,该桥键导致“锁”构象,该构象赋予含有LNAs的寡核苷酸增强的热稳定性。或者,所述反义寡核苷酸可以包含肽核酸(PNAs),其包含基于肽的主链而非糖-磷酸主链。反义寡核苷酸可以包含的其它化学修饰包括,但不局限于,糖修饰,如2’-O-烷基(如2’-O-甲基、2’-O-甲氧基乙基),2’-氟和4’硫修饰和主链修饰,如一种或多种硫代磷酸酯、吗啉代或膦酰基羧酸酯连接(参见,例如,美国专利第6,693,187和7,067,641号,将它们全部通过题述整体并入本文)。在一些实施方案中,合适的反义寡核苷酸是在中心具有至少10个脱氧核糖核苷酸的、在5’和3’末端均包含2’-O-甲氧乙基-修饰的核糖核苷酸的2’-O-甲氧基乙基“缺口聚物(gapmer)”。这些“缺口聚物”能够触发RNA酶H依赖性的RNA靶降解机制。增强稳定性和提高效力的反义寡核苷酸的其它修饰(如那些在美国专利第6,838,283号所描述的那些,将其全部通过题述整体并入本文)是本领域已知的并且适用于本发明的方法。可用于抑制miRNAs活性的优选的反义寡核苷酸长度是大约19至大约25个核苷酸。反义寡核苷酸可以包含与成熟miRNA序列至少部分互补的序列,如至少与成熟miRNA序列大约75%、80%、85%、90%、95%、96%、97%、98%或99%互补。在一个实施方案中,反义寡核苷酸包含与成熟miRNA序列100%互补的序列。
在一些实施方案中,miRNA抑制剂包含与hsa-miR-125b(SEQ ID NO:2)、hsa-miR-195(SEQ ID NO:4)、hsa-miR-100(SEQ ID NO:7)、hsa-miR-23a(SEQID NO:10)、hsa-miR-342(SEQ ID NO:14)、hsa-miR-28(SEQ ID NO:24)、hsa-miR-199a(SEQ ID NO:25)、hsa-miR-181b(SEQ ID NO:26)、hsa-miR-26b(SEQ ID NO:30)或hsa-miR-382(SEQ ID NO:33)互补的序列。在一个优选实施方案中,miRNA抑制剂包含与hsa-miR-100(SEQ ID NO:7)互补的序列。
在又一实施方案中,miRNA抑制剂是与成熟miRNA序列至少具有部分序列同一性的抑制性RNA分子。该抑制性RNA分子可以是双链的、小干扰RNA(siRNA)或包含茎环结构的短发夹RNA分子(shRNA)。抑制性RNA分子的双链区可以包含与成熟miRNA序列具有至少大约75%、80%、85%、90%、95%、96%、97%、98%或99%同一性的序列。在一些实施方案中,抑制性RNA分子的双链区可以与靶mRNA序列含有100%同一性的序列。抑制性RNA分子可以包含与hsa-miR-125b(SEQ ID NO:2)、hsa-miR-195(SEQ ID NO:4)、hsa-miR-100(SEQ ID NO:7)、hsa-miR-23a(SEQ ID NO:10)、hsa-miR-342(SEQ ID NO:14)、hsa-miR-28(SEQ ID NO:24)、hsa-miR-199a(SEQ ID NO:25)、hsa-miR-181b(SEQ ID NO:26)、hsa-miR-26b(SEQ ID NO:30)或hsa-miR-382(SEQ ID NO:33)具有至少部分序列同一性的序列。
所述miRNA抑制剂可以在活体内自表达载体产生。在一个实施方案中,表达miRNA抑制剂的表达载体包含与编码反义寡核苷酸的多核苷酸可操作地连接的启动子,其中表达的反义寡核苷酸的序列与成熟miRNA序列互补。在另一个实施方案中,表达miRNA抑制剂的表达载体包含与编码shRNA或siRNA的多核苷酸可操作地连接的一种或多种启动子,其中表达的shRNA或siRNA包含与成熟miRNA序列完全相同或基本相同的序列。“基本相同”指的是与靶多核苷酸序列至少大约75%、80%、85%、90%、95%、96%、97%、98%或99%相同的序列。
在本发明的另一个实施方案中,治疗或预防受试者心力衰竭或与心力衰竭相关的疾病或状况的方法还包括向受试者施用第二心脏治疗。心脏治疗包括通常用于治疗心力衰竭或与心力衰竭相关的疾病或状况的一种或多种症状任何疗法。第二心脏治疗可以包括,那不局限于,所谓“β阻断剂”,抗高血压药、强心剂、抗血栓药、血管扩张剂、激素拮抗剂、肌力药(iontropes)、利尿剂、内皮素受体拮抗剂(endothelin receptor antagonist)、钙通道阻断剂、磷酸二酯酶抑制剂,ACE抑制剂、血管紧张肽2型拮抗剂和细胞因子阻断剂/抑制剂、HDAC抑制剂和组蛋白乙酰基转移酶(HAT)调节剂。在一个实施方案中,所述第二心脏治疗是肌力药、神经介质阻断剂、醛固酮拮抗剂、利尿剂、血管扩张药、内皮素受体拮抗剂或HDAC抑制剂。
“肌力药(inotropic agent)”指的是增加或降低肌肉收缩的力或能量的药物,特别是增加心肌收缩性的强度的药物。正性肌力药(positive inotropic agent)可以包括钙;钙增敏剂左西孟旦(calcium sensitizer levosimendan);强心苷(cardiacglycoside)、地高辛(digoxin);儿茶酚胺类(catecholamines),如多巴胺、多巴酚丁胺(dobutamine)、多培沙明(dopexamine)、肾上腺素(epinephrine)、异丙肾上腺素(isoproterenol)和去甲肾上腺素(norepinephrine);以及磷酸二酯酶抑制剂,如依诺西蒙(enoximone)、米力农(milrinone)和茶碱(theophylline)。负性肌力药(negative inotropic agent)包括β肾上腺素能阻断剂(beta adrenergicblocker),如拉贝洛尔(labetalol)和卡维地洛(carvedilol);以及钙通道阻断剂,如地尔硫卓(diltiazam)和维拉帕米(verapamil)。
“神经介质阻断剂”指的是阻断神经介质应答的药物且包括β肾上腺素能阻断剂,如比索洛尔(bisoprolol)、卡维地洛和美托洛尔(metoprolol);以及血管紧张肽转变酶抑制剂,如依那普利(enalapril)、赖诺普利(lisinopril)、雷米普利(ramipril)和卡托普利(captopril)。
“利尿剂”指的是规定用于液体潴留(fluid retention)和脚、腿及腹部肿胀的药物。利尿剂促进肾从血液中滤过更多的钠和水。体内液体变少,心脏就能用更小的力量来泵送和循环血液。另外,利尿药能够减少肺、踝、腿和身体其它部位中的液体潴留。利尿剂包括,例如呋塞米(furosemide)、布美他尼(bumetanide)、美托拉宗(metolazone)、螺内酯(spironolactone)和依普利酮(eplerenone)。
“醛固酮拮抗剂”指的是对抗醛固酮在盐皮质激素受体上的作用的药物。这组药物通常与其他药物组合作为辅助治疗用于处理慢性心力衰竭。醛固酮拮抗剂包括,例如螺内酯和依普利酮。
“血管扩张药”指的是扩张或引起血管的直径增加的药物。血管扩张剂包括,例如肼屈嗪(hydralazine)、硝酸异山梨酯(isosorbide dinitrate)和单硝酸异山梨酯(isosorbide mononitrate)、卡托普利(captopril)和较长效因子如麦他明(metamine)、二氧林(paveril)、硝酸甘油(nitroglyn)和长效硝酸甘油(peritrate)。
内皮素(ET)是21-氨基酸肽,其具有似乎与心力衰竭发展相关的强力生理学和病理生理学效应。ET的作用是通过与两类细胞表面受体的相互作用介导的。A型受体(ET-A)与血管收缩和细胞生长有关,而B型受体(ET-B)与内皮细胞介导的血管舒张和其它神经激素(如醛固酮)的释放有关。本领域已知能够抑制ET的产生或其刺激相关细胞的能力的药理制剂。抑制ET的产生涉及阻断称为内皮素转化酶的酶的作用物的施用,该酶涉及自其前体加工活性肽。抑制ET刺激细胞的能力涉及使用阻断ET与其受体的相互作用的作用物。内皮素受体拮抗剂(ERA)的非限制性实例包括波生坦(Bosentan)、恩拉生坦(Enrasentan)、安贝生坦(Ambrisentan)、达卢生坦(Darusentan)、替唑生坦(Tezosentan)、阿曲生坦(Atrasentan)、阿伏生坦(Avosentan)、克拉生坦(Clazosentan)、艾多南坦(Edonentan)、西他生坦(sitaxsentan)、TBC 3711、BQ 123和BQ 788。
可以将第二心脏治疗与所述miRNA模拟物或miRNA抑制剂向受试者共施用(例如同时)。或者,第二心脏治疗可以在miRNA模拟物或miRNA抑制剂之前或之后施用。施用miRNA模拟物或miRNA抑制剂和施用第二心脏治疗之间的时间间隔的范围可以是从数分钟到数周。在一些实施方案中,两种治疗可以在彼此的12-24小时内,更优选在彼此的6-12小时内,并且最优选在彼此的12小时内施用。然而,在一些情况下,显著延长治疗时间可能是理想的,其中在各施用时间之间经过若干天(2、3、4、5、6或7)至若干周(1、2、3、4、5、6、7或8)。
本发明也包括治疗和预防受试者心力衰竭或与心力衰竭相关的疾病或状况的方法,其包括向受试者施用药物组合物,其中所述药物组合物包含至少一种miRNA模拟物或至少一种miRNA抑制剂和药学上可接受的载体。在一个实施方案中,所述至少一种miRNA模拟物或至少一种miRNA抑制剂是选自下组的miRNA中的一种或多种的模拟物或抑制剂:hsa-miR-542-5p(SEQID NO:1)、hsa-miR-125b(SEQ ID NO:2)、hsa-miR-197(SEQ ID NO:3)、hsa-miR-195(SEQ ID NO:4)、hsa-miR-92a(SEQ ID NO:5)、hsa-miR-139(SEQID NO:6)、hsa-miR-100(SEQ ID NO:7)、hsa-miR-483(SEQ ID NO:8)、hsa-miR-22(SEQ ID NO:9)、hsa-miR-23a(SEQ ID NO:10)、hsa-miR-486(SEQID NO:11)、hsa-miR150(SEQ ID NO:12)、hsa-miR-30c(SEQ ID NO:13)、hsa-miR-342(SEQ ID NO:14)、hsa-miR-133a(SEQ ID NO:15)、hsa-miR-422b(也称为miR-378;SEQ ID NO:16)、hsa-miR-221(SEQ ID NO:17)、hsa-let-7f(SEQ ID NO:18)、hsa-miR-133b(SEQ ID NO:19)、hsa-miR-222(SEQ ID NO:20)、hsa-miR-224(SEQ ID NO:21)、hsa-let-7a(SEQ ID NO:22)、hsa-miR-1(SEQ ID NO:23)、hsa-miR-28(SEQ ID NO:24)、hsa-miR-199a(SEQ ID NO:25)、hsa-miR-181b(SEQ ID NO:26)、hsa-miR-20a(SEQ ID NO:27)、hsa-let-7c(SEQ ID NO:28)、hsa-miR-484(SEQ ID NO:29)、hsa-miR-26b(SEQ ID NO:30)、hsa-let-7d(SEQ ID NO:31)、hsa-miR-10b(SEQ ID NO:32)、hsa-miR-382(SEQ ID NO:33)、hsa-miR-92b(uauugcacucgucccggccucc;SEQ ID NO:48)、hsa-let-7b(ugagguaguagguugugugguu;SEQ ID NO:49)、hsa-let-7e(ugagguaggagguuguauaguu;SEQ ID NO:50)和hsa-let-7g(ugagguaguaguuuguacaguu;SEQ ID NO:51)。所述药物组合物可以包含如上所述通常规定用于治疗心力衰竭的第二治疗药物。在一个实施方案中,所述药物组合物进一步包含肌力药、神经介质阻断剂、醛固酮拮抗剂、利尿剂、血管扩张药、内皮素受体拮抗剂或HDAC抑制剂中的一种或多种。
在另一个实施方案中,所述药物组合物包含至少一种miRNA模拟物,其中所述的miRNA模拟物包含hsa-miR-197(SEQ ID NO:3)、hsa-miR-92a(SEQID NO:5)、hsa-miR-139(SEQ ID NO:6)、hsa-miR-483(SEQ ID NO:8)、hsa-miR-22(SEQ ID NO:9)、hsa-miR-486(SEQ ID NO:11)、hsa-miR-150(SEQID NO:12)、hsa-miR-30c(SEQ ID NO:13)、hsa-miR-133a(SEQ ID NO:15)、hsa-miR-422b(也称为miR-378;SEQ ID NO:16)、hsa-miR-221(SEQ ID NO:17)、hsa-let-7f(SEQ ID NO:18)、hsa-miR-133b(SEQ ID NO:19)、hsa-miR-222(SEQ ID NO:20)、hsa-miR-224(SEQ ID NO:21)、hsa-let-7a(SEQ ID NO:22)、hsa-miR-1(SEQ ID NO:23)、hsa-miR-20a(SEQ ID NO:27)、hsa-let-7c(SEQ IDNO:28)、hsa-miR-484(SEQ ID NO:29)、hsa-let-7d(SEQ ID NO:31)或hsa-miR-10b(SEQ ID NO:32)的序列。而在另一个实施方案中,所述药物组合物包含至少一种miRNA抑制剂,其中所述miRNA抑制剂包含与hsa-miR-125b(SEQ ID NO:2)、hsa-miR-195(SEQ ID NO:4)、hsa-miR-100(SEQ ID NO:7)、hsa-miR-23a(SEQ ID NO:10)、hsa-miR-342(SEQ ID NO:14)、hsa-miR-28(SEQ ID NO:24)、hsa-miR-199a(SEQ ID NO:25)、hsa-miR-181b(SEQ ID NO:26)、hsa-miR-26b(SEQ ID NO:30)或hsa-miR-382(SEQ ID NO:33)互补的序列。
所述药物组合物可以以任何合适的手段施用用于在此所述的任何用途,所述手段,例如口服,如以片剂、胶囊、颗粒或粉末的形式;舌下地;含服;胃肠外地,如通过皮下、静脉内、肌内、或脑池内(intracistemal)注射或灌注技术(如作为无菌可注射水或非-水溶液或悬液);经鼻地,包括向鼻粘膜施用,如用吸入喷雾;局部地,如以乳剂(cream)或软膏(ointment)的形式;或经直肠如以栓剂的形式;以含有无毒的、药学上可接受的载体后稀释剂的剂量单位制剂的形式。如此处所应用的,“药学上可接受的载体”包括用于配制药物,例如适合于施用给人的药物时可接受的溶剂、缓冲液、溶液、分散介质、包衣(coating)、抗细菌剂和抗真菌剂、等渗和吸收延迟剂(isotonic and absorptiondelaying agent)等。
包含miRNA抑制剂或miRNA模拟物的药物组合物也可以通过导管系统或分离冠状循环用于向心脏递送治疗剂的系统来给药。用于给心脏递送治疗剂的不同的导管系统和冠状血管系统在本领域是已知的。美国专利第6,416,510号;美国专利第6,716,196号;美国专利第6,953,466号;WO2005/082440,WO 2006/089340,美国专利公开第2007/0203445号,美国专利公开第2006/0148742号和美国专利公开第2007/0060907号公开了适用于本发明的一些基于导管递送方法或冠状分离法的非限制性实例,将他们全部通过提述并入本文。miRNA模拟物或miRNA抑制剂可以,例如,以适合于立即释放或延长释放的形式施用。立即释放或延长释放可以通过使用包含miRNA模拟物或miRNA抑制剂的合适的药物组合物来实现,或者,特别是在延长释放的情况下,通过使用装置如皮下植入物或渗透泵来实现。
胶体分散体系,如大分子复合物、纳米胶囊(nanocapsule)、微球、珠和包括水包油乳液、微团(micelle)、混合的微团和脂质体的基于脂质的体系可以被用作miRNA模拟物和miRNA抑制物的递送运载体。适用于将本发明的核酸递送至心脏和骨骼肌组织的市售脂肪乳剂包括II、III、Nutrilipid和其它类似脂质乳剂。在体内用作递送运载体的优选的胶体体系是脂质体(即,人工膜囊)。这些体系的制备和用途在本领域是已知的。美国5,981,505;美国6,217,900;6,383,512;美国5,783,565;美国7,202,227;美国6,379,965;美国6,127,170;美国5,837,533;美国6,747,014和WO 03/093449中也公开了示例性的制剂,将它们全部通过题述并入本文。
用于口服施用的示例性的组合物包括悬液,其可以包括,例如,用于赋予(imparting)大体积的微晶纤维素、作为助悬剂的海藻酸或海藻酸钠、作为粘性增强剂的甲基纤维素和增甜剂或调味剂如本领域已知的那些;以及立即释放的片剂,其可以含有,例如,微晶纤维素、磷酸二钙、淀粉、硬脂酸镁和/或乳糖和/或其它赋形剂、粘合剂、增充剂(extender)、崩解剂、稀释剂和润滑剂,如本领域已知的那些。所述miRNA模拟物和miRNA抑制剂也可以经过口腔通过舌下和/或口腔含服施用来递送。模压片剂、压缩片剂或冷冻干燥片剂是可以使用的示例性的形式。示例性的组合物包括将本发明的化合物与快速溶解的稀释剂(如甘露醇、乳糖、蔗糖和/或环糊精)一起配制的那些。高分子量赋形剂如纤维素(微晶粉末纤维素(avicel))或聚乙二醇(PEG)也可以包含在这些制剂中。这些制剂还可以包括以帮助粘膜粘附的赋形剂,如羟丙基纤维素(HPC)、羟丙基甲基纤维素(HPMC)、羧甲基纤维素钠(SCMC)、顺丁烯二酸酐共聚物(如Gantrez);和控制释放的作用剂,如聚丙烯酸共聚物(如Carbopol934)。为了便于配制和使用也可以加入润滑剂、助流剂、香味剂(flavor)、着色剂和稳定剂。
用于鼻的气溶胶或吸入施用的示例性的组合物包括盐水溶液,其含有,例如苯甲醇或其它合适的防腐剂、增强生物利用度的吸收促进剂和/或其它增溶剂或分散剂如本领域已知的那些。
用于胃肠外施用的示例性的组合物包括可注射溶液或悬浮液,其含有,例如,合适的非毒性、胃肠外可接受的稀释剂(parenterally acceptable diluent)或溶剂,如甘露醇、1,3-丁二醇、水、林格氏液(Ringer’s solution)、等渗氯化钠溶液、或其它合适的分散剂或湿润剂和悬浮剂,包括合成的甘油一酯或甘油二酯,和脂肪酸,包括油酸或Cremaphor。
用于直肠施用的示例性的组合物包括栓剂,其包含,例如,合适的非灌注-赋形剂,如可可脂、合成的甘油酯或聚乙二醇,其在常温下是固体,但在直肠腔内液化和/或溶解以释放药物。用于局部施用的示例性的组合物包括局部的载体如Plastibase(用聚乙烯凝胶化的矿物油)。
可以理解的是对任何特定的受试者来说具体的剂量水平和用药频率可以变化,并会取决于包括采用的具体化合物(如特殊的miRNA模拟物或miRNA抑制剂)的活性、代谢的稳定性和化合物的作用的时长、物种、年龄、体重、总体健康状况、受试者的性别和饮食、施用的模式和时间、排泄的频率、药物组合和特定状况的严重度在内的多种因素。
本发明也考虑了调节细胞中胎儿心脏基因程序(FGP)再活化的方法。在一个实施方案中,所述方法包括提供具有一种或多种miRNA抑制剂和miRNA模拟物的心脏细胞,其中一种或多种miRNA抑制剂或miRNA模拟物调整胎儿心脏基因程序的至少一种标志物的表达。术语“心脏细胞”指的是心肌细胞和/或心内皮细胞。根据本发明的某些实施方案,所述术语包括心成纤维细胞和/或心脏中存在的其它细胞类型如平滑肌细胞(如在心血管壁中),心脏神经中的神经元和神经胶质细胞等。胎儿心脏基因程序(FGP)的再活化是与心脏功能受损和不良预后相关的肥大和衰竭心脏的特有特征。(Kuwahara等,2003,EMBO J.22(23):6310-6321)β-肌球蛋白重链(βMyHC)、α-肌球蛋白重链(αMyHC)、心房钠尿因子(atrial natriuretic factor)(ANF)、脑钠肽(brainnatriuretic peptide)(BNP)、SERCA2a和α-骨骼肌肌动蛋白(Sk α-肌动蛋白)是反映FGP的活化和在心力衰竭中表达改变的标志物基因,且可能不利地影响疾病的进展。在一些实施方案中,调节胎儿心脏程序的至少一种标志物的表达,其中所述胎儿心脏基因程序的至少一种标志物选自βMyHC、αMyHC、ANF、BNP、SERCA2a和骨骼肌α-肌动蛋白(skeletal α-actin)中。
用于调节FGP的再活化的合适的miRNA抑制剂和miRNA模拟物包括选自下组的miRNA的一种或火种miRNA抑制剂或miRNA模拟物:hsa-miR-542-5p(SEQ ID NO:1)、hsa-miR-125b(SEQ ID NO:2)、hsa-miR-197(SEQ ID NO:3)、hsa-miR-195(SEQ ID NO:4)、hsa-miR-92a(SEQ ID NO:5)、hsa-miR-139(SEQ ID NO:6)、hsa-miR-100(SEQ ID NO:7)、hsa-miR-483(SEQID NO:8)、hsa-miR-22(SEQ ID NO:9)、hsa-miR-23a(SEQ ID NO:10)、hsa-miR-486(SEQ ID NO:11)、hsa-miR150(SEQ ID NO:12)、hsa-miR-30c(SEQ ID NO:13)、hsa-miR-342(SEQ ID NO:14)、hsa-miR-133a(SEQ ID NO:15)、hsa-miR-422b(也称为miR-378;SEQ ID NO:16)、hsa-miR-221(SEQ IDNO:17)、hsa-let-7f(SEQ ID NO:18)、hsa-miR-133b(SEQ ID NO:19)、hsa-miR-222(SEQ ID NO:20)、hsa-miR-224(SEQ ID NO:21)、hsa-let-7a(SEQID NO:22)、hsa-miR-1(SEQ ID NO:23)、hsa-miR-28(SEQ ID NO:24)、hsa-miR-199a(SEQ ID NO:25)、hsa-miR-181b(SEQ ID NO:26)、hsa-miR-20a(SEQ ID NO:27)、hsa-let-7c(SEQ ID NO:28)、hsa-miR-484(SEQ ID NO:29)、hsa-miR-26b(SEQ ID NO:30)、hsa-let-7d(SEQ ID NO:31)、hsa-miR-10b(SEQID NO:32)、hsa-miR-382(SEQ ID NO:33)、hsa-miR-92b(uauugcacucgucccggccucc;SEQ ID NO:48)、hsa-let-7b(ugagguaguagguugugugguu;SEQ ID NO:49)、hsa-let-7e(ugagguaggagguuguauaguu;SEQ ID NO:50)和hsa-let-7g(ugagguaguaguuuguacaguu;SEQ ID NO:51)。在一个实施方案中,所述一种或多种miRNA抑制剂或miRNA模拟物是hsa-miR-92a(SEQ ID NO:5)、hsa-miR-100(SEQ ID NO:7)或hsa-miR-133b(SEQ ID NO:19)的模拟物或抑制剂。
本发明还考虑监测对受试者心力衰竭进行的治疗的方法。在一个实施方案中,所述方法包括在第一时间点从受试者获得第一生物样品,其中所述第一时间点是在心力衰竭的药物治疗方案开始之前;在第二时间点从受试者中获得第二生物样品,其中所述第二时间点是在心力衰竭的药物治疗方案开始之后;用miRNA阵列分析、mRNA阵列分析和定向蛋白质组学表达分析处理第一生物样品和第二生物样品;并且比较来自第一生物样品的miRNA、mRNA和蛋白质表达数据和来自第二生物学样品的表达数据以确定基因表达模式的改变,其中基因表达的改变指示所述药物治疗方案的效力。所述第一时间点和第二时间点可以间隔大约3至大约18个月、间隔大约3至大约12个月,或优选间隔大约3至大约9个月。额外的生物样品可以在药物治疗方案开始之后在多个时间点从各患者获得。在一些实施方案中,在药物治疗方案开始之后收集两个或更多的样品。在其它的实施方案中,在药物治疗方案开始之后收集三个或更多的样品。这些样品可以大约每3个月收集一次、大约每6个月收集一次、大约每9个月收集一次或大约每12个月收集一次。
用于治疗受试者的药物治疗方案可以是治疗心力衰竭的任何标准药物治疗方案,如ACE抑制剂(如卡托普利、赖诺普利等)、β-阻断剂(如美托洛尔、卡维地洛)、醛固酮拮抗剂、利尿剂(如呋塞米、螺内酯(spirolactone)、依普利酮)和地高辛。药物治疗方案也可以是包括施用在本文中所披露的一种或多种miRNA模拟物或miRNA抑制剂的任何治疗方法。药物治疗的剂量或形式可以根据在治疗后一个或多个时间点获得的表达谱数据来调整。本发明方法允许为治疗特定的患者中的心力衰竭而进行药物治疗方案的调整。
通过如下的附加实施例进一步示例本发明,而不应解释成对本发明的限制。本申请通篇引用的所有参考文献、专利和公开的专利申请的内容,以及附图,全部通过题述并入本文。
实施例
实施例1.miRNA在衰竭和非衰竭的人心脏中差异性地表达
使用基于阵列的技术,比较来自非衰竭(NF)和衰竭的人心脏的人心脏组织中心源miRNAs(cardiac derived miRNA)的表达水平。衰竭心脏包括由特发性扩张性心肌病(IDC)和缺血性心肌病(ISC)导致的那些。470个微小RNA的生物信息学分析(Sanger miRBase Release 9.0)被用于鉴定在非衰竭相对于衰竭心脏中差异表达的多种miRNAs。揭示了一种独特的miRNA表达“指纹”,其中特定miRNAs在表达上增加或者降低。这些miRNAs代表心力衰竭的生物标志物,也是心力衰竭治疗的新的治疗靶。
如图1A和图2所示,比较NF与IDC心脏之间的miRNA表达,注意到22种miRNAs的表达发生改变,其中6种与NF心脏相比在IDC心脏中表达增加(hsa-miR-125b、hsa-miR-195、hsa-miR-100、hsa-miR-181b、hsa-miR-23a和hsa-miR-382),且其中16种与NF心脏相比在IDC心脏中表达降低(hsa-miR-133a、hsa-miR-133b、hsa-miR-221、hsa-miR-197、hsa-miR-30c、hsa-miR-92、hsa-miR-22、hsa-miR-486、hsa-miR-542-5p、hsa-miR-139、hsa-miR-150、hsa-miR-10b、hsa-miR-483、hsa-miR-594、hsa-miR-422b(也称为miR-378)和hsa-miR-20a)。基于Sanger miRBase 9.2版,目标成熟miRNAs的序列示于表1中(下文)。
如图1B和图2所示,比较NF与ISC心脏之间的miRNA表达,发现22种miRNAs表达发生改变,与NF心脏相比,其中6种在ISC心脏中表达增加(hsa-miR-195、hsa-miR-100、hsa-miR-342、hsa-miR-28、hsa-miR-199a和hsa-miR-26b),以及16种在ISC中表达下降(hsa-miR-92、hsa-miR-221、hsa-miR-486、hsa-miR-133a、hsa-miR-150、hsa-miR-422b(也称为miR-378)、let-7f、let-7a、hsa-miR-1、hsa-miR-222、let-7c、hsa-miR-133b、hsa-miR-224、hsa-miR-484、hsa-miR-594和let-7d)。基于Sanger miRBase ver.9.2,选择的成熟的miRNAs序列示于表1中(下文)。
**hsa-miR-594是miRBase中注释的tRNA基因的片段。
在Sanger miRBase数据库中hsa-miR-422b已被重命名为miR-378(版本11.0)
在Sanger miRBase数据库中has-miR-92已被重命名为miR-92a(版本11.0)
图3显示NF与IDC以及NF与ISC之间人心脏miR表达数据的比较,p<0.05(不成对t-检验)。3种miRNAs的表达在IDC中比NF中有显著增加(hsa-miR-125b、hsa-miR-195和hsa-miR-100),而6种miRNAs的表达在IDC中比NF心脏中显著降低(hsa-miR-22、hsa-miR-92、hsa-miR-221、hsa-miR-486、hsa-miR-30c和hsa-miR-133a)。5种miRNAs的表达在ISC中比NF心脏中显著增加(hsa-miR-195、hsa-miR-100、hsa-miR-342、hsa-miR-28和hsa-miR-199a),而10种miRNAs在ISC心脏中比NF中的表达显著增加(hsa-miR-92、hsa-miR-221、hsa-miR-486、hsa-miR-133a、hsa-miR-150、hsa-miR-422b(也称为miR-378)、let-7f、let-7a、hsa-miR-1和hsa-miR-222)。
比较NF与IDC心脏以及NF与ISC心脏的miRNA表达水平的综合分析结果表明几种miRNA的一致性调节(concordant regulation),和以唯一的、心力衰竭亚型特异性的方式调节的miRNA(图2)。miRNAs hsa-miR-92、hsa-miR-221、hsa-miR-486、hsa-miR-150和hsa-miR-422b在IDC和ISC心脏中都显著下调,而hsa-miR-195和hsa-miR-100在IDC和ISC心脏中都是上调的。后一结果与Rooij等人,2006的研究是一致的,Rooij等人先前证明了在衰竭的(IDC)人心脏中以及用机械-或遗传诱导病理性肥大的转基因小鼠中的miR-195都是上调的。
从完整的数据集中得到来自Sanger人miRBase 9.0数据集的每一个可检测的miRNAs的相对表达的详细分析。所有的心脏具有介于87到238之间(在有可能的454中)的可定量检测的miR转录物(没有显示数据)。
具体的方法
微小RNA提取:从来自6例非衰竭(NF)、5例特发性扩张性心肌病(IDC)和5例缺血性心肌病(ISC)患者的人心脏的左心室提取了miRNA。所有患者都是50-65岁之间的男性。在心脏移植的时候,患者接受包括ACE抑制剂、利尿剂、β-阻断剂、洋地黄、抗凝剂、胰岛素、β-激动剂和硝酸盐的多种药物制剂。通过超声波心动描记法评估在心脏移植时各患者的心脏表现。每个组的射血分数百分比如下:NF:64±4.3%;IDC:12±3.1%;以及ISC:18.6±3.5(平均值±SEM)。提取使用mirVanaTM试剂盒(Ambion)根据厂商的建议进行。
阵列分析:样品提交给LC Sciences,LLC,休斯顿,德克萨斯进行微小RNA的检测微阵列服务。用生物信息学分析鉴定470个微小RNA的相对表达(Sanger miRBase Release 9.0)。这种分析鉴定了多种在非衰竭(NF)与衰竭心脏(ISC和/或IDC)中差异性表达的miRNA。检验了两种衰竭心脏类型,其源自那些患有特发性扩张性心肌病(IDC)的患者或源自患有缺血性心肌病(ISC)的那些患者。在NF组中,检验了6例心脏(n=6)。比较NF与IDC心脏(n=5),发现12种miRNA在统计学上显著变化(p<0.05,不成对t-检验),其中3种增加及9种降低,如图1A所示。在NF与ISC心脏(n=5)之间的类似分析揭示了16种miRNAs的表达的变化,其中5种增加,及11种下降,如图1B所示。在图2中显示了在微阵列上的平均miR表达,其为在人心脏中,所有与IDC或者ISC比较在NF中的miR表达的显著变化。
miRNA RT-PCR:使用TaqMan微小RNA逆转录试剂盒(ABI)根据厂家的建议进行miRNA的逆转录。简言之,将5ng的miRNA与dNTP、MultiScribe逆转录酶和对靶miRNA特异性的引物混合。将得到的cDNA稀释15倍,并用于PCR反应中。根据厂家(Applied Biosystems)的建议进行PCR。简言之,将cDNA与对靶miRNA特异性的TaqMan测定组合,并使用ABI7300进行PCR反应。
图4显示RT-PCR确认(validation)的6例非衰竭(NF)、5例特发性心肌病(IDC)和5例缺血性心肌病(ISC)患者的人心脏的miRNA表达。由于这种微小RNA的表达水平没有变化,所以将数据标准化为miRNA-24。当与NF心脏比较时,IDC和ISC衰竭心脏的miR-92和miR-133b的表达显著降低,而当与NF心脏比较时,miR-100和miR-195的表达显著增加。
由于miR-133a和miR-133b的序列高度相似,进行其中将miR-133a或者miR-133b抑制剂或模拟物过表达的实验。通过RT-PCR得到的数据证实了检测对于每种miR是特异性的。因此,miR-133a的过表达表明miR-133a的表达增加,而miR-133b的丰度却没有变化。转化实验的结果得到相同程度的特异性。
实施例2.通过调节特异性的miRNA可以调节胎儿心脏基因程序
胎儿心脏基因程序(FGP)的再活化为与受损的心脏功能和不良预后相关的肥大和心力衰竭的典型特征。(Kuwahara等,2003,EMBO J.22(23):6310-6321)心脏α-肌球蛋白重链(αMyHC)、β-肌球蛋白重链(βMyHC)、心房钠尿因子(ANF)、脑钠肽(BNP)、骨骼肌α-肌动蛋白(Sk-肌动蛋白)和钙ATP酶属于其表达指示FGP活化并在心力衰竭中有变化的基因,且可能不利地影响疾病的进展。例如,β-肾上腺素能信号传导在扩张性心肌病的天然历史中起到重要的作用。在心脏应激(cardiac stress)期间β-肾上腺素能受体(β1-AR和β2-AR)的慢性活化通过包括基因表达的改变的机制最终损害衰竭的心脏。Sucharov等人之前在2006年报道了新生大鼠心室肌细胞中用异丙肾上腺素(β-肾上腺素能受体激动剂)刺激β-AR引起人心脏胎儿和/或成人基因启动子的相对活性的“胎儿”应答,其包括人和大鼠α-肌球蛋白重链(α-MyHC)启动子的阻抑,和同时的人心房钠尿肽(ANP)和大鼠βMyHC启动子的活化(Sucharov等,2006,A beta1-adrenergic receptor CaM kinase II-dependent pathway mediatescardiac myocyte fetal gene induction Am J Physiol Heart Circ Physiol 291:H1299-H1308)。其之前也显示了启动子的变化与内源基因表达的变化相关联,所述内源基因表达是通过mRNA表达测量的。
在本实施例中描述的实验的目的是在不存在和存在β-肾上腺素能受体刺激时测定特定的miRNA的表达或抑制对胎儿基因程序的活化的影响。
使用Amaxa技术(Amaxa AG,Cologne,德国)将miRIDIANTM微小RNA模拟物和抑制剂(Dharmacon)转染至新生大鼠心室肌细胞(NRVM)。使用的Dharmacon模拟物和抑制剂的产品编号如下:hsa-miR-92模拟物:C-00030-02;基于miR-92的Sanger序列的双链miRNA;hsa-miR-92抑制剂:I-300030-02,基于miR-92的基于Sanger的序列的单链miRNA:uauugcacuugucccggccugu(SEQ ID NO:5)。hsa-miR-100模拟物:C-300036-01;基于miR100的Sanger序列的双链miRNA;hsa-miR-100抑制剂;I-300036-01;基于miR-100的基于Sanger的序列的单链miRNA:aacccguagauccgaacuugug(SEQ ID NO:7)。hsa-miR-133b模拟物:C-300199-01;基于miR133b的Sanger序列的双链miRNA;hsa-miR-133b抑制剂:I-300199-01;基于miR133b的基于Sanger的序列的单链miRNA:uugguccccuucaaccagcua(SEQ ID NO:19)。
将2.4x106NRVM悬浮在用于心肌细胞的合适的Amaxa溶液中,并与20微摩尔的模拟物或抑制剂混合。使用心肌细胞程序对细胞进行电穿孔,其导致短RNA的95%的转染效力。将含有血清的培养基加入细胞。24小时后,用无血清培养基置换所述培养基。在转染72小时后收获细胞。在转染后48小时用β-肾上腺素能激动剂异丙肾上腺素(10-7M)处理细胞亚组。
使用TRIZol(Invitrogen)从收获的细胞提取mRNA。基本按照厂家的说明使用iScript(Bio-Rad)制备含有多聚腺苷酸的mRNA的cDNA。通常,在RT-PCR反应中使用0.1ng的cDNA、12.5nM的各种引物和Power Syber GreenPCR Master Mix(ABI)。使用ABI7300系统进行反应。使用的引物示于表2中(见下面)。根据如在实施例1中所述的ABI合成miRNA cDNA,并进行RT-PCR反应。
如上所述,用具有受体激活剂异丙肾上腺素(ISO)的β-肾上腺素能受体进行的刺激活化胎儿基因程序(FGP)。FGP的ISO介导的诱导是通过对成年基因、α-肌球蛋白重链(α-MyHC)和SERCA的阻抑,以及胎儿基因、b-型钠尿肽(BNP)、心房利纳因子(ANF)、骨骼肌α-肌动蛋白和β-肌球蛋白重链(β-MyHC)的上调表明的。图5显示在不存在和存在异丙肾上腺素时用miR-92模拟物或miR-92抑制剂转染的新生儿心肌细胞中的胎儿基因程序(FGP)表达。模拟物和抑制剂乱序的miR(Dharmacon)被用作对照(分别为Con-M和Con-I)。用模拟物或抑制剂对照转染的ISO-处理的NRVM显示了αMyHC和SERCA降低的表达和ANF、BNP、α-骨骼肌肌动蛋白和βMyHC增加的表达,其为FGP的活化的特征。
由于在心力衰竭中,miR-92是下调的,我们假定对miR-92的抑制(即,用miR-92抑制剂治疗)会导致诱导对胎儿基因程序的诱导,并假设miR-92的上调(即,用miR-92模拟物治疗)会防止对胎儿基因程序的诱导。如与对照组相比,观察到用miR-92模拟物转染的细胞中ANF、BNP和α-骨骼肌肌动蛋白的表达的轻微增加,以及Serca mRNA的降低(图5)。然而,一般而言,对miR-92的抑制或上调对胎儿或成人基因表达调节具有最低限度的影响。这些结果表明在心力衰竭中miR-92的下调可能涉及调节心力衰竭表型的其它途径。
图6显示在存在和不存在异丙肾上腺素时用miR-100模拟物或miR-100抑制剂转染的新生儿心肌细胞中的胎儿基因程序(FGP)表达。模拟物和抑制剂乱序miR被用作对照(分别为Con-M和Con-I,Dharmacon)。用miR-100模拟物转染心室肌细胞导致对成人基因αMyHC和SERCA的阻抑,以及增强的ISO-介导的对胎儿基因ANF和βMyHC的上调。令人感兴趣的是,对miR-100的下调防止了对αMyHC和SERCA的ISO-介导的阻抑,但是没有防止对胎儿亚型的ISO-诱导作用(图6),表明miR-100的抑制作用特异性地调节涉及成人亚型的ISO-介导的阻抑的基因的表达。
图7显示了在存在和不存在异丙肾上腺素时,用miR-133b模拟物或miR-133b抑制剂转染的新生儿心肌细胞中的胎儿基因程序(FGP)表达。模拟物和抑制剂乱序miR(Dharmacon)被用作对照(分别为Con-M和Con-I)。对miR-133b的阻抑导致与对照(Con-I)相比小但是普遍的对所分析基因的上调。然而,用miR-133b模拟物转染的miR-133b的上调防止了对αMyHC和SERCA的ISO-介导的下调,ISO-介导的对MyHC的上调,以及减弱的ISO-介导的对BNP的上调(图7)。当与对照(Con-M)比较时,miR-133b的过表达也导致对骨骼肌α-肌动蛋白和βMyHC的阻抑,而BNP和ANF的表达没有变化。这些结果表明miR-133b表达的变化在调节胎儿基因程序中具有重要的作用,其类似于在Care等(Nat Med(2007),Vol.13(5):613-618)中所描述的那样,Care等人证明了对miR-133的上调防止或降低了α-肾上腺素能对肥大基因程序的诱导。这些发现表明miR-133家族可为心脏病中基因表达的全局调节子(globalregulator)。
为了确认各miRNA模拟物或抑制剂分别对靶miRNA产生了上调或下调,如上所述将miR-92、miR-100和miR-133b的模拟物和抑制剂转染入NRVM。通过RT-PCR测试各miRNA的表达水平。示于图8的结果显示miRNA模拟物导致靶miRNA的过表达,而miRNA抑制剂阻抑特异性miRNA的表达,并且miR这种改变的表达水平保持72小时。
实施例3.miR-133b和细胞肥大
由于对miR-133b的抑制诱导胎儿亚型BNP、骨骼肌α-肌动蛋白和ANF的表达,并且miR-133b的过表达阻断ISO-介导的对胎儿基因程序的活化(实施例2),所以测试了miR-133b的过表达或抑制对细胞肥大的影响。如在实施例2中所述,用模拟物和抑制剂转染NRVM,并用抗-α-辅肌动蛋白(anti-actinin)抗体染色。在转染后用异丙肾上腺素处理一些细胞。根据Harrison等(Molecular & Cellular Biology(2004)Vol.24(24):10636-10649)实施免疫荧光法。简而言之,用TBST洗涤细胞,并用10%甲醛固定20分钟。再次用TBST洗涤细胞,并用0.1%Triton-X温育额外的30分钟。然后用TBST中的1%BSA阻断细胞1小时,接着与抗-辅肌动蛋白抗体的1∶500的稀释一起温育1小时。接着,与Alexa 594的抗-小鼠抗体的1∶1000稀释和2g/ml的Hoechest染色剂一起温育1小时。用装配有数码相机(Zeiss AxioCam)和Zeiss AxioVision3.0.6.36版成像软件的荧光显微镜(Nikon E800)在40倍放大率下进行拍照。使用Image J软件程序(NIH)定量各种状况的3个不同区域的30个细胞的细胞表面积。
如图9和10所示,miR-133b的下调导致细胞尺寸的增加,而miR-133b的过表达显著地降低ISO-介导的心肌细胞细胞尺寸的增加。这些结果进一步支持了这样的观点:miR-133b可为心肌细胞肥大的全局调节子。
实施例4.发现基于miR的靶的SAGE方法
图11中的示意图描述了用于鉴定心力衰竭的治疗性靶的基因表达系列分析(SAGE)方法。与心力衰竭的临床试验相关的患有心力衰竭(HF)的患者,在三个不同的时间点进行任择的心内膜心肌活检。第一时间点为β-阻断剂(BB)治疗开始之前,其它时间点为治疗开始之后的3个月和12个月。对于每位患者,用miRNA阵列分析、mRNA(Afflymetrix)阵列分析和(定向)蛋白质组学表达分析处理各个时间点的活检。来自相同样品的miR/mRNA/蛋白质表达的唯一组合同时表示在多种水平检验基因表达模式中的变化的唯一时机。因为miRNA表达影响mRNA表达,其接着又影响蛋白质表达,由此(here-to-for),可以检测到调节性对照的控制点之间的不明确的关联(linkage)。此外,来自相同患者的不同时间的样品分析消除了与个人与个人之间的遗传变异相关的显著问题。通过比较来自相同患者的不同时间点(例如治疗后3个月对治疗前等)的基因表达水平,可以鉴定治疗性靶。基因表达水平也可以与临床结果相关联。
实施例5.用β-阻断剂治疗处理的人患者中miRNA表达的改变
为了进一步说明心力衰竭中miRNA的重要性,在用β-阻断剂治疗之前或之后评估人患者中miRNA的表达。在心力衰竭末期(治疗之前),用β-阻断剂治疗处理后3个月和处理后12个月从5位患者获得了系列心脏活检。对每一个患者的每一个样品进行miRNA阵列分析以确定几种在衰竭的心脏中是下调的miRNAs的表达水平,包括miR-1、miR-19b、miR-133b、miR-133a、miR-30d、miR-92a、miR-208b、miR-499和miR-let-7g的表达水平。通过如实施例1所述的RT-PCR确认在miRNA阵列分析中的表达水平。RT-PCR的结果示于图12-16中。将特定miRNA的表达水平标准化为用作对照的miR-370(右侧的组)或小RNA RNU66(左侧的组)。在心力衰竭末期(A)、治疗后3个月(B)和治疗后12个月(C)评估miRNA的表达。在4个对β-阻断剂治疗有响应的患者(20、21、25和34)的治疗后样品中miRNA的表达是上调的。在获得自对治疗无应答的患者(患者103)的样品中特定的miRNAs没有上调。结果显示miRNAs的亚组的表达水平与心力衰竭的严重性有关,并且可以被用作生物标志物以如实施例4中所述的监测患者对具体药物治疗的应答。可以根据这些miRNAs中一种或多种的表达水平调整药物治疗的剂量或类型。
实施例6.miR-92和miR-133的靶是蛋白激酶Cε、磷酸二酯酶1A和钙调蛋白
Target Scan 4.2(可在Target Scan(靶扫描)网页得到)对蛋白激酶Cε(PKCε)和磷酸二酯酶1A(PDE1A)mRNAs进行的3’UTR分析显示miR-92a(以前称为miR-92)的可能的靶位点。为了测试miR-92a是否靶向这两种mRNAs,将新生大鼠心肌细胞用miR-92a模拟物转染,导致miR-92a过表达,或用miR-92a抑制剂转染,导致miR-92a的下调。转染后72小时收获细胞,并用Western印迹分析细胞溶解物。通过miR-92a抑制剂对miR-92a的下调导致PKCε(图17A)和PDE1A(图17B)蛋白水平的上调。通过miR-92a模拟物进行的对miR-92a的上调导致了对PKCε(图17A)和PDE1A(图17B)表达的阻抑。
通过靶扫描对钙调蛋白mRNA的3’UTR进行了类似的分析。该分析显示了miR-133b可能的靶位点。为了测试miR-133b是否靶向钙调蛋白,将新生大鼠心肌细胞用miR-133b模拟物(导致miR-133b过表达)或miR-133b抑制剂(导致miR-133b下调)转染。转染后72小时收获细胞,并用RT-PCR分析钙调蛋白mRNA表达(图18A)。用Western印迹分析了钙调蛋白的蛋白质表达(图18C)。通过miR-133b抑制剂进行的对miR-133b的下调导致了钙调蛋白mRNA和蛋白质水平的上调,而通过miR-133b模拟物进行的对miR-133b的上调对钙调蛋白表达的影响很小。
为了测定miR-133b是否对钙调蛋白3’UTR有直接的影响,将新生大鼠心脏细胞用miR-133b模拟物或miR-133b抑制剂和包含与萤光素酶在3’UTR连接的钙调蛋白的构建体共转染(图18B)。萤光素酶测定的结果证实了mRNA和蛋白表达分析的发现。用miR-133b模拟物的转染在萤光素酶水平方面没有产生显著的改变。然而,miR-133b抑制剂的转染导致了萤光素酶的上调,证明钙调蛋白是miR-133b的直接的靶。
综上,这些结果显示PKCε和PDE1A是miR-92a的靶,而钙调蛋白是miR-133b的靶。所有这三个靶都与导致心力衰竭的心肌病的发展有关。
应该理解到披露的本发明不仅仅限于描述的特定的方法学、方案和物质,因为这些均可变化。还应该理解这里所用的术语仅仅是为了描述特定的实施方案的目的,而不是试图限制本发明的范围,本发明的范围仅受限于所附的权利要求。
本领域的技术人员将认识到,或者能够确认使用不超过常规实验,在本文中所述的本发明的具体的实施方案的许多等价物。这种等价物意欲包含在所附的权利要求中。
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Claims (4)
1.结合或检测一种或多种靶miRNA的作用剂在制备用于在受试者中诊断或预后缺血性心肌病的诊断剂中的用途,其中所述作用剂是与一种或多种靶miRNA互补的寡核苷酸,且其中所述诊断或预后包括:
a.测定来自所述受试者的生物样品中一种或多种靶miRNA的表达水平,其中所述一种或多种靶miRNA选自:hsa-miR-195(SEQ ID NO:4),hsa-miR-100(SEQ ID NO:7),hsa-miR-342(SEQ ID NO:14),hsa-miR-28(SEQID NO:24),hsa-miR-199a(SEQ ID NO:25),hsa-miR-92a(SEQ ID NO:5),hsa-miR-221(SEQ ID NO:17),hsa-miR-486(SEQ ID NO:11),hsa-miR-133a(SEQ ID NO:15),hsa-miR-150(SEQ ID NO:12),hsa-miR-422b(SEQ ID NO:16),hsa-let-7f(SEQ ID NO:18),hsa-let-7a(SEQ ID NO:22),hsa-miR-1(SEQID NO:23),和hsa-miR-222(SEQ ID NO:20);和
b.比较在上述生物样品中一种或多种靶miRNA的表达水平与在所述标准样品中一种或多种靶miRNA的水平;
其中上述生物样品中至少一种或多种靶miRNA的表达水平与在所述标准样品中一种或多种靶miRNA的水平相比的差异对缺血性心肌病是诊断性或预测性的。
2.权利要求1的用途,其中所述一种或多种靶miRNA进一步包括hsa-miR-26b(SEQ ID NO:30),hsa-let-7c(SEQ ID NO:28),hsa-miR-133b(SEQID NO:19),hsa-miR-224(SEQ ID NO:21),hsa-miR-484(SEQ ID NO:29),hsa-let-7d(SEQ ID NO:31),hsa-miR-92b(SEQ ID NO:48),hsa-let-7b(SEQ IDNO:49),hsa-let-7e(SEQ ID NO:50),或hsa-let-7g(SEQ ID NO:51)。
3.权利要求1或2的用途,其中选自hsa-miR-195(SEQ ID NO:4),hsa-miR-100(SEQ ID NO:7),hsa-miR-342(SEQ ID NO:14),hsa-miR-28(SEQID NO:24),hsa-miR-199a(SEQ ID NO:25),和hsa-miR-26b(SEQ ID NO:30)的一种或多种靶miRNA的增加对缺血性心肌病是诊断性或预测性的。
4.权利要求1或2的用途,其中选自hsa-miR-92a(SEQ ID NO:5),hsa-miR-221(SEQ ID NO:17),hsa-miR-486(SEQ ID NO:11),hsa-miR-133a(SEQ ID NO:15),hsa-miR-150(SEQ ID NO:12),hsa-miR-422b(SEQ ID NO:16),hsa-let-7f(SEQ ID NO:18),hsa-let-7a(SEQ ID NO:22),hsa-miR-1(SEQID NO:23),hsa-miR-222(SEQ ID NO:20),hsa-let-7c(SEQ ID NO:28),hsa-miR-133b(SEQ ID NO:19),hsa-miR-224(SEQ ID NO:21),hsa-miR-484(SEQ ID NO:29),和hsa-let-7d(SEQ ID NO:31)的一种或多种靶miRNA的减少对缺血性心肌病是诊断性或预测性的。
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HK1143612A1 (en) | 2011-01-07 |
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KR20100049079A (ko) | 2010-05-11 |
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AU2008275877B2 (en) | 2015-01-22 |
US20100267804A1 (en) | 2010-10-21 |
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