CN111471679A - 干预eif4h剪接亚型的寡聚核苷酸及其应用 - Google Patents

干预eif4h剪接亚型的寡聚核苷酸及其应用 Download PDF

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CN111471679A
CN111471679A CN201811569563.7A CN201811569563A CN111471679A CN 111471679 A CN111471679 A CN 111471679A CN 201811569563 A CN201811569563 A CN 201811569563A CN 111471679 A CN111471679 A CN 111471679A
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王勇波
王泽峰
包煜芳
沈先锋
肖曼
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Abstract

本发明属于生物技术领域，涉及干预EIF4H特定剪接亚型的干扰小RNA及(siRNA)反义寡聚核苷酸(ASO)序列成分以及检测EIF4H不同剪接亚型RNA及蛋白表达的方法。本发明提供的干预EIF4H剪接亚型表达的寡聚核苷酸序列为SEQ ID NO 6‑152，或与SEQ ID NO 6‑152中任意一项的序列重复超过5个核苷酸的干扰小RNA或者反义寡聚核苷酸，或者是其各种类似物、化学修饰形式或带有连接物的衍生核苷酸序列。本发明还提供了使用干预EIF4H剪接亚型表达的siRNA或者ASO在制备肿瘤治疗药物，及其在制备肿瘤诊断、治疗制品中的应用。

Description

干预EIF4H剪接亚型的寡聚核苷酸及其应用
技术领域
本发明属于生物技术领域，提供了干预EIF4H特定剪接亚型的干扰小RNA及反义寡聚核苷酸序列成分，检测EIF4H不同剪接亚型RNA及蛋白表达的方法，以及通过干预EIF4H特定剪接亚型从而治疗恶性肿瘤的潜在寡聚核苷酸药物。
背景技术
恶性肿瘤是危害人类健康的主要因素之一，是疾病导致人类死亡的主要原因(Siegel et al.,2017)。肺癌是世界范围内癌症致死的首要原因，其中约85％的肺癌为非小细胞肺癌(non-small cell lung cancer,NSCLC)，主要包括肺腺癌(lungadenocarcinoma,LUAD)与肺鳞癌(lung squamous cell carcinoma,LUSC)，前者在肺癌中的占比超过40％(Chen et al.,2014；Herbst et al.,2018)。随着诊断、治疗技术的不断进步，恶性肿瘤的发生率和死亡率都得到了一定程度的缓解(Siegel et al.,2017)。肿瘤靶向治疗、免疫治疗等新技术、新方法的临床应用显著提升了恶性肿瘤的治疗效果。然而，一部分恶性肿瘤病人缺乏临床上可靶向的癌基因，且大量患者对靶向治疗、免疫治疗等治疗手段的反应不完全或容易产生耐受复发(Rotow and Bivona,2017；Siegel et al.,2017)。因此，提升恶性肿瘤的治疗效果迫切需要开发新的分子标记物、治疗靶点及干预方法。
RNA剪接(RNA splicing)，即将RNA前体(pre-RNA)中内含子(intron)移除并将外显子(exon)连接的过程，是高等真核生物基因表达的关键一环。RNA剪接是一个多步骤、高度动态的过程，由剪接复合体(spliceosome)催化完成(Shi,2017)。可变剪接(alternativesplicing,AS)是多外显子基因在特定条件下通过剪接位点的选择与配对，形成不同的RNA剪接亚型(splice variant)的过程。可变剪接普遍存在于高等真核生物中，极大扩展了其转录组与蛋白组功能的多样性，人类超过95％的多外显子基因存在可变剪接现象(Roy etal.,2013)。剪接调控在多种生理条件下发挥着重要的作用，如不同细胞类型、不同发育阶段、对外部刺激的应激反应等(Kalsotra and Cooper,2011)；相应的，剪接异常是导致人类遗传疾病的主要原因之一(Scotti and Swanson,2016)，在多种肿瘤中起关键作用(Zhangand Manley,2013)。
大量研究表明，RNA剪接异常通过影响肿瘤细胞各个标志性生物学特征相关的基因，包括细胞增殖、凋亡、迁移与侵袭、能量代谢、新血管生成等，从而在肿瘤发生、发展及药物耐受过程中发挥驱动作用，被认为是肿瘤细胞的一个新的标志性分子特征(Oltean andBates,2014；Siegfried and Karni,2018；Zhang and Manley,2013)。导致肿瘤相关剪接异常的原因主要包括：1)、RNA前体上顺式序列元件突变，从而引起癌基因的剪接异常；2)、反式调控因子的突变或表达异常，从而导致受其调控的靶标基因的剪接异常。肿瘤相关的剪接异常不仅具有十分重要的病理学功能，而且是潜在的肿瘤诊断、预后评价的新分子标记物以及治疗新靶标(Agrawal et al.,2018；Lee and Abdel-Wahab,2016；Yoshimi andAbdel-Wahab,2017)。
剪接异常的干预策略主要包括：1)、针对特定基因剪接异常的反义寡聚核苷酸(antisense oligonucleotide,ASO)或干扰小RNA(small interfering RNA,siRNA)等基于RNA的治疗策略；2)、针对剪接调控因子或特定基因剪接异常的小分子化合物。基于RNA的治疗策略理论上可以特异性靶向任何目标基因，特别是小分子化合物难以靶向的癌基因或其特定的剪接亚型，具有巨大的应用价值。值得注意的是，近年来干预RNA的策略已在疾病治疗中取得突破性进展。例如，干预剪接异常的ASO药物已被美国FDA批准用于治疗遗传疾病：脊髓性肌萎缩症(spinal muscle atrophy,SMA)、杜氏肌营养不良(Duchenne musculardystrophy,DMD)；首个siRNA药物被美国FDA批准用于治疗遗传性甲状腺素运载蛋白介导的淀粉样病变(hereditary transthyretin-mediated amyloidosis,hATTR)。此外，基于ASO及siRNA的基因或其剪接干预在癌症治疗研究中已取得了非常好的结果(Agrawal et al.,2018；Chakraborty et al.,2017)。
EIF4H(eukaryotic translation initiation factor 4H)编码一个蛋白翻译起始调节因子eIF4H，与另一个蛋白翻译起始调节因子eIF4B同源(Marintchev et al.,2009)。EIF4H有两个主要的剪接亚型，编码的蛋白有20个氨基酸的差异。研究表明，eIF4H在特定癌症中具有促进癌症发生发展的功能(Vaysse et al.,2015)，然而其两个剪接亚型在癌症中表达及功能是否有差异未知，且相应的干预手段缺乏。
与本发明相关的参考文献有：
Agrawal,A.A.,Yu,L.,Smith,P.G.,and Buonamici,S.(2018).Targetingsplicing abnormalities in cancer.Current Opinion in Genetics&Development 48,67-74.
Chakraborty,C.,Sharma,A.R.,Sharma,G.,Doss,C.G.P.,and Lee,S.-S.(2017).Therapeutic miRNA and siRNA:Moving from Bench to Clinic as Next GenerationMedicine.Molecular Therapy-Nucleic Acids 8,132-143.
Chen,Z.,Fillmore,C.M.,Hammerman,P.S.,Kim,C.F.,and Wong,K.-K.(2014).Non-small-cell lung cancers:a heterogeneous set of diseases.Nat Rev Cancer14,535-546.
Ding,Y.,Chan,C.Y.,and Lawrence,C.E.(2004).Sfold web server forstatistical folding and rational design of nucleic acids.Nucleic AcidsResearch 32,W135-W141.
Elbashir,S.M.,Harborth,J.,Lendeckel,W.,Yalcin,A.,Weber,K.,and Tuschl,T.(2001).Duplexes of 21-nucleotide RNAs mediate RNA interference in culturedmammalian cells.Nature 411,494.
Herbst,R.S.,Morgensztern,D.,and Boshoff,C.(2018).The biology andmanagement of non-small cell lung cancer.Nature 553,446.
Kalsotra,A.,and Cooper,T.A.(2011).Functional consequences ofdevelopmentally regulated alternative splicing.Nat Rev Genet 12,715-729.
Lee,S.C.-W.,and Abdel-Wahab,O.(2016).Therapeutic targeting ofsplicing in cancer.Nat Med 22,976-986.
Marintchev,A.,Edmonds,K.A.,Marintcheva,B.,Hendrickson,E.,Oberer,M.,Suzuki,C.,Herdy,B.,Sonenberg,N.,and Wagner,G.(2009).Topology and Regulationof the Human eIF4A/4G/4H Helicase Complex in Translation Initiation.Cell 136,447-460.
Oltean,S.,and Bates,D.O.(2014).Hallmarks of alternative splicing incancer.Oncogene 33,5311-5318.
Reynolds,A.,Leake,D.,Boese,Q.,Scaringe,S.,Marshall,W.S.,and Khvorova,A.(2004).Rational siRNA design for RNA interference.Nature Biotechnology 22,326.
Rotow,J.,and Bivona,T.G.(2017).Understanding and targeting resistancemechanisms in NSCLC.Nature Reviews Cancer 17,637.
Roy,B.,Haupt,L.M.,and Griffiths,L.R.(2013).Review:AlternativeSplicing(AS)of Genes As An Approach for Generating Protein Complexity.CurrentGenomics 14,182-194.
Scotti,M.M.,and Swanson,M.S.(2016).RNA mis-splicing in disease.NatRev Genet 17,19-32.
Shao,Y.,Wu,Y.,Chan,C.Y.,McDonough,K.,and Ding,Y.(2006).Rationaldesign and rapid screening of antisense oligonucleotides for prokaryotic genemodulation.Nucleic Acids Research 34,5660-5669.
Shi,Y.(2017).Mechanistic insights into precursor messenger RNAsplicing by the spliceosome.Nature Reviews Molecular Cell Biology 18,655.
Siegel,R.L.,Miller,K.D.,and Jemal,A.(2017).Cancer statistics,2017.CA:A Cancer Journal for Clinicians 67,7-30.
Siegfried,Z.,and Karni,R.(2018).The role of alternative splicing incancer drug resistance.Current Opinion in Genetics&Development 48,16-21.
Vaysse,C.,Philippe,C.,Martineau,Y.,Quelen,C.,Hieblot,C.,Renaud,C.,Nicaise,Y.,Desquesnes,A.,Pannese,M.,Filleron,T.,et al.(2015).Key contributionof eIF4H-mediated translational control in tumor promotion.Oncotarget 6,39924-39940.
Yoshimi,A.,and Abdel-Wahab,O.(2017).Molecular Pathways:Understandingand Targeting Mutant Spliceosomal Proteins.Clinical Cancer Research 23,336-341.
Zhang,J.,and Manley,J.L.(2013).Misregulation of Pre-mRNA AlternativeSplicing in Cancer.Cancer Discovery 3,1228-1237.。
发明内容
本发明要解决的问题是提供干预EIF4H剪接亚型的寡聚核苷酸，尤其是干预EIF4H特定剪接亚型的干扰小RNA及反义寡聚核苷酸序。
本发明的另一个要解决的问题是提供通过干预EIF4H特定剪接亚型从而治疗恶性肿瘤的潜在寡聚核苷酸药物。
本发明的再一个要解决的问题是提供新的检测EIF4H不同剪接亚型RNA及蛋白表达的方法。
本发明提供了干预EIF4H剪接亚型的干扰小RNA、反义寡聚核苷酸序列。
本发明提供了一种寡聚核苷酸，该寡聚核苷酸具有干预EIF4H剪接亚型表达的活性，
并且是下列中的任意一项：
(1)SEQ ID NO 6-152中任意一项，或与SEQ ID NO 6-152中任意一项的序列重复超过5个核苷酸的RNA或者反义寡聚核苷酸；
(2)上述(1)中核苷酸的各种化学修饰形式；
(3)上述(1)中核苷酸带有连接物的衍生核苷酸序列；
本发明中，所述的寡聚核苷酸是干扰小RNA、微小RNA、双链RNA、短发卡RNA或其化学修饰形式。
本发明中，所述的寡聚核苷酸是反义寡聚核苷酸或其类似物或其化学修饰形式。
本发明中，针对EIF4H长、短两种剪接亚型特异的核苷酸序列(附件序列文件)，根据干扰小RNA(siRNA)的设计原则(Elbashir et al.,2001；Reynolds et al.,2004)，设计了特异性靶向两种EIF4H剪接亚型的siRNA(附件序列文件)。基于RNA沉默的原理可以判断，与这些siRNA序列重复超过5个核苷酸的单链siRNA，微小RNA(miRNA)，双链RNA(doublestranded RNA,dsRNA)，短发卡RNA(short hairpin RNA,shRNA)，以及相关核苷酸序列的各种化学修饰形式及带有连接物的衍生核苷酸序列能不同程度的沉默EIF4H剪接亚型的表达，均属于本发明的范围。
针对EIF4H pre-mRNA中长剪接亚型特异的外显子及其相邻的内含子中距离剪接位点50个核苷酸以内的序列(附件序列文件)，根据反义寡聚核苷酸(ASO)设计原则(Dinget al.,2004；Shao et al.,2006)，设计了抑制EIF4H长剪接亚型的反义寡聚核苷酸序列(附件序列文件)。基于ASO干预mRNA剪接的原理可以判断，与这些ASO序列重复超过5个碱基的反义寡聚核苷酸及相关反义寡聚核苷酸的类似物、各种化学修饰形式及带有连接物的衍生核苷酸序列能不同程度抑制EIF4H pre-mRNA中长剪接亚型特异的外显子剪接，均属于本发明范围。
本发明提供了检测EIF4H不同剪接亚型RNA及蛋白表达的方法。
在细胞、组织中提取总RNA，进行反转录，运用能够检测两种EIF4H剪接亚型的引物进行PCR，凝胶电泳分离，通过与不同剪接亚型对应的PCR产物强度分析，检测EIF4H不同剪接亚型的比例(percent-spliced-in,PSI)及表达水平。不同电泳和条带强度分析方法，如毛细管电泳及基于荧光染料的PCR产物定量，均属于该发明的范围。此外，对于总RNA样本，通过RNA测序(RNA sequencing,RNA-Seq)，获得支持EIF4H特定外显子剪接进入和剪接排除的测序序列(sequencing reads)，计算其PSI，从而检测EIF4H不同剪接亚型RNA比例。
在细胞、组织中提取总蛋白，运用eIF4H抗体，进行Western blot分析，根据不同剪接亚型蛋白分子量大小差异，区分不同蛋白亚型。运用不同种的eIF4H抗体，基于分子大小差异，检测eIF4H不同剪接亚型蛋白表达的方法均属于该发明范围。
在本发明的一个优选例中，采用反转录-聚合酶链反应检测EIF4H不同剪接亚型mRNA表达。
在本发明的一个优选例中，采用eIF4H抗体检测EIF4H不同剪接亚型的蛋白表达产物。
EIF4H不同剪接亚型RNA及蛋白的检测可应用于包括肿瘤在内的疾病诊断、治疗及药物开发等方面，均属于该发明范围。
本发明提供了通过干预EIF4H特定剪接亚型从而治疗恶性肿瘤的潜在寡聚核苷酸药物。
运用EIF4H剪接亚型特异的siRNA，在肿瘤细胞中分别敲低EIF4H长、短剪接亚型，进行细胞增殖、凋亡等表型分析，发现EIF4H长剪接亚型敲低能显著抑制肿瘤细胞增殖、促进凋亡，与EIF4H基因整体敲低对细胞生长的抑制效应相似。这些结果表明，针对EIF4H长剪接亚型的siRNA可作为肿瘤治疗的潜在药物，可应用于EIF4H长剪接亚型高表达的不同肿瘤的治疗中。
运用EIF4H剪接亚型特异的ASO，在肿瘤细胞中抑制EIF4H长剪接亚型，进行细胞增殖等表型分析，发现抑制EIF4H长剪接亚型表达能显著抑制肿瘤细胞增殖。这些结果表明，针对EIF4H长剪接亚型的ASO可作为肿瘤治疗的潜在药物，可应用于EIF4H长剪接亚型高表达的不同肿瘤的治疗中。
本发明提供了上述寡聚核苷酸的应用，即所述的寡聚核苷酸在制备诊断试剂或者治疗药物中的应用。
所述的药物可以是通过抑制EIF4H特定剪接亚型表达从而治疗恶性肿瘤的潜在寡聚核苷酸药物。
本发明还包括干预EIF4H剪接亚型表达的siRNA或者ASO在包括肿瘤在内的疾病诊断、治疗、药物开发中的应用。
本发明提供了干预EIF4H特定剪接亚型的干扰小RNA及反义寡聚核苷酸序列，检测EIF4H不同剪接亚型RNA及蛋白表达的方法，以及通过干预EIF4H特定的剪接亚型从而治疗恶性肿瘤的寡聚核苷酸药物。
附图说明
图1显示EIF4H剪接亚型RNA的反转录-聚合酶链反应(RT-PCR)检测；图中显示为肺支气管上皮细胞系BEAS-2B(B2B),肺癌细胞系A549,PC9中，运用总RNA的反转录产物为模板，能够扩增EIF4H长(long,L)、短(short,S)两种剪接亚型的引物，进行PCR，对PCR产物进行琼脂糖凝胶电泳分离，通过与不同剪接亚型对应的条带强度分析，检测EIF4H不同剪接亚型的比例(percent-spliced-in,PSI)；ACTB为内参。
图2显示EIF4H剪接亚型蛋白表达的Western blot检测；图中显示为肺支气管上皮细胞系BEAS-2B(B2B),肺癌细胞系A549,PC9中，运用eIF4H抗体，进行Western blot分析的结果图，根据eIF4H长、短剪接亚型蛋白(eIF4H-L,eIF4H-S)的分子量大小差异，区分eIF4H不同剪接亚型；β-actin为内参。
图3显示siRNA介导的EIF4H及其剪接亚型敲低对肿瘤细胞增殖的影响；图3A显示在肺癌细胞PC9中，用不同浓度、针对EIF4H长剪接异构体的siRNA(siEIF4H-L)处理细胞后，对细胞生长进行CCK-8检测的结果图；图3B显示用阴性对照siRNA(siRNA againstnegative control,siNC)及分别针对EIF4H基因(siEIF4H)，长、短剪接亚型(siEIF4H-L,siEIF4H-S)的siRNA处理肺癌PC9细胞48h后，对细胞生长进行CCK-8检测的结果图；Errorbar:±SEM；n＝2；ns:not significant,***P<0.001,Two-way ANOVA followed byBonferroni’s multiple comparisons tests。
图4显示siRNA介导的EIF4H剪接亚型敲低对肿瘤细胞凋亡的影响；图4A显示用阴性对照siRNA(siRNA against negative control,siNC)及分别针对EIF4H基因的(siEIF4H)，长、短剪接亚型(siEIF4H-L,siEIF4H-S)的siRNA处理肺癌PC9细胞48h后，对细胞凋亡进行Annexin V-PI染色及流式细胞检测的结果示意图；图4B为三次生物重复实验的统计分析结果；Error bar:+SD；n＝3；*P<0.05,**P<0.01,One-way ANOVA followed byDunnett’s multiple comparisons tests。
图5显示ASO介导的EIF4H长剪接亚型抑制对肿瘤细胞增殖的影响；图5A显示用阴性对照ASO(negative control,NC)及针对EIF4H长剪接亚型特异的外显子5‘剪接位点(5’splice site,5’ss)的ASO处理肺癌A549细胞48h后，对细胞增殖进行CCK-8检测的结果图；Error bar:±SEM；n＝3；***P<0.001,Two-way ANOVA followed by Bonferroni’smultiple comparisons tests；图5B为Western blot检测ASO对EIF4H长剪接亚型的抑制效果，β-actin为内参。
具体实施方式
实施例1
针对EIF4H不同剪接亚型的干扰小RNA设计
针对EIF4H长、短两种剪接亚型特异的核苷酸序列，根据siRNA设计原则(Elbashiret al.,2001；Reynolds et al.,2004)，包括：序列基元AA(N19)TT,NA(N21),或NAR(N17)YNN,其中N表示任意碱基,R表示嘌呤(A,G)，Y表示嘧啶(C,U)；GC含量；不包含4个连续相同的碱基；NCBI BLAST序列分析，设计了特异性靶向两种EIF4H剪接亚型的siRNA(附件序列文件)；基于这些siRNA序列，可衍生出序列部分或全部重复的siRNA，微小RNA(miRNA)，双链RNA(double-stranded RNA,dsRNA)，短发卡RNA(short hairpin RNA,shRNA)，以及这些核苷酸序列的各种化学修饰形式。
实施例2
针对EIF4H长剪接亚型的ASO设计
针对EIF4H pre-mRNA中长剪接亚型特异的外显子及其相邻的内含子中距离剪接位点300个核苷酸以内的序列，根据根据反义寡聚核苷酸(ASO)设计原则(Ding et al.,2004；Shao et al.,2006)，包括：不包含“GGGG”序列；GC含量；ASO靶向位点的碱基配对概率；ASO与靶向位点的结合强度，设计了抑制EIF4H长剪接亚型的反义寡聚核苷酸序列(附件序列文件)。根据这些ASO序列，可衍生出序列部分或全部重复的反义寡聚核苷酸及这些反义寡聚核苷酸的各种化学修饰形式。
实施例3
EIF4H剪接亚型的反转录-聚合酶链反应(RT-PCR)检测
细胞培养
人肺癌细胞系A549、PC9用含10％胎牛血清的1640培养基，人肺支气管上皮细胞BEAS-2B用含10％胎牛血清的DMEM高糖培养基，于37℃、5％CO2的培养箱中培养。取处于对数生长期的细胞进行后续实验。
RNA提取
使用Trizol(Thermo Scientific)提取细胞总RNA，对于不超过6cm培养皿中100％覆盖率的细胞数，吸弃培养基后用1mL Trizol吹打收集，用200μL三氯甲烷震荡混匀，室温静置5min，4℃、15000rpm离心15min。吸取上层水相，加入等比例异丙醇，室温静置10min，4℃、15000rpm离心10min。在离心管底部可见白色RNA沉淀，弃上清，加入1mL 75％乙醇漂洗，4℃、7500rpm离心5min，弃上清，室温放置若干分钟至残留乙醇彻底挥发，待RNA沉淀干燥后加入20-50μL RNase-free无菌水，枪尖吹打数次溶解RNA。
对于组织样本RNA的提取，需要将新鲜切下的组织(大小适中，约为20-100mg)加入1mL Trizol中，立即用匀浆仪均质化样本，到肉眼见不到小碎组织块为止。
用TURBO DNA-free Kit试剂盒(Thermo Scientific)去除基因组DNA，以逆转录酶HiScript II Reverse Transcriptase(Vazyme)和oligo dT20或random primer将500ng-5μg RNA(20μL反应体系)反转录成cDNA。
RT-PCR分析
使用与可变外显子两侧邻近外显子结合的特异性引物通过PCR检测可变外显子包含及排除的剪接亚型。PCR扩增体系为：10×PCR buffer缓冲液2μL，10mM dNTP 0.2μL，Taq聚合酶0.1μL，模板cDNA 1μL(根据用于反转录的起始总RNA的量稀释模板)，10μM正反向引物0.5μL，本实施例中使用的EIF4H上游引物为：5’-TCACTTCGTGTGGACATTGC-3’(SEQ ID NO1)，
下游引物为：5’-CCTGCCCCCTAAGAAGTCAT-3’(SEQ ID NO 2)，用双蒸水补足20μL。将PCR产物与含有GelRed荧光染料(GENEray)的上样缓冲液混合，在1.5或2％琼脂糖凝胶中电泳，并使用UV成像系统进行成像，使用ImageJ软件定量条带的信号强度。我们将剪接百分比(PSI)定义为：包含外显子的条带的强度/(包含外显子的条带的强度+排除外显子的条带的强度)×100％。
RT-PCR结果显示，EIF4H长的剪接亚型在肺癌细胞中的比例高于正常肺支气管上皮细胞(图1)。
RNA-Seq数据分析外显子剪接
通过RNA测序数据，获得支持EIF4H特定外显子剪接进入和剪接排除的测序序列，计算其剪接百分比(PSI)。我们将剪接百分比定义为：支持包含外显子的reads数/(支持包含外显子的reads数+支持排除外显子的reads数)×100％。
对The Cancer Genome Atlas(TCGA)不同肿瘤组织及癌旁正常组织的RNA-Seq数据分析结果表明，与癌旁正常组织相比，EIF4H长剪接亚型的比例在多种肿瘤组织中明显升高(https://bioinformatics.mdanderson.org/TCGASpliceSeq/)。
实施例4
EIF4H剪接亚型的Western blot检测
当细胞接近铺满培养皿时，吸去培养基，用预冷的PBS清洗一次。胰酶消化并离心收集细胞。加入含有蛋白酶、磷酸化酶抑制剂cocktail(Millipore)的RIPA强裂解液，超声后提取细胞总蛋白，BCA试剂盒定量总蛋白浓度。提取的蛋白质用10％SDS-PAGE电泳，转膜，采用溶于TBST的5％牛奶室温封闭2h，一抗兔抗人eIF4H(CST)，1∶1000稀释，鼠抗人β-actin(Abmart)，1∶5000稀释，4℃孵育过夜，TBST洗膜后3次后加二抗过氧化物标记的山羊抗兔或抗小鼠IgG，1∶5000稀释，室温孵育2h。TBST洗膜3次，滴加ECL发光液，用Bio-Rad凝胶成像仪曝光，用Image-J半定量靶蛋白质的表达水平。
与正常肺支气管上皮细胞BEAS-2B相比，肺癌细胞A549和PC9中EIF4H长剪接亚型蛋白表达量及比例增加(图2)，与RNA水平一致。
实施例5
针对EIF4H剪接亚型的siRNA转染及效率分析
通常情况下，不同的siRNA浓度会影响靶标基因的沉默效率。因此，为选择合适的siRNA浓度检测对细胞表型的影响，我们采取20nM，30nM，40nM的针对EIF4H长剪接亚型的siRNA处理PC9细胞，并用CCK-8(Cell Counting Kit-8，Byotime)检测处理后的细胞活力。CCK-8试剂盒，是一种基于WST-8而快速、灵敏的检测细胞增殖与细胞毒性的试剂盒。WST-8类似于MTT，在电子耦合剂的存在下，可以被线粒体氧化还原脱氢酶催化形成橙黄色的甲臜。在相同的条件下，颜色越深，则细胞生长越快，颜色越浅，则细胞生长越慢。
1)、肺癌PC9细胞在10cm培养皿中，使用10mL含10％FBS的1640培养基，在37℃、5％CO2的培养箱中培养；
2)、6孔板中，加入Lipofectamine RNAiMax-siRNA混合物，室温孵育15min；
Figure BDA0001911881150000121
3)、弃掉10cm培养皿中PC9细胞的培养基，加入2mL的0.25％的胰酶，消化10min，将消化后的细胞离心收集，用1xPBS漂洗后，混悬在新鲜培养基中，以每孔3×105个细胞的量加入到置有Lipofectamine RNAiMax-siRNA混合物的6孔板中，轻晃混匀，37℃、5％CO2的培养箱中培养6hrs后，更换新鲜的培养基，继续培养48h；
4)、0.25％的胰酶消化siRNA处理组与对照组的PC9细胞，1xPBS漂洗后重悬在1mL培养基中，用自动细胞计数仪进行细胞计数；96孔板中，每孔以100μL培养基中包含1000个细胞，为保证实验误差造成的影响，每个条件设置5个生物技术重复，37℃、5％CO2的培养箱中培养，每隔为24h用CCK-8检测细胞活力，包含第0天的结果；
5)、在检测时间点，将CCK-8按1:9比例加入培养基中，配置好培养基-CCK8混合液，弃掉96孔板中的培养基，每孔加入100μL混合液，37℃避光孵育1.5h，孵育完成后，运用多功能酶标仪在450nm处测量吸光度值(optical density,OD)，以吸光度值为纵坐标，时间为横坐标，将测量结果汇成生长曲线。
针对EIF4H长剪接亚型、不同浓度siRNA对细胞生成影响无明显差异(图3A)，基于此选择较低浓度进行实验，减少siRNA可能的脱靶效应。
实施例6
肿瘤细胞系中EIF4H剪接亚型的siRNA干预及细胞表型分析
细胞增殖
基于不同siRNA浓度处理细胞的实验结果，在肺癌细胞中选择20nM siRNA终浓度处理细胞。
1)、6孔板中，加入Lipofectamine RNAiMax-siRNA混合物，室温孵育15min；
Figure BDA0001911881150000131
2)、弃掉10cm培养皿中PC9细胞的培养基，加入2mL的0.25％的胰酶，消化10min，将消化后的细胞离心收集，用1xPBS漂洗后，混悬在新鲜培养基中，以每孔3×105个细胞的量加入到置有Lipofectamine RNAiMax-siRNA混合物的6孔板中，轻晃混匀后，37℃、5％CO2的培养箱中培养6hrs后，更换新鲜的培养基，继续培养48h；
3)、弃掉10cm培养皿中PC9细胞的培养基，加入2mL的0.25％的胰酶，消化10min，将消化后的细胞离心收集，用1xPBS漂洗后，混悬在新鲜培养基中，以每孔3×105个细胞的量加入到置有Lipofectamine RNAiMax-siRNA混合物的6孔板中，轻晃混匀，37℃、5％CO2的培养箱中培养6hrs后，更换新鲜的培养基，继续培养48h；
4)、0.25％的胰酶消化siRNA处理组与对照组的PC9细胞，1xPBS漂洗后重悬在1mL培养基中，用自动细胞计数仪进行细胞计数。96孔板中，每孔以100μL培养基中包含1000个细胞，为保证实验误差造成的影响，每个条件设置5个生物技术重复，37℃、5％CO2的培养箱中培养，每隔为24h用CCK-8检测细胞活力，包含第0天的结果；
5)、在检测时间点，将CCK-8按1:9比例加入培养基中，配置好培养基-CCK8混合液，弃掉96孔板中的培养基，每孔加入100μL混合液，37℃避光孵育1.5h，孵育完成后，运用多功能酶标仪在450nm处测量吸光度值(optical density,OD)，以吸光度值为纵坐标，时间为横坐标，将测量结果汇成生长曲线。
在肿瘤细胞中运用siRNA分别敲低EIF4H基因及其长、短剪接亚型，进行细胞增殖分析，发现EIF4H长剪接亚型敲低能显著抑制肿瘤细胞增殖，与EIF4H基因整体敲低对细胞生长的抑制效应相似(图3B)。
细胞凋亡
通常情况下，磷脂酰丝氨酸(phosphatidylserine,PS)只存在于脂质双分子层的内侧，而在细胞发生凋亡时，磷脂酰丝氨酸就会由细胞膜内侧翻转至外侧；Annexin-V是一种磷脂结合蛋白，它与PS具有很强的亲和力，将Annexin-V用绿色荧光染料FITC进行标记，通过流式细胞仪检测早期凋亡的发生；而核酸染料碘化丙啶(Propidium Iodide,PI)不能通过完整的细胞膜，只有在细胞处于晚期凋亡或坏死的情况下，PI才能将细胞核染成红色，所以Annexin-V和PI的联合使用可以将正常细胞、凋亡早期细胞、凋亡晚期及坏死细胞区分开。
1)、Lipofectamine RNAiMax转染siRNA：
Figure BDA0001911881150000141
以6孔板中一个孔为例，将胰酶消化后的细胞以每孔3×105个细胞的混悬至至2mL培养基，加入到含有Lipofectamine RNAiMax-siRNA混合物的6孔板中，轻轻摇晃混匀，37℃、5％CO2的培养箱中培养8h后，更换新鲜的培养基，继续培养至48h；
2)、收集细胞：将培养基中漂浮的细胞收集到EP管中，同时加入不含EDTA胰酶，将贴壁细胞消化下来，4℃，1000rpm/5min，弃去培养基；
3)、洗涤：加入预冷的1xPBS，4℃，1000rpm/5min，洗涤3次；
4)、向每个样品中加入100μL的Binding buffer，充分混匀，再加入5μL的Annexin-V和5μL的PI，充分混匀后避光孵育10min；
5)、孵育完毕后向每个EP管中加入400μL的Binding buffer，上下颠倒混匀后，避光可保存1h；
6)、流式细胞仪检测：激发光波长为488nm，绿色的FITC荧光可以在FL1通道中被检测到，红色的PI荧光在FL2通道中被检测到。
在肿瘤细胞中运用siRNA分别敲低EIF4H基因及其长、短剪接亚型，进行细胞凋亡分析，发现EIF4H长剪接亚型敲低能显著促进肿瘤细胞凋亡，与EIF4H基因整体敲低对细胞生长的抑制效应相似(图4)，表明针对EIF4H长剪接亚型的siRNA可作为潜在的肿瘤治疗药物。
实施例7
针对EIF4H长剪接亚型的ASO转染及效率分析
转染ASO方法采用以下两种方式进行：使用Lipofectamine RNAiMAXTransfection Reagent或Lipofectamine 2000Transfection Reagent(ThermoScientific)，前者将2μL 100μM的ASO、5μL RNAiMAX和200μL的opti-MEM混匀，加入6孔板中，室温静置15min，然后加入3×105个混悬在2mL培养基中的细胞，混匀；后者需要将3×105个细胞铺至6孔板中，待细胞贴壁后，将2μL 100μM的ASO、5μL Lipofectamine2000和200μL的opti-MEM混匀，室温静置15min后缓慢滴加入6孔板，补加培养基至2mL。为了检测不同浓度ASO的效应，我们设计了50nM和100nM两种浓度梯度，在48h收集细胞计数，同时种入96孔板中检测细胞增殖活力，剩余细胞收集用于提取RNA和提取细胞总蛋白。
根据EIF4H剪接亚型RNA和蛋白检测结果，针对EIF4H长剪接异构体的ASO的效应依赖于浓度，100nM的效率大于50nM。
实施例8
肿瘤细胞系中EIF4H长剪接亚型的ASO干预及细胞表型分析
1)、Lipofectamine RNAiMax转染ASO：
Figure BDA0001911881150000151
以6孔板中一个孔为例，将胰酶消化后的细胞以每孔3×105个细胞的量加培养基至2mL，37℃、5％CO2的培养箱中培养贴壁后，缓慢滴入Lipofectamine2000-ASO混合物，轻轻摇晃混匀后，37℃、5％CO2培养箱中继续培养至48h；
2)、铺板：将实验组与对照组细胞全部消化下来，自动细胞计数仪计数，以每孔100μL/1000个细胞的量将均匀的铺在96孔板中，每一组细胞均需要铺30个孔，以每一横行的5个生物技术重复孔的平均值作为吸光度值，以减少实验误差，37℃、5％CO2培养箱中培养，每隔一天进行细胞生长检测；
3)、细胞完全贴壁后，将培养基全部吸弃，CCK-8与培养基按1:9的比例混和，充分混匀后，以每孔100μL的量加入混合液，同时选取5个空白孔，加入100μL的混合液(以去除培养基颜色对吸光度值的影响)；37℃、5％CO2培养箱中避光孵育1.5h；
4)、吸光度的测量：孵育1.5h后，用多功能酶标仪在450nm的波长下测量吸光度值，将不同时间点的数据绘制成细胞生长曲线图。
在肿瘤细胞中运用ASO抑制EIF4H长剪接亚型，进行细胞增殖分析，发现EIF4H长剪接亚型敲低能显著抑制细胞生长(图5)，表明针对EIF4H长剪接亚型的ASO可作为潜在的肿瘤治疗药物。
本发明主要相关序列如下：
1、EIF4H基因序列及不同剪接亚型mRNA序列EIF4H基因序列(大写字母：外显子；小写字母：内含子。)
>hg19_ncbiRefSeqCurated_NM_022170.1range＝chr7:73588706-736114295’pad＝0 3’pad＝0strand＝+repeatMasking＝none(SEQ ID NO 3)
GACGGCAAATGGCGGACTTCGACACCTACGACGATCGGGCCTACAGCAGCTTCGGCGGCGGCAGAGGgtgaggcgggcgtgcgcgggccccgtcgggggctgcgggaccggcggagtcggggccgtcagggtggcggccgtcctgcgctcagccggggcggcgggaggcgggggcccgcggaggcctaggagcggcgcctggagggccggggcctggaaatggcgcgctcggagacggggcggcggcggctgggctcccggggtcttgcttgcggggcggatcccttccactgcctcccgccaacgacaacgtgttggtttttgccctgccttggccgcggcggcgccgatgcccggagacgagcgcccgcttccagccccatcccccctacggtaggaccagtgcggggctgagctgtggcgctggggtacgggccacacggcctgccccgctccggtcccggcctctttcgcggccctctgttgcctgcccgccttgcaggcccctggccccttcgccacgaggccggccgactggggaaggtcggcagccaagaggggcgattcccttccccttacgtgtgaggcagggatttggcggctctggcccggggatggtacgactccttgcgggcggggggagaccaaggagataaatccatcgtagcttctttgtattaatctttcttgggtcacagtgccttgacaggtgctacgtgctctttggcacaatttcatttctccttggtttctcgctctgatctggagaaggaaccgcatttagcgtcgtcctgattgttgtcaaatgcagacaactatgtataattgaaacactttgcttaaaagaaacatctttgaatagagagctcatggtgtttaattccaggactctgcacttttttttattttattttttaaatacaagttgcctcgttagaatataggctatgaacatcttgctgtttgtcaaggacacacttgggctttgtgttttcttgcagaaaggattactttcccaggataacaccacaataatacacactgaggtgttcagtctcctgggttgcagtccaaaagtatctttgtttggtaaagaatgatcacttttaataccttggtttgggaacggctcagacaaaagcttgtttcggggcaggagtgtagctaatgttcatgatactgtaacgtggaagtgtgccggtgtaggtggagctattgtcattccaaagaacattttatttgctaataaaaaaagaagtaaatgttggatgacatgaaattatcgcctttgggttaggtttctaaacatcaaatttgttctcaagtcatttcgaaatttaataactagggtttttttttttttcctagttccaaaaaacgttcacagaagtacccagaaaaattcttcttaaacagcgtggtcaatttaagttttagttgcattgggactacaataaggtgaccttattaaactgagtgtggggatttaaaaaaatttccttcagacacatttgccagctcttctggtgtcaaattttgacatttgggggtgccaggaacaatcagaagtaaaattctattaagatgctattaatttcaaatctccacagcagatgaaaatattagccatttgccccaggtgtactccaaaaactgataggagagtttcctaggtagcactgtgcatcacttgaacttcctaatagaaagtctctttactgttaagcatcttctatgtctcacttacttggaattttctatttggcttcatattaatgtgaaactacataaagtattgctactttcagaagtagttttaaaaacttgttggtgaatttacatattcatttatacatgtgtattctttttttttttttttgagatggagtttcactcttgttgcccaagctggagtgcaatggtgtgatctcggctcactgcaacctccacctcccgggtttaagcgattctcctgcctcagcctcccaaagtgctgggattacaggcattagccaccgcgcccggcctcaattattttaattatgtccacatgaacaaatgtcactgctttacaatgcttaagcatcatagaagattgccacctatggaaagcagttttaacactgcattgtttggtacttgtgtaggcaccttctcccttgtctaaaatgcctcagacaggctcgtacatctcacctctgaattacttgggtcacaggtgaaagaaatggtaggcctaaaatgaacagttatcatcataaaacatagtttggtagttatacatcttcctaagtttatttcgtagttgtgtttaataaccagtgcagttgtcaggaaacagtattgaaccactgcttttttcccccgagcctgtaagggcatgtccccagcagccagggagagaaccagggactgcattgttcagcaagcaaatcaaaacaaaattccaaatgatcaagagttgattgtaccagatcttgaatggactttgttttgcagtttgctaaattatctttttgaggttccagtttctcatctaccattggcagtaaagtgcagagtaatactgattttatatttctaattgcaacaaagctatgagtgcttagtggaaaaccaccgatcctgagaattagggtggaaattggaggtggcaagaattgaggtggctgaaatggtgccatctaaaaactggcaaaactccaaatttattctttatcaggattcagttagggagtttagaattcttttttaaatagagatgggtttcactatgttgcccaggctggtcttgaactcttggactcaagtgatcctcccgcctcagcctcccaaagtgttgggattacaggcatgagctaccacgcccagcctaggagtttagaattctaacctttgaatctttaaacttagcatgtctgtctaaatttctgagcatatttttatccaaaatttttatggtggtaaaatatacataacataaaatgtgccactttacccatttttcagtatacatttcagggatattgagtgtattcgtaatgtataaccatcaccactatccagctccttaactcttacagcttgtaaaactaaaaccctgtcccctttgaatagtaattccctattgccctccttcccccaggtccctggcaaccaccattctacttcctgtctctgagcatattttaagatttgtatgtaataggaaggaaagcagtgtataaaaactacagctagttttcactgatgctagggcctgtaaagctattgaaaagcttattttaaaatgctgtttacatgtcaaatgtcaagtcgtttaggtttgttatccttcttcactccaagtgttccattgcttagttccaaaccattatcctctatgaaaagtgttaccagaaatatttggatttgctaagaggtctctgcatgacggttcatgtaaaagccctggctttggaataaggcagaccagagtttcatcagttcagccatgtgttagttgtgtgatctgaagtaagtttcttcacctctctgagcctcgttttccccacatatgagttagaaacatgtatttatttatgaaataggctctccttctgtcacccaggctggagtgcagtggcaggatcatagctcagtgcagcctcgacttcccaggctcaagccatcctcccacctcagcctcccaagtagctgggattacagggtgcgccaccatgcccagctaatttttgtatttttttgtagagacagagtgtctctatgttgcccaggctagtctcaaacccctgggctcaagggatcttcctgcctcagcctcccaaggctcattgtgtgagccacagtacccagccttcaatttatgtttctattagaaattaagctttgtggctgggtgcagtggttcatgcctgttatcccagcactttggaaggccgaagcaggcggatcacttgtggccagaagtttgagaccaccctgaccaacgtggcaaaaccccatgtctactaaaaatacaaaacgggtgtggcggcacatacctgtaatcctagctacttgggaggctgaggcaggagaattgcttgaaccagggaggcggaggttgcattgagctgagatcatgccactgcactcctccagcctgggcgacagagagagactgtctccaaaaaaaaaaaaagaaaaattaagcttagtgttctggaaagacttcacaattttaaggagtctccctggaagcttagaacgtacaaagcagactctgaatgtctttccaggatcacactctggcttccagcttcccctggaaggttcaggagagtatttgtcgcctggttgcttttacatcatgctgattagacctgcacacccgggcatgaaatgagtgtgattgcatcattttcatagaacctaatggcagctgtatgtacgtctaagacttcttttgtgtaagtcgaatgttcttcgaagtttccccacgtcatgatttatgtgattttccgtcttgatcaggaccttctgaaggagctcaagtatgtggctgctccacggttgatgagtctctggttaagcatggccttccagttcagggctaggggggccgtcacctccctttgttctgcacattcttctcctgtctttcatgtatactcacatttttggagccttctgtgtactcacatttttggagccttctaaatgtctggcttaatagaacacagctggattctcatacctgcatctgtctccactcttggaatgtgttgctttggttgacttttatggagcagctcaatccttgcacagataaatagttggaaatgggaggagtaatcaaataattctggatattcatatttgattctataccaaaactcaataagcagtagtttctatgaaggttagttgcaatgtgaaacctaaaaccttatcaataaactcttcatacttttacattagagttcatcatttggctgggcgcggtggctcactcctgt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tctgcagatggcctgagaaaggtttgaatattatggttaaaacattcaagaagaaagctttctagaaacgcagctgctatttcagtgcatgttcagttagcattttctgactgataaaatagaattgagagggaggaatgccagtcagggttctatctattgaaaggctctcctagtctgccatcatctagacaacagaatggtagttgtgacgtggagaagtagtatgaataggatcttcccacatctttaacccagaattacttgaggaaatcggggtcttcttttcttttccagGATTCTGCTATGTAGAATTCGATGAAGTGGATTCCCTTAAGGAAGCCTTGACATACGATGGTGCAgtaagtatcctcgggttttctctgtcatagcagttgagattgttttctttgccaatacttacactattttccctttatcattagCTGTTGGGCGATCGGTCACTTCGTGTGGACATTGCAGAAGGCAGAAAACAAGATAAAGGTGGCTTTGGATTCAGAAAAGGTGGACCAGATGACAGAGgtgattggttcttctaaagctgaacatcataaagatttgcagtatgcctaccaattccaactcgtgaacgttcctagtagaactcgattatctcataggtgtgataggttctcgtgagctcttaaaagtttgcgtaaaatcctagaaagaatggctgatgcttctgcaagcctggcgttttttgttttctgttggaagcaaaagctcttaaaatgatttcattgaaccttccatcacttgagttgtatgtcttccaaatgaatttttccatgtttgcgcagcaaggtaatgacacgacctcaactttatcttttttgtgtatcctcagGAATGGGTAGCTCTCGAGAATCTAGAGGTGGATGGGATTCCCGGGATGACTTCAATTCTGgtatcagtatttaaagtatcaccacttaatttttcctaggagccttgctgctgttctctgtttcttacgagtagcccgcctggccggactacttatttagttcctttaacaaatacaactctcctgcagggtgcccaggcatgcaagtaagcctctgttagagcctgtgggggcaggacttgagccccatgcaccttgtgtggtcacctttgttagcctgcagctccttggatgtgtacgggcctgggtccttgagctcgctcagcagtctttctcagactctagcccagggaaactcctctgtggttttgtgtatggcaccaagagaagtactgttccttctggaaaacataggatagaggtttgggttcttacatggtgaaaggtagtcctgatggcccttttttttcttggaggattgtggggaggtccagtcccttgatggtggacctgatgcacttcgccgttctctttcttagccaacccaggcagagcagctggtggtatagcacaaagcatcagtccacacgccaggcctctgccctgcagggtgattttaagcatttttatcatctttactttctgtttttaaattacaaaagtataattttttatatatgcacatgataaaaacttcagttgtaacccagaaaggtaggaagagaaaaacaaatttgttttctttcccgcaaacttgggatgtgctgtcaatgctgtgctgtatagaacgtcagcaccctccccccatcaaaacaaaaccggtgtaaagtagtgggtgagtatgggagggactggtgcacgctgtcttgtgagtctgagatttttaaaaactcagctttggctgaacaggtgacgactacctgttgaataatatagttcttaaaagtcctgtagccagtcagaaatgagcttattcataaaagtgcagtatggtgaagtcaatctgtaattttatgtataagctagtctctgattgaaacatgcagcagctgtcttcttagagccaaccagcagctcagggaacggcctttcccctgccagtttggtattcgcaggtgttatagaagttgcccacatactaacgtctttctacccttggcttagcttggccactggcgctcaagcagagacacagtgaacacggccatggcctggccgccaccagttctggtgggaactgcagtgttttggtgacatctcaaaccctatgtgggctcactcgatggagtaatggctaatttgatactttatacgttatttgcgagagtagatcttttttactttttaataggatggtgctattttttttaatgatacctgctcatgtaaaaaacaaaatagagcaacagaaaagtaccaagaaagcaaaaacatttcatcatcagaaatagccagtgttaaactaagctttgtaaagaaaactctgaagattctcatgaatagtttgcttcctagaaacccaccaacagattttttgttgttgttttttttgagacggagtcttgctctgttgcccaggctggagtgcagtggtgcgatctcggctcactgcaagctctgcctcctgggttcatgccattctcctgcctcagcctcccaagtagctgggactacaggcacccaccaccacgcccggctaattttttgtatttttagcagagacggggtttcatcatgttagccaggatggtctcgatctcctgacctcgtgatccacctgcctcggcctcccaaagtgctgggattacaggcatgagccaccgcgcccggcccccaccaacagatttgatgagagatccttgttcatctgcttcaccagcgtggtgtgagctatttgtttcacgctagacattggtggtcgaaatcatttaatctcatagtaaagggtgtaaatttactttgtactcacgtggaaaggtacacttcacagtttagtttgggggaaacgtgaagttaaatgttttaatactaactaggcttgttcactgaactgtgtaatgtgggtaaaatactttgtaaaggaccttatactcgactgtatgcctcccagtgtatttcccctaaagcagtgctcttattggtctaaatctgttcagacttgctttcattacttggtttgttaaaatttattatttaactcagtctctgaactgggtgtgagatcccttggtttccaaaacccttgccacctttaaagaatagaaatcggcccccaccacaggtgatgcagtcagcgcttatcattggaggtggttctgtggagttgctagaacactgaatgagcccgtgcggtgctgctcctgtgtgctacagatcacaacattttcgtcagctgatcatacctaacattgctttatgtgacttttttttttttcttttttttttttttgagacagaggctcactctgttgcccaggctggagtgcagtggcatgatttcggttcactgcaatctccgcctccagggttcaagcgattctcctgcctcagcctcccaagtagctgggattacaggcgcacaccaccacacccagctaatttttatatttttagtagagacggggtctctccgtgttggtcaggctggtctcgaactcccagcctcaggtgatctgcccacctctgcctcccagagtgctgggattacaggcgtgagccaccacacccagcctatgtgaccttgattttaaagacgcctcatttaatgttagtgttgattcatttaatagtgttaacattgaactcagggccagctgtagtaagtagtctgtggtgccatcatttcagcctgttagaaagctcatcccacaccggttttgtctgtaagacacctgacagccagcctgcacttaagaacagcagacagcacttcacactgcacctggggccacttcagacagcgatgtcaccagaaaaagcccaaagggcaaaacacccgacactcatcgtgaaaagacttgacagctaataactgaaacaggcagagcattatcgttttctgcctcagctgggatcgtcgcatctgtgcaggtccaggactgactggagaaatgctgctggtatctagcttgggattacacatgaattttagcgactgggtggatttgcaaatatgggatctgtggataatgaggatgactgtatagtgcatactgcgggctctgggggttcttctcaaagacaagctccctgaaagcttggagccttggcaccactctcgcagagagtagcagtggcaccccctgagtgcgcaaccaacagcagcaagctcatttgtttaattatggtttaacttcttttcgccagttttttttttttttccttgagacagagtctcacttcatcacccaggctgaagtgtaatggcacgatcttggctcactgcaccctctaccttccaggttcgagcgattatcctgcctcagcctcatgagtagctggaaccccaggcgtgcgccaccatgcccagctaatttttgtgttttttgtagagagtgagtctcactgtgttgcccagtctggtcctgaactcctatactcacctcccacctcagcctcccaaagtgctgggattacaggcgtgaaccagtctttttaccaaattttaacatcagtaggttatacagttaaagtttagctttcactgaagtacaagcctgaaggccccttaagaccattgagaaaatactctttcctcctacacaccagtgggaattcagcatttctaaatgagatgcagtttacttctgccaagagcccacttagcaggcacacaaatgaatggggaaagaacgctttgcccccacaggaggcgccggtgggggtgaggcgctgtgctctctgctcatgcccctctgtgtagggcatggccaatttcggataactgctttgtttaccacacaggtgctttgcttgtagaatggcccacacctaaaacgcagccaaaacaggaaaattgggactttgtgtttttattttgtcttcatatagcgttttcttgtgatttttgtgttctgagttgtaaatacttctaaatttcaggtttatcttttaaaatagctgtatagatttctattagaaaatagagttcaacctgctttgaggctttcttgttttgcttaagccgtattaaaatacagtttcgtggtattaatatttttctacttttcatgcaagctgagcatgtaaacagctcgtttgtttgctctcctaagatcttgattcctttttctttgggaagtctgttagcgctgtcattactccacacattggcatttggacccacttttaacacactttattttggagagacttcagatagtggacaaactaaagaatttaaaatgaaactcaaacaattcccagaacattattctataagcagcttggagacgatagtttgaaaagtaattcagtgtcctgtttcttcctcagGCTTCAGGGATGACTTCTTAGGGGGCAGGGGAGGTAGTCGCCCAGGCGACCGGCGAACAGGCCCCCCCATGGGCAGCCGCTTCAGAGATGGCCCTCCCCTCCGTGGATCCAACATGGATTTCAGAGAACCCACAGAAGgtacgggctcatgtgtcagtggagggcatcttgtcctgatgggatgatcatggccggttcacaccccgtggggacttggcgttctacggcacacagagttgtcggcatagatccccacgttctctggaatagcaagttcaccttggtcattagagcatctgctgttggggtagcaggccaggtgaattttagaaagtgggctggtttgctgacagcaacatcagcatggtcgttttgctggatatggaatgggatccacactctgacaaactctgctttttctcccccagAGGAAAGAGCACAGAGACCACGACTCCAGCTTAAACCTCGAACAGTCGCGACGCCCCTCAATCAAGTAGCCAATCCCAACTCTGCTATCTTCGGGGGTGCCAGGCCTAGAGAGGAAGTCGTTCAAAAGGAGCAAGAATGAGCCTGCGGTTGGGAGGGAATGGGGCGTGGGGGGTTAGAGCAGGACCACAGCCTGGTGAGTCCCCGGGCAGCCGTCCTGCAGCCGCCACTCCTGCGCCTGCCATTGGCCTCCTCACAGCGGAAACACAGCTTGTGAGTGCATGTCAGCTGTTAACAAGTGGTTTTTAGTACATTCTGGGCTTTGCTGTATCTATCTAGTGCCTGTTTGTGCGTTTTTTTCTTTCTTCCGCTGCTTCCCCATTTTCCTTCTGTCCTTTTTCTCCTGCTCCTTGTTTTCCCAGCAGCACATGGGGTTCCTCGGAGGAGCAGAGGTGGCCGCCGTGGGGGGGCGTTTGGGCTGCGGTGCTGCGTCATTTTTCCTTTGCTTTCTCTTTACTTTAGACACTGGCCCAACTCCAGGCGTTTCCTTTCATTCCCTCAGTGCTTCTCTTCTGACCTGCATGTTGAGTTCTGTATTGCTGGGGCTTCCAACAAAAACCAGAGTCACTGACAGAGGGAACAGCAGAGACCTTGTTGGTATTCAGCTGTGATGGATATAGAGAATCAGAGGCACCTTGTTTTCACAACTAGGATAAAAATATCTGCAGGGTCCTTTCCATTCCTATTTAGAGGGAGTCCTGGCTCCATGACCCCCTCCCGAGTGGACTGTCCAAGCAGATAGGCTCACACGAGAAACAGTGAGGCTGAAAGGGGGGGCTATGGAAGAGCGGTAGGGAGTCCACGGAGAAGATGCAGTGAATGCTTGCATGCATTCACACGTGTGTGTGTCCCAGCTAGTTCACTCCTTTCGCCGTGCGTGGTGGAGGCTGGCCTCTCTGGCTGGGTGCAGTGAATGGCCAGCGGGTTTCTTTTCTGCTGGGCCAAGGCGCTTTGGGGGTGGAGGGGGTGGTGCTGGTGCTGCACTGGGCTGACTGCGGCGCTGACGCAGCGTTTCCCCCCATCCCTGTTGCCTGTGTGTTGTGTGGATCTGTTCCTAGTATAGGCAACATAATGAGATACTGTGCTTCCCACCTCCCCTTCAGTTCAGAGCCAAAATGGGTCTAGAATCTGGCACTTTACTCATTTCCTTTGATAAATTGTACTATGCAGAGCTGTCAGGAACCTTCAGATAGCAGTAGAGGACTGCAGCTGTCTAGGTCTGCGGCCACATCTTGGGGACACACTGGACTGTTCCCATGTGCAGGGTTCAGCAGTTATGTGGGAGTGCTAGGGGTTAGGCTTTTGAGCTTGAACGCCTGCGTGTGAACAGATGAAAAATCCTTCAGTACCCAAGTCCCAGTCTGTCCTATGGGGAGCAGTTTGGGGGCGGCCGGCAGCAGGAGCCTGGGAAAGAGGCCCTCGCCAGGTGATGGCAGGGCCAGGGTGGCCTGGGGCACCCAGCGGAATGTGCTTAGTATTTGGTCACCAGCCGTCATCCTGGGCTTTTCCTACTGTGTCTTGTTACAAGGCCTCAGCAATCCACAGAACTCTCTCTCCTTCCTTCCACCTGTCAGCTTCTCTGCTTCTGAGATAAGAACCATTTGTGTAACACCAACACTTAACTTCAGAAAGACATGCATTATGTGGTGTAATCAAACCCGATGCTTTCAGATGACCTACTTACATCTTCAATGTGGATAAGATAAAGAACAAAACACATGCATCTAAACTGCTGGGCAATCCAGTTGACTTTTAAATGTAAGAATGGAATTCCAAACACTTAACACATTCAGCTATATGACAGAAAGTAAATCTATGGATATGGTATTTTGTGAATGATCTTTTAAATAAAAGAAAACCTTACGTAATATTT
EIF4H长剪接亚型mRNA序列(序列中U以T替代)
>NM_022170.1(SEQ ID NO 4)
gacggcaaatggcggacttcgacacctacgacgatcgggcctacagcagcttcggcggcggcagagggtcccgcggcagtgctggtggccatggttcccgtagccagaaggagttgcccacagagcccccctacacagcatacgtaggaaatctacctttcaatacggttcagggcgacatagatgctatctttaaggatctcagcataaggagtgtacggctagtcagagacaaagacacagataaatttaaaggattctgctatgtagaattcgatgaagtggattcccttaaggaagccttgacatacgatggtgcactgttgggcgatcggtcacttcgtgtggacattgcagaaggcagaaaacaagataaaggtggctttggattcagaaaaggtggaccagatgacagaggaatgggtagctctcgagaatctagaggtggatgggattcccgggatgacttcaattctggcttcagggatgacttcttagggggcaggggaggtagtcgcccaggcgaccggcgaacaggcccccccatgggcagccgcttcagagatggccctcccctccgtggatccaacatggatttcagagaacccacagaagaggaaagagcacagagaccacgactccagcttaaacctcgaacagtcgcgacgcccctcaatcaagtagccaatcccaactctgctatcttcgggggtgccaggcctagagaggaagtcgttcaaaaggagcaagaatgagcctgcggttgggagggaatggggcgtggggggttagagcaggaccacagcctggtgagtccccgggcagccgtcctgcagccgccactcctgcgcctgccattggcctcctcacagcggaaacacagcttgtgagtgcatgtcagctgttaacaagtggtttttagtacattctgggctttgctgtatctatctagtgcctgtttgtgcgtttttttctttcttccgctgcttccccattttccttctgtcctttttctcctgctccttgttttcccagcagcacatggggttcctcggaggagcagaggtggccgccgtgggggggcgtttgggctgcggtgctgcgtcatttttcctttgctttctctttactttagacactggcccaactccaggcgtttcctttcattccctcagtgcttctcttctgacctgcatgttgagttctgtattgctggggcttccaacaaaaaccagagtcactgacagagggaacagcagagaccttgttggtattcagctgtgatggatatagagaatcagaggcaccttgttttcacaactaggataaaaatatctgcagggtcctttccattcctatttagagggagtcctggctccatgaccccctcccgagtggactgtccaagcagataggctcacacgagaaacagtgaggctgaaagggggggctatggaagagcggtagggagtccacggagaagatgcagtgaatgcttgcatgcattcacacgtgtgtgtgtcccagctagttcactcctttcgccgtgcgtggtggaggctggcctctctggctgggtgcagtgaatggccagcgggtttcttttctgctgggccaaggcgctttgggggtggagggggtggtgctggtgctgcactgggctgactgcggcgctgacgcagcgtttccccccatccctgttgcctgtgtgttgtgtggatctgttcctagtataggcaacataatgagatactgtgcttcccacctccccttcagttcagagccaaaatgggtctagaatctggcactttactcatttcctttgataaattgtactatgcagagctgtcaggaaccttcagatagcagtagaggactgcagctgtctaggtctgcggccacatcttggggacacactggactgttcccatgtgcagggttcagcagttatgtgggagtgctaggggttaggcttttgagcttgaacgcctgcgtgtgaacagatgaaaaatccttcagtacccaagtcccagtctgtcctatggggagcagtttgggggcggccggcagcaggagcctgggaaagaggccctcgccaggtgatggcagggccagggtggcctggggcacccagcggaatgtgcttagtatttggtcaccagccgtcatcctgggcttttcctactgtgtcttgttacaaggcctcagcaatccacagaactctctctccttccttccacctgtcagcttctctgcttctgagataagaaccatttgtgtaacaccaacacttaacttcagaaagacatgcattatgtggtgtaatcaaacccgatgctttcagatgacctacttacatcttcaatgtggataagataaagaacaaaacacatgcatctaaactgctgggcaatccagttgacttttaaatgtaagaatggaattccaaacacttaacacattcagctatatgacagaaagtaaatctatggatatggtattttgtgaatgatcttttaaataaaagaaaaccttacgtaatattt
EIF4H短剪接亚型mRNA序列(序列中U以T替代)
>NM_031992.1(SEQ ID NO 5)
gacggcaaatggcggacttcgacacctacgacgatcgggcctacagcagcttcggcggcggcagagggtcccgcggcagtgctggtggccatggttcccgtagccagaaggagttgcccacagagcccccctacacagcatacgtaggaaatctacctttcaatacggttcagggcgacatagatgctatctttaaggatctcagcataaggagtgtacggctagtcagagacaaagacacagataaatttaaaggattctgctatgtagaattcgatgaagtggattcccttaaggaagccttgacatacgatggtgcactgttgggcgatcggtcacttcgtgtggacattgcagaaggcagaaaacaagataaaggtggctttggattcagaaaaggtggaccagatgacagaggcttcagggatgacttcttagggggcaggggaggtagtcgcccaggcgaccggcgaacaggcccccccatgggcagccgcttcagagatggccctcccctccgtggatccaacatggatttcagagaacccacagaagaggaaagagcacagagaccacgactccagcttaaacctcgaacagtcgcgacgcccctcaatcaagtagccaatcccaactctgctatcttcgggggtgccaggcctagagaggaagtcgttcaaaaggagcaagaatgagcctgcggttgggagggaatggggcgtggggggttagagcaggaccacagcctggtgagtccccgggcagccgtcctgcagccgccactcctgcgcctgccattggcctcctcacagcggaaacacagcttgtgagtgcatgtcagctgttaacaagtggtttttagtacattctgggctttgctgtatctatctagtgcctgtttgtgcgtttttttctttcttccgctgcttccccattttccttctgtcctttttctcctgctccttgttttcccagcagcacatggggttcctcggaggagcagaggtggccgccgtgggggggcgtttgggctgcggtgctgcgtcatttttcctttgctttctctttactttagacactggcccaactccaggcgtttcctttcattccctcagtgcttctcttctgacctgcatgttgagttctgtattgctggggcttccaacaaaaaccagagtcactgacagagggaacagcagagaccttgttggtattcagctgtgatggatatagagaatcagaggcaccttgttttcacaactaggataaaaatatctgcagggtcctttccattcctatttagagggagtcctggctccatgaccccctcccgagtggactgtccaagcagataggctcacacgagaaacagtgaggctgaaagggggggctatggaagagcggtagggagtccacggagaagatgcagtgaatgcttgcatgcattcacacgtgtgtgtgtcccagctagttcactcctttcgccgtgcgtggtggaggctggcctctctggctgggtgcagtgaatggccagcgggtttcttttctgctgggccaaggcgctttgggggtggagggggtggtgctggtgctgcactgggctgactgcggcgctgacgcagcgtttccccccatccctgttgcctgtgtgttgtgtggatctgttcctagtataggcaacataatgagatactgtgcttcccacctccccttcagttcagagccaaaatgggtctagaatctggcactttactcatttcctttgataaattgtactatgcagagctgtcaggaaccttcagatagcagtagaggactgcagctgtctaggtctgcggccacatcttggggacacactggactgttcccatgtgcagggttcagcagttatgtgggagtgctaggggttaggcttttgagcttgaacgcctgcgtgtgaacagatgaaaaatccttcagtacccaagtcccagtctgtcctatggggagcagtttgggggcggccggcagcaggagcctgggaaagaggccctcgccaggtgatggcagggccagggtggcctggggcacccagcggaatgtgcttagtatttggtcaccagccgtcatcctgggcttttcctactgtgtcttgttacaaggcctcagcaatccacagaactctctctccttccttccacctgtcagcttctctgcttctgagataagaaccatttgtgtaacaccaacacttaacttcagaaagacatgcattatgtggtgtaatcaaacccgatgctttcagatgacctacttacatcttcaatgtggataagataaagaacaaaacacatgcatctaaactgctgggcaatccagttgacttttaaatgtaagaatggaattccaaacacttaacacattcagctatatgacagaaagtaaatctatggatatggtattttgtgaatgatcttttaaataaaagaaaaccttacgtaatattt
2、针对EIF4H不同剪接亚型的siRNA序列
EIF4H长剪接亚型siRNAs
基于RNA沉默的原理可以判断，与这些siRNA序列重复超过5个核苷酸的siRNA，微小RNA(miRNA)，双链RNA(double stranded RNA,dsRNA)，短发卡RNA(short hairpin RNA,shRNA)，以及相关核苷酸序列的各种化学修饰形式和带有连带物的形式能不同程度的沉默EIF4H剪接亚型的表达，均属于该发明范围。实施例中使用SEQ ID NO 6与SEQ ID NO 28作为代表。
Figure BDA0001911881150000261
EIF4H短剪接亚型siRNAs
Figure BDA0001911881150000262
3、针对EIF4H长剪接亚型的寡聚核苷酸序列
ASO通常为一段RNA或DNA序列，采用特定骨架和碱基修饰，以提高其细胞或体内的稳定性。以下序列中U以T代替，相关修饰形式未作标注。基于ASO干预mRNA剪接的原理可以判断，与这些ASO序列重复超过5个碱基、长度为5-85的反义寡聚核苷酸及相关反义寡聚核苷酸的类似物(包括morpholino)、各种化学修饰形式和带有连带物的形式能不同程度抑制EIF4H pre-mRNA中长剪接亚型特异的外显子剪接，均属于该发明范围。实施例中使用SEQID NO 111，硫代磷酸骨架，2’-甲氧基修饰。
ASO-3’ss-1(SEQ ID NO 34)
AGGTCGTGTCATTACCTTGCTG
ASO-3’ss-2(SEQ ID NO 35)
GAGGTCGTGTCATTACCTTGCT
ASO-3’ss-3(SEQ ID NO 36)
TGAGGTCGTGTCATTACCTTGC
ASO-3’ss-4(SEQ ID NO 37)
TTGAGGTCGTGTCATTACCTTG
ASO-3’ss-5(SEQ ID NO 38)
GTTGAGGTCGTGTCATTACCTT
ASO-3’ss-6(SEQ ID NO 39)
AGTTGAGGTCGTGTCATTACCT
ASO-3’ss-7(SEQ ID NO 40)
AAGTTGAGGTCGTGTCATTACC
ASO-3’ss-8(SEQ ID NO 41)
AAAGTTGAGGTCGTGTCATTAC
ASO-3’ss-9(SEQ ID NO 42)
TAAAGTTGAGGTCGTGTCATTA
ASO-3’ss-10(SEQ ID NO 43)
ATAAAGTTGAGGTCGTGTCATT
ASO-3’ss-11(SEQ ID NO 44)
GATAAAGTTGAGGTCGTGTCAT
ASO-3’ss-12(SEQ ID NO 45)
AGATAAAGTTGAGGTCGTGTCA
ASO-3’ss-13(SEQ ID NO 46)
AAGATAAAGTTGAGGTCGTGTC
ASO-3’ss-14(SEQ ID NO 47)
AAAGATAAAGTTGAGGTCGTGT
ASO-3’ss-15(SEQ ID NO 48)
AAAAGATAAAGTTGAGGTCGTG
ASO-3’ss-16(SEQ ID NO 49)
AAAAAGATAAAGTTGAGGTCGT
ASO-3’ss-17(SEQ ID NO 50)
AAAAAAGATAAAGTTGAGGTCG
ASO-3’ss-18(SEQ ID NO 51)
CAAAAAAGATAAAGTTGAGGTC
ASO-3’ss-19(SEQ ID NO 52)
GGATACACAAAAAAGATAAAGT
ASO-3’ss-20(SEQ ID NO 53)
CTGAGGATACACAAAAAAGATA
ASO-3’ss-21(SEQ ID NO 54)
CCTGAGGATACACAAAAAAGAT
ASO-3’ss-22(SEQ ID NO 55)
TCCTGAGGATACACAAAAAAGA
ASO-3’ss-23(SEQ ID NO 56)
TTCCTGAGGATACACAAAAAAG
ASO-3’ss-24(SEQ ID NO 57)
ATTCCTGAGGATACACAAAAAA
ASO-3’ss-25(SEQ ID NO 58)
CATTCCTGAGGATACACAAAAA
ASO-3’ss-26(SEQ ID NO 59)
CCATTCCTGAGGATACACAAAA
ASO-3’ss-27(SEQ ID NO 60)
CCCATTCCTGAGGATACACAAA
ASO-3’ss-28(SEQ ID NO 61)
ACCCATTCCTGAGGATACACAA
ASO-3’ss-29(SEQ ID NO 62)
TACCCATTCCTGAGGATACACA
ASO-3’ss-30(SEQ ID NO 63)
CTACCCATTCCTGAGGATACAC
ASO-3’ss-31(SEQ ID NO 64)
GCTACCCATTCCTGAGGATACA
ASO-3’ss-32(SEQ ID NO 65)
AGCTACCCATTCCTGAGGATAC
ASO-3’ss-33(SEQ ID NO 66)
GAGCTACCCATTCCTGAGGATA
ASO-3’ss-34(SEQ ID NO 67)
AGAGCTACCCATTCCTGAGGAT
ASO-3’ss-35(SEQ ID NO 68)
GAGAGCTACCCATTCCTGAGGA
ASO-3’ss-36(SEQ ID NO 69)
CGAGAGCTACCCATTCCTGAGG
ASO-3’ss-37(SEQ ID NO 70)
TCGAGAGCTACCCATTCCTGAG
ASO-3’ss-38(SEQ ID NO 71)
CTCGAGAGCTACCCATTCCTGA
ASO-3’ss-39(SEQ ID NO 72)
TCTCGAGAGCTACCCATTCCTG
ASO-3’ss-40(SEQ ID NO 73)
TTCTCGAGAGCTACCCATTCCT
ASO-3’ss-41(SEQ ID NO 74)
ATTCTCGAGAGCTACCCATTCC
ASO-3’ss-42(SEQ ID NO 75)
GATTCTCGAGAGCTACCCATTC
ASO-3’ss-43(SEQ ID NO 76)
AGATTCTCGAGAGCTACCCATT
ASO-3’ss-44(SEQ ID NO 77)
TAGATTCTCGAGAGCTACCCAT
ASO-3’ss-45(SEQ ID NO 78)
CTAGATTCTCGAGAGCTACCCA
ASO-3’ss-46(SEQ ID NO 79)
TCTAGATTCTCGAGAGCTACCC
ASO-3’ss-47(SEQ ID NO 80)
CTCTAGATTCTCGAGAGCTACC
ASO-3’ss-48(SEQ ID NO 81)
CCTCTAGATTCTCGAGAGCTAC
ASO-3’ss-49(SEQ ID NO 82)
ACCTCTAGATTCTCGAGAGCTA
ASO-3’ss-50(SEQ ID NO 83)
CACCTCTAGATTCTCGAGAGCT
ASO-3’ss-51(SEQ ID NO 84)
CCACCTCTAGATTCTCGAGAGC
ASO-3’ss-52(SEQ ID NO 85)
TCCACCTCTAGATTCTCGAGAG
ASO-3’ss-53(SEQ ID NO 86)
ATCCACCTCTAGATTCTCGAGA
ASO-3’ss-54(SEQ ID NO 87)
CATCCACCTCTAGATTCTCGAG
ASO-3’ss-55(SEQ ID NO 88)
CCATCCACCTCTAGATTCTCGA
ASO-5’ss-1(SEQ ID NO 89)
ATCCCGGGAATCCCATCCACCT
ASO-5’ss-2(SEQ ID NO 90)
CATCCCGGGAATCCCATCCACC
ASO-5’ss-3(SEQ ID NO 91)
TCATCCCGGGAATCCCATCCAC
ASO-5’ss-4(SEQ ID NO 92)
GTCATCCCGGGAATCCCATCCA
ASO-5’ss-5(SEQ ID NO 93)
AGTCATCCCGGGAATCCCATCC
ASO-5’ss-6(SEQ ID NO 94)
AAGTCATCCCGGGAATCCCATC
ASO-5’ss-7(SEQ ID NO 95)
GAAGTCATCCCGGGAATCCCAT
ASO-5’ss-8(SEQ ID NO 96)
TGAAGTCATCCCGGGAATCCCA
ASO-5’ss-9(SEQ ID NO 97)
TTGAAGTCATCCCGGGAATCCC
ASO-5’ss-10(SEQ ID NO 98)
ATTGAAGTCATCCCGGGAATCC
ASO-5’ss-11(SEQ ID NO 99)
AATTGAAGTCATCCCGGGAATC
ASO-5’ss-12(SEQ ID NO 100)
GAATTGAAGTCATCCCGGGAAT
ASO-5’ss-13(SEQ ID NO 101)
AGAATTGAAGTCATCCCGGGAA
ASO-5’ss-14(SEQ ID NO 102)
CAGAATTGAAGTCATCCCGGGA
ASO-5’ss-15(SEQ ID NO 103)
CCAGAATTGAAGTCATCCCGGG
ASO-5’ss-16(SEQ ID NO 104)
ACCAGAATTGAAGTCATCCCGG
ASO-5’ss-17(SEQ ID NO 105)
TACCAGAATTGAAGTCATCCCG
ASO-5’ss-18(SEQ ID NO 106)
ATACCAGAATTGAAGTCATCCC
ASO-5’ss-19(SEQ ID NO 107)
GATACCAGAATTGAAGTCATCC
ASO-5’ss-20(SEQ ID NO 108)
TGATACCAGAATTGAAGTCATC
ASO-5’ss-21(SEQ ID NO 109)
CTGATACCAGAATTGAAGTCAT
ASO-5’ss-22(SEQ ID NO 110)
ACTGATACCAGAATTGAAGTCA
ASO-5’ss-23(SEQ ID NO 111)
TACTGATACCAGAATTGAAGTC
ASO-5’ss-24(SEQ ID NO 112)
ATACTGATACCAGAATTGAAGT
ASO-5’ss-25(SEQ ID NO 113)
AATACTGATACCAGAATTGAAG
ASO-5’ss-26(SEQ ID NO 114)
AAATACTGATACCAGAATTGAA
ASO-5’ss-27(SEQ ID NO 115)
TAAATACTGATACCAGAATTGA
ASO-5’ss-28(SEQ ID NO 116)
TTAAATACTGATACCAGAATTG
ASO-5’ss-29(SEQ ID NO 117)
TTTAAATACTGATACCAGAATT
ASO-5’ss-30(SEQ ID NO 118)
CTTTAAATACTGATACCAGAAT
ASO-5’ss-31(SEQ ID NO 119)
ACTTTAAATACTGATACCAGAA
ASO-5’ss-32(SEQ ID NO 120)
TACTTTAAATACTGATACCAGA
ASO-5’ss-33(SEQ ID NO 121)
ATACTTTAAATACTGATACCAG
ASO-5’ss-34(SEQ ID NO 122)
GATACTTTAAATACTGATACCA
ASO-5’ss-35(SEQ ID NO 123)
TGATACTTTAAATACTGATACC
ASO-5’ss-36(SEQ ID NO 124)
GTGATACTTTAAATACTGATAC
ASO-5’ss-37(SEQ ID NO 125)
GGTGATACTTTAAATACTGATA
ASO-5’ss-38(SEQ ID NO 126)
TGGTGATACTTTAAATACTGAT
ASO-5’ss-39(SEQ ID NO 127)
GTGGTGATACTTTAAATACTGA
ASO-5’ss-40(SEQ ID NO 128)
AGTGGTGATACTTTAAATACTG
ASO-5’ss-41(SEQ ID NO 129)
AAGTGGTGATACTTTAAATACT
ASO-5’ss-42(SEQ ID NO 130)
TAAGTGGTGATACTTTAAATAC
ASO-5’ss-43(SEQ ID NO 131)
TTAAGTGGTGATACTTTAAATA
ASO-5’ss-44(SEQ ID NO 132)
ATTAAGTGGTGATACTTTAAAT
ASO-5’ss-45(SEQ ID NO 133)
AATTAAGTGGTGATACTTTAAA
ASO-5’ss-46(SEQ ID NO 134)
AAATTAAGTGGTGATACTTTAA
ASO-5’ss-47(SEQ ID NO 135)
AAAATTAAGTGGTGATACTTTA
ASO-5’ss-48(SEQ ID NO 136)
AAAAATTAAGTGGTGATACTTT
ASO-5’ss-49(SEQ ID NO 137)
GAAAAATTAAGTGGTGATACTT
ASO-5’ss-50(SEQ ID NO 138)
GGAAAAATTAAGTGGTGATACT
ASO-5’ss-51(SEQ ID NO 139)
AGGAAAAATTAAGTGGTGATAC
ASO-5’ss-52(SEQ ID NO 140)
TAGGAAAAATTAAGTGGTGATA
ASO-5’ss-53(SEQ ID NO 141)
CTAGGAAAAATTAAGTGGTGAT
ASO-5’ss-54(SEQ ID NO 142)
CCTAGGAAAAATTAAGTGGTGA
ASO-5’ss-55(SEQ ID NO 143)
TCCTAGGAAAAATTAAGTGGTG
ASO-5’ss-56(SEQ ID NO 144)
CTCCTAGGAAAAATTAAGTGGT
ASO-5’ss-57(SEQ ID NO 145)
GCTCCTAGGAAAAATTAAGTGG
ASO-5’ss-58(SEQ ID NO 146)
GGCTCCTAGGAAAAATTAAGTG
ASO-5’ss-59(SEQ ID NO 147)
AGGCTCCTAGGAAAAATTAAGT
ASO-5’ss-60(SEQ ID NO 148)
AAGGCTCCTAGGAAAAATTAAG
ASO-5’ss-61(SEQ ID NO 149)
CAAGGCTCCTAGGAAAAATTAA
ASO-5’ss-62(SEQ ID NO 150)
GCAAGGCTCCTAGGAAAAATTA
ASO-5’ss-63(SEQ ID NO 151)
AGCAAGGCTCCTAGGAAAAATT
ASO-5’ss-64(SEQ ID NO 152)
CAGCAAGGCTCCTAGGAAAAAT
序列表
<110> 复旦大学
<120> 干预EIF4H剪接亚型的寡聚核苷酸及其应用
<130> 20181203
<160> 152
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> Artificial
<400> 1
tcacttcgtg tggacattgc 20
<210> 2
<211> 20
<212> DNA
<213> Artificial
<400> 2
cctgccccct aagaagtcat 20
<210> 3
<211> 22724
<212> DNA
<213> Artificial
<400> 3
gacggcaaat ggcggacttc gacacctacg acgatcgggc ctacagcagc ttcggcggcg 60
gcagagggtg aggcgggcgt gcgcgggccc cgtcgggggc tgcgggaccg gcggagtcgg 120
ggccgtcagg gtggcggccg tcctgcgctc agccggggcg gcgggaggcg ggggcccgcg 180
gaggcctagg agcggcgcct ggagggccgg ggcctggaaa tggcgcgctc ggagacgggg 240
cggcggcggc tgggctcccg gggtcttgct tgcggggcgg atcccttcca ctgcctcccg 300
ccaacgacaa cgtgttggtt tttgccctgc cttggccgcg gcggcgccga tgcccggaga 360
cgagcgcccg cttccagccc catcccccct acggtaggac cagtgcgggg ctgagctgtg 420
gcgctggggt acgggccaca cggcctgccc cgctccggtc ccggcctctt tcgcggccct 480
ctgttgcctg cccgccttgc aggcccctgg ccccttcgcc acgaggccgg ccgactgggg 540
aaggtcggca gccaagaggg gcgattccct tccccttacg tgtgaggcag ggatttggcg 600
gctctggccc ggggatggta cgactccttg cgggcggggg gagaccaagg agataaatcc 660
atcgtagctt ctttgtatta atctttcttg ggtcacagtg ccttgacagg tgctacgtgc 720
tctttggcac aatttcattt ctccttggtt tctcgctctg atctggagaa ggaaccgcat 780
ttagcgtcgt cctgattgtt gtcaaatgca gacaactatg tataattgaa acactttgct 840
taaaagaaac atctttgaat agagagctca tggtgtttaa ttccaggact ctgcactttt 900
ttttatttta ttttttaaat acaagttgcc tcgttagaat ataggctatg aacatcttgc 960
tgtttgtcaa ggacacactt gggctttgtg ttttcttgca gaaaggatta ctttcccagg 1020
ataacaccac aataatacac actgaggtgt tcagtctcct gggttgcagt ccaaaagtat 1080
ctttgtttgg taaagaatga tcacttttaa taccttggtt tgggaacggc tcagacaaaa 1140
gcttgtttcg gggcaggagt gtagctaatg ttcatgatac tgtaacgtgg aagtgtgccg 1200
gtgtaggtgg agctattgtc attccaaaga acattttatt tgctaataaa aaaagaagta 1260
aatgttggat gacatgaaat tatcgccttt gggttaggtt tctaaacatc aaatttgttc 1320
tcaagtcatt tcgaaattta ataactaggg tttttttttt tttcctagtt ccaaaaaacg 1380
ttcacagaag tacccagaaa aattcttctt aaacagcgtg gtcaatttaa gttttagttg 1440
cattgggact acaataaggt gaccttatta aactgagtgt ggggatttaa aaaaatttcc 1500
ttcagacaca tttgccagct cttctggtgt caaattttga catttggggg tgccaggaac 1560
aatcagaagt aaaattctat taagatgcta ttaatttcaa atctccacag cagatgaaaa 1620
tattagccat ttgccccagg tgtactccaa aaactgatag gagagtttcc taggtagcac 1680
tgtgcatcac ttgaacttcc taatagaaag tctctttact gttaagcatc ttctatgtct 1740
cacttacttg gaattttcta tttggcttca tattaatgtg aaactacata aagtattgct 1800
actttcagaa gtagttttaa aaacttgttg gtgaatttac atattcattt atacatgtgt 1860
attctttttt tttttttttg agatggagtt tcactcttgt tgcccaagct ggagtgcaat 1920
ggtgtgatct cggctcactg caacctccac ctcccgggtt taagcgattc tcctgcctca 1980
gcctcccaaa gtgctgggat tacaggcatt agccaccgcg cccggcctca attattttaa 2040
ttatgtccac atgaacaaat gtcactgctt tacaatgctt aagcatcata gaagattgcc 2100
acctatggaa agcagtttta acactgcatt gtttggtact tgtgtaggca ccttctccct 2160
tgtctaaaat gcctcagaca ggctcgtaca tctcacctct gaattacttg ggtcacaggt 2220
gaaagaaatg gtaggcctaa aatgaacagt tatcatcata aaacatagtt tggtagttat 2280
acatcttcct aagtttattt cgtagttgtg tttaataacc agtgcagttg tcaggaaaca 2340
gtattgaacc actgcttttt tcccccgagc ctgtaagggc atgtccccag cagccaggga 2400
gagaaccagg gactgcattg ttcagcaagc aaatcaaaac aaaattccaa atgatcaaga 2460
gttgattgta ccagatcttg aatggacttt gttttgcagt ttgctaaatt atctttttga 2520
ggttccagtt tctcatctac cattggcagt aaagtgcaga gtaatactga ttttatattt 2580
ctaattgcaa caaagctatg agtgcttagt ggaaaaccac cgatcctgag aattagggtg 2640
gaaattggag gtggcaagaa ttgaggtggc tgaaatggtg ccatctaaaa actggcaaaa 2700
ctccaaattt attctttatc aggattcagt tagggagttt agaattcttt tttaaataga 2760
gatgggtttc actatgttgc ccaggctggt cttgaactct tggactcaag tgatcctccc 2820
gcctcagcct cccaaagtgt tgggattaca ggcatgagct accacgccca gcctaggagt 2880
ttagaattct aacctttgaa tctttaaact tagcatgtct gtctaaattt ctgagcatat 2940
ttttatccaa aatttttatg gtggtaaaat atacataaca taaaatgtgc cactttaccc 3000
atttttcagt atacatttca gggatattga gtgtattcgt aatgtataac catcaccact 3060
atccagctcc ttaactctta cagcttgtaa aactaaaacc ctgtcccctt tgaatagtaa 3120
ttccctattg ccctccttcc cccaggtccc tggcaaccac cattctactt cctgtctctg 3180
agcatatttt aagatttgta tgtaatagga aggaaagcag tgtataaaaa ctacagctag 3240
ttttcactga tgctagggcc tgtaaagcta ttgaaaagct tattttaaaa tgctgtttac 3300
atgtcaaatg tcaagtcgtt taggtttgtt atccttcttc actccaagtg ttccattgct 3360
tagttccaaa ccattatcct ctatgaaaag tgttaccaga aatatttgga tttgctaaga 3420
ggtctctgca tgacggttca tgtaaaagcc ctggctttgg aataaggcag accagagttt 3480
catcagttca gccatgtgtt agttgtgtga tctgaagtaa gtttcttcac ctctctgagc 3540
ctcgttttcc ccacatatga gttagaaaca tgtatttatt tatgaaatag gctctccttc 3600
tgtcacccag gctggagtgc agtggcagga tcatagctca gtgcagcctc gacttcccag 3660
gctcaagcca tcctcccacc tcagcctccc aagtagctgg gattacaggg tgcgccacca 3720
tgcccagcta atttttgtat ttttttgtag agacagagtg tctctatgtt gcccaggcta 3780
gtctcaaacc cctgggctca agggatcttc ctgcctcagc ctcccaaggc tcattgtgtg 3840
agccacagta cccagccttc aatttatgtt tctattagaa attaagcttt gtggctgggt 3900
gcagtggttc atgcctgtta tcccagcact ttggaaggcc gaagcaggcg gatcacttgt 3960
ggccagaagt ttgagaccac cctgaccaac gtggcaaaac cccatgtcta ctaaaaatac 4020
aaaacgggtg tggcggcaca tacctgtaat cctagctact tgggaggctg aggcaggaga 4080
attgcttgaa ccagggaggc ggaggttgca ttgagctgag atcatgccac tgcactcctc 4140
cagcctgggc gacagagaga gactgtctcc aaaaaaaaaa aaagaaaaat taagcttagt 4200
gttctggaaa gacttcacaa ttttaaggag tctccctgga agcttagaac gtacaaagca 4260
gactctgaat gtctttccag gatcacactc tggcttccag cttcccctgg aaggttcagg 4320
agagtatttg tcgcctggtt gcttttacat catgctgatt agacctgcac acccgggcat 4380
gaaatgagtg tgattgcatc attttcatag aacctaatgg cagctgtatg tacgtctaag 4440
acttcttttg tgtaagtcga atgttcttcg aagtttcccc acgtcatgat ttatgtgatt 4500
ttccgtcttg atcaggacct tctgaaggag ctcaagtatg tggctgctcc acggttgatg 4560
agtctctggt taagcatggc cttccagttc agggctaggg gggccgtcac ctccctttgt 4620
tctgcacatt cttctcctgt ctttcatgta tactcacatt tttggagcct tctgtgtact 4680
cacatttttg gagccttcta aatgtctggc ttaatagaac acagctggat tctcatacct 4740
gcatctgtct ccactcttgg aatgtgttgc tttggttgac ttttatggag cagctcaatc 4800
cttgcacaga taaatagttg gaaatgggag gagtaatcaa ataattctgg atattcatat 4860
ttgattctat accaaaactc aataagcagt agtttctatg aaggttagtt gcaatgtgaa 4920
acctaaaacc ttatcaataa actcttcata cttttacatt agagttcatc atttggctgg 4980
gcgcggtggc tcactcctgt aaccccagga ctttgggagg ccaaggcggg cggattacct 5040
gaggtcagta gtctgagacc agcctggcta acatggcaaa gccccatctc tactaaaaac 5100
acaaaaacta gctgggcatg gtggtgggcc cctgtagtcc cagctacttg ggaggctgaa 5160
gccatagaat cacttgaacc tgggaggtgg agtttgcagt gagcccagat tgcgccactg 5220
cactcccgcc tgggcgacag ggtgagactc tgtatcaaaa aaaaaaaaaa aaagtcactt 5280
tatcttgtac tccgactgga tcctttacca gtgcatacat ttgtaaggtg ttggtgattt 5340
ggaaaggacc tgttcactga gccaggcaga gccatgcaga tgttccagat gttggtacat 5400
tttgttatac agtatcaaag tcacattcac catagctgtt agaaaagtct ctgagtattg 5460
aaagactgtc aggcttatgt tgatggatac aggttttccg aaactctttt ttgcttgaaa 5520
gcttaaattt taccattggc aacaaatact gttattttgg ccaggtgctg tggctcatgt 5580
ctgtaatccc aacactttgg gaggccatgg caggaggatc actctagccc agtctggaca 5640
atatgtccag acctggtctt ttcaaaaata aaattagctg gtgtggtggt atatgcctat 5700
agtcccacct acttgggagg ctgaggctag aggatcactt gagtcctgga gattgaggct 5760
gcagtgagca gtgattgcac cacagcactc aaatctgggc aacagagcaa gaccctgtct 5820
caaaacaaaa aacccaaata ctgttctttt acttcaagga acagacttaa cttcattttt 5880
aaaaaactta gtacccatgt ctgaattaac catagtttgt tctatagaaa gaacagctag 5940
ttcagcttgt agctcaagtg cttttcctgc agcagccatc atgcttgtgt atgcagcaga 6000
agtgctttgc gtgtttcata acatagggta ctaaaaagtg ggtcacgatt catttttact 6060
gctttaccaa ggatattcct aattgaagct gtgatttttc ttcctatttt aaatacttca 6120
agtgcatggt gggaaagcat gagtaatgta gtttgatgcc cctgccttcg tttgtattaa 6180
cacaaagacc tgttgtgaga tcagtatagg tctttgcttt ataaatgtgg aatattgtga 6240
accattctgg gaaccatatt ttaaggcatc agcacactgg agcctatgaa gaagaaggtg 6300
atagtgggga tggccaggct ctgaaacttc atcttaggag gaagggctga gcagcttcag 6360
tgaggacctt cgaaagaagc ctcagcagca gtgatacagc agctcttaag tctctgggac 6420
tgctgagtgg aagaaaggtg tggccctcca ggccagaata ggaccaaagg attacatctt 6480
ggccttcttc agagactgca caaatgtaaa gaactgctgc ttgagctaaa taattgtttt 6540
tttaatagag acagggtctc cctttgttgc ctaagctggt cttgaactag cctcaaggga 6600
tactcctgcc ttgacctcct aaagtgctgg gattgcaagc atgagccacc acgcccagcc 6660
tcaaccattt ttaagtgtac aatttagtgg catggagtac cttcatagtg ttgtgtaacc 6720
attgccacta tctagttcca gaaccttttc ataatccaaa cagaaactct atacccacta 6780
aacaactcca ttcctttccc agttttaacc tcataaacat tttggctatt tctgttacat 6840
ctatcttccc tacttttggg tggaggtgtg ggctaggata atttaaggct aatcctagtc 6900
acctatcctt ctaaaatagt tcttattttt ttaaatttta atcctcatct ttgtgttggt 6960
aaatattaca tactttagag caatttttgt ttcaattttt atggctaccg tagtagatgt 7020
atcttatata ttctttcttt gttttttttg ttttttgttt tgttttgttt tctgaggcag 7080
agttttactc ttgttgccca ggctggaatg cagtggcact atctcggctc accacaacct 7140
ccgcctcctg ggttcaagcg attctcctgc ctcagcctcc cgagtagctg ggattacagg 7200
catgtgccac cacacctggc aaattttgta tttttagcag agatagagca gaaacagagg 7260
gtttctacat gttggtcagg ctggtctcga actcccgacc tcaggtaatc tgcctgcctc 7320
ggcctcccaa agtgctggga ttacaggtgt gagccaccga gcccggcctt tttttttgag 7380
atggagtctc actctgcctc ccaggctaga gctcagtggc gcgatctcag ctcactgcag 7440
cctccgccac cacccctcag cctcccgagt agctgggatt acaggcacct gccactgtgc 7500
ccagctgatt tttgtactgt tagtagagat ggggtttcac catgttggcc gggttggtct 7560
tgaactcctg accttgtgat tcatccgctt cagcctccca aagtgctggg attataggtg 7620
tgagccacca cacccggcct cttacatact tttcatactt gagtatgttt ctaacagata 7680
agggcttctt ttttttcttt aaccaacagt ttttccagta cccagtgttc agtttttccc 7740
agtttttgca aagatgactt tgtatagttg gattgtttga atatgcaaaa cattacagaa 7800
aaatgtagaa aatttagaac acgaaccaaa gcataatata gtgacctgta tacccattac 7860
ctactcttta agtgaccact cttctctata ctactatggt ttataatagg tagtataaat 7920
ggtatttatc aaaggcttga cctttgaaag cttaaccatt tttgatgatt gtcaaaaacc 7980
caaatgttac ctgtttgtag atatggggtt ttgccgtgtt ccccacgctg gtctcaaact 8040
cctgagctca agtgatctcc ccaccttggc ctcccgaagt gccgggattg caggtgtgag 8100
ccatggcgtc cggccagtca ttttctttgg tttacactac tttacccttc tgggccctac 8160
tttccccaaa tgagagctag aaactcctct gttgggaggt ttaaatgaga tgtgtctcat 8220
aatttggagg catagtcttg ccctgttgcc caggctggag tgcagttgca caatcacagt 8280
cccaaagtgc tgggactaca ggccggagcc acatgcccat gcccagcctt gagcactcaa 8340
ctttctgttg catctactta ctggatacat ccctaccctc ataacatcta ctctgtacat 8400
tggcttcttt ttatagttac cctctctcag tctcctttga cacctacaga tcttttctgg 8460
ctcaccctaa gggttcagta ttggccccat ctgccatctg gaagtggagt cccacttctg 8520
ttgctctctg ctgtgctgca tatcctggct cccaactgct ccaccctggt gaaactgttc 8580
tcattgctat cttccagatt ttctgcagtc ctttgcttgt gacgtgattt ctcctttcca 8640
aattatcagc tccttgaggg cagaaaccga gtcttaggtt tgtgccttct ctacattctc 8700
tccacccttc atagacatgc agatacgtgg ttgatttgag taaggaaaag tgatttgcct 8760
gtcaaggatt ttagtcacac ttggagccga aagtagggtt tcctaaatta ctgaggctgt 8820
aattgactac tttaatcttt cataatcaat agactgtccc taatgggcag tagaaaggac 8880
actgctcagt agtggcaggg agtttgcagc acagaccttg acttcaggtg tcctgaattg 8940
gcacgctctg ctgatctgtt cctgttctac acgcactcct gccagcccag tgtagcctca 9000
ggcctgttgg gtctacccag gagcatggca gaattatata gcacttggta cttaggaaat 9060
gtttttatgg tcagttttaa acttataaaa tcaagattga aatataacag acaagaaaac 9120
ataggtacag aggtaatttg gcagaggtcc tacatcaagc tagctaaggt gtcaagtgaa 9180
cctgttctcc catgtgttgt tattgatctg tgtaggaatg tactgaattt cacgaaacac 9240
agcttattta tttaggtagt agtcccccaa acttgaatct agtgaaaggt ggcagacatt 9300
ttagctcaga gttttctgac atttgatttt gtaaatgaat cggtgggtaa cagatctgtg 9360
tcatcctatg tgatgtgatg tgatgtcatc ctatgtgatg tgtactctgg ggtactcaca 9420
tcctatgtga tgtgtctcaa gtgactgtca cacagtggac actttaccag tgctagcttg 9480
aatgaggtct acctccatgg cccagccctg ctgcccccta gtgatttcat tgaatgggat 9540
gaaaaggtaa aaagtgctta agacctgtag aagggactac atcaggtagg gtatgggaat 9600
atttaggact actttggtaa ttattgaaaa gtctagagta ttaagccact ggtggttttg 9660
tgaggcacga gtgagttgaa cagtttggtg ggctttggtg tatctttgct ccagaattat 9720
atcaccagtt agctgttagg tgctgccatt tggtaacgtg ccaacttttc aaaaatggtt 9780
tgccccaaac tagatttttc tatcttctcc aaagccagag ccaccttcct gtcctagtgc 9840
atttttagca gtgccccgtc atcttactgg cattcacctg ggcttcctgt gctcaatcag 9900
gggtcaagtc tggcctgatc tcagttccac agttcctcta ctctgtaagt tcgagcacaa 9960
ttagcaagcc ttcgcacctc cattttcctt acttagcaac tacatcaaac acccgagagt 10020
ttatcatctc tcttcatcct caccctcctt tttagttttt atgtatatgt tacacattca 10080
tacacacaaa catacctgca tatatacatg tgttttatac gtcccacaca actgtgagct 10140
gcttacatac aagacagtct tttaacatca ttacgcatca ctagatgttt aatgttgagt 10200
aatgaattta aactgttttt aacattgacc ctccattttt atggttttcc acatgtttat 10260
actgtatctt gttttttgtt tgtttggttt tgttgttgtt ttttgtgctt ttttgtttgt 10320
tttttgtgtt tttttgtttt tgtttttttg agatggagtc tcgctctgta gcccaggctg 10380
aagcgcagtg acacagtctc agctcactgc aagctccacc tcagcctctg aagtagctgg 10440
gattacaagt gtgtgctacc acacctggct agtttttgta tttttagtag agacggagtt 10500
ttaccatgtt ggccaggctg gtcttgaact tctggcctca agtgatccac ccaccttgac 10560
ctcccgaagt gctgggatta caggtatgag ccactgtgcc ccgccttttt gttgttgttg 10620
ttgtttcttc agttggagac agtctccttc tgtcacccag gctggagtgc agtggtgcca 10680
tcacagttca ctgcagcaac aacctcctgg gctcaggtga tcctcccacc tcagcctccc 10740
aggtagcttg ggactacaag tgtggaccat cactcccggc taattttttg tattttttat 10800
agagatgggg tcttactgtg ttgcccaggc tggtcttgaa ctcctggctt taagtgatcc 10860
tcccgcctca gcctcccaaa ctgttgggat tacaggcatg agccactggg cccggcttta 10920
tactgtaact catatattta ttatcccgtc ctaaatagta tcttactgaa aatattttag 10980
gatttatgat ttcggagata tcagcgatag agtactattg gcaaaggtta ttgaaatgca 11040
aagtaagtca catttactag gcccgaaaag atgggagaag tgggcaggta ggatgcaatt 11100
cacttgtaaa cctattttct ggtggtctta gaacagaacg tggaacttcc tattggaaag 11160
taccggaatt gtgaattatg aaacttaaaa gctttttttt ttttaaccat caaagaatca 11220
tcacagcttg gttgaccctt ctactttaga aaaatgcttt aatgaataat atttgtctgc 11280
aagacacctg gaatttatct tcaggaagaa gtggaatttc cattgtgttt ctggcagtat 11340
gtagatgaaa gaccaggttg ctctctaaga tggatattaa tatttcccct gcaaagaggg 11400
tgactttggg caggtcttgg gttgttgtga aggatctcat ttatttgaac gcatttcccg 11460
tttagaaaaa aaaactgcag ttggctgcca gcacggtatt cttggagcaa acaggaaatg 11520
ggttaaatgt ctgtttcaaa aaaaaatcac ccccttaatt tttttttcct cctgagtttt 11580
ccaaacgttt tattttggaa attttcaaat ctacaataag gttgaaataa tggtacagtt 11640
ggatgacgct agtggatgca gaagtgacta ttctgccttg tttgctgaat ccttgctgta 11700
taccggcaca catgcccttt atgctgacct gcttgaaaac aaatgttggc catcttggca 11760
cttcacttct aacgacttta agtcgttcct ggggcattct tatggcaaat atcgtttaat 11820
ataataatct atttatacta tattctgatg tctttttttt ttttttgaga tggactctcg 11880
ctctgtcgcc caggctggag tgcaatgggc tcactacaac ctccacctcc tgggttcaag 11940
cgattctcct gcctcagcct gcccagcagc tgggattaca ggcatgcacc accacaccaa 12000
gctaattttg tatttttagt agagacaggg ttttaccaca ttggtcaggc tgatcttgaa 12060
ctcctgacct caagtgatcc acccacctcg gcctcccaaa atgctgtatt aggattaact 12120
cagttttaca aataaaataa agcttagaga agttgaataa cttctcaagg tcatatgcta 12180
gtaagtgaca gaggctagat ttaaaccaat acctatgcag agtaaacgac gcttgataca 12240
gctgaggaga tgacatcgag gttgaaatgg attggtaatg cacgccatac gcatgtattt 12300
gaggaccctg tgaagtattg cacgagagtg ttaggtacgt cagtacttgc tgtgtgacat 12360
ctaaaatttt tattaggaca ttcttatgcc cataatcagg caacaaggag aaattttttt 12420
tttttttttt tttttttttt tttttttttt tttttttttt tttttgagac ggagtctctg 12480
tcgcgtgagc tgaagtgcag tggtgcgatc ttggctcact gcagcctccg cttcccaggt 12540
tcaagcggta ggagaaataa ttctattatg tatgaatgtg ttaaacgtaa acaacttcag 12600
taatccttct tctgaataca acattacttc tagactgtcc atttctgtat ttcttagaac 12660
cacggtgtgt gaaggagggt acttgcttag gttttaggat gtaaatactg cagatacaga 12720
ttctgagtga ataggagaga agcagttgtc ttaggaaggt gtgttacgtt tgggctgggc 12780
atggtggctc acacctgtaa tcccagcact ttgggagggt gaggcaggca gaccacctga 12840
ggtcagaagt tggagaccag cctggccaac atggtgaaac cctgtttcta ctaaaaatgc 12900
acaaattagc caggtgtggt ggcacatgcc tgtaatctca gcatccttag gaggctgaga 12960
caggagaatc tcttgaaccc gggaagcaga ggttccagtg agccaagatt gtgccactgc 13020
actccagcct gggtgacaga gtgacagtct gtctcaaaaa atttttcttt tccttttttt 13080
ttgtttctcc agccattctc tgtaacagtc ttgtggtaca tcgagcagag tgcctcacct 13140
ggggcgctgg cggcgtagct cagcggaaga ccgctttact tgttttgctt attccactct 13200
gggcacactt gaatcctgtt tgtctcccct cctaggtccc gcggcagtgc tggtggccat 13260
ggttcccgta gccagaagga gttgcccaca gagcccccct acacagcata cgtaggaaat 13320
ctacctttca atacggttca gggcgacata gatgctatct ttaaggatct cagcataagg 13380
agtgtacggc tagtcagaga caaagacaca gataaattta aaggtgagtt tgggggattc 13440
ttattgtata ttactgtaaa gtgtagggga ggatgggaag gggttctaaa taaaaataga 13500
tttgtgaacc agaggctgta atcaagaaag tgttttaaac atttgcctgg tctaagtggc 13560
cgaaagaaac ataactcttc aactcatagg tcttcagttc acttagggga aaatgttttg 13620
tacacagtgt agttcataga aatacatggc atatgcccag gtttgtgagc ctgacctcta 13680
cagcagtcct gtctgccagt aaaggagttc cccaggagag agaggaatag agccgaggac 13740
tcttaggcac attcctcctg gaatgactcg tagattttcc ttggtagtta atccagtaat 13800
catatggtac tgtcatccag taatcataaa acccagaaga gaagcagcca gtgctgtcag 13860
tatgcatttg taaatttgga gagtgtgctt gggagggctg gctgagattt cagatatggt 13920
gtctataggt cgtgtgtctg cagggtgcgg gagcaaagag caggaagaag aagggatccg 13980
agcagcattc tcctgcctgc agggcttcct gaaggaagtg tgcctgctgt gtaagtgcac 14040
gatgctagga cccaggatgt ttggcccgca taatccaccc aaaagcacat ctgtgacaag 14100
tatggggtaa cttggggcgg ggagctgtgc cagcagtcga gaaggccttg ctgacctggg 14160
tggttttgtg tgtcggtggc actcttactc tttttgactt cagaatctga agccacatgt 14220
agtgtgtaat caaacagtga aaacaaatat atcagttaca cttgtgaagt aagctccagg 14280
aagcattagg aactaaactt tgggttggag tgtgtgggtg agcctagcct ttgggctggg 14340
tcagtcgctg caggctttgg agtttggctc atgaaggacc atcttcattt gagcagctct 14400
agaagatgag attgggtgtc tgttgcttct gtgaagttgg gggtagtttt ctccattatt 14460
ctcaacattg aatccactta gggaggaaac ctggggggac atccctccag ccaaggcagt 14520
atgttcttaa cacacagttg gtaaacaggg aaagtttgga ctcaggcctt tagagcactt 14580
gcggtctagt gggcttcaaa gatgaagtgt gattattaag ttattataca cagggcaagg 14640
acgctggggg agaggggatg cagggtgtag tgccagcatc tggggagtaa tgaactagat 14700
ttagactcca gacagcaagg aaggggcaat gtcagacccc tggcatcagg gacacaggtt 14760
gtcagtaaca ggtacttgga cccaggctgc ctgtctttta attaacattc agagaagaat 14820
gccctgattg ccaaggatat attttcagct ttgtgaagca aattttatgt cagttacctg 14880
agtagtaagt taaatcggca ctttaagcct ctttcttcag agcattttag gtaccaagtt 14940
tgaaatctac gaaatctaat cctcacttag attaaatagt cattcagaag tcagaagagc 15000
tctgcagatg gcctgagaaa ggtttgaata ttatggttaa aacattcaag aagaaagctt 15060
tctagaaacg cagctgctat ttcagtgcat gttcagttag cattttctga ctgataaaat 15120
agaattgaga gggaggaatg ccagtcaggg ttctatctat tgaaaggctc tcctagtctg 15180
ccatcatcta gacaacagaa tggtagttgt gacgtggaga agtagtatga ataggatctt 15240
cccacatctt taacccagaa ttacttgagg aaatcggggt cttcttttct tttccaggat 15300
tctgctatgt agaattcgat gaagtggatt cccttaagga agccttgaca tacgatggtg 15360
cagtaagtat cctcgggttt tctctgtcat agcagttgag attgttttct ttgccaatac 15420
ttacactatt ttccctttat cattagctgt tgggcgatcg gtcacttcgt gtggacattg 15480
cagaaggcag aaaacaagat aaaggtggct ttggattcag aaaaggtgga ccagatgaca 15540
gaggtgattg gttcttctaa agctgaacat cataaagatt tgcagtatgc ctaccaattc 15600
caactcgtga acgttcctag tagaactcga ttatctcata ggtgtgatag gttctcgtga 15660
gctcttaaaa gtttgcgtaa aatcctagaa agaatggctg atgcttctgc aagcctggcg 15720
ttttttgttt tctgttggaa gcaaaagctc ttaaaatgat ttcattgaac cttccatcac 15780
ttgagttgta tgtcttccaa atgaattttt ccatgtttgc gcagcaaggt aatgacacga 15840
cctcaacttt atcttttttg tgtatcctca ggaatgggta gctctcgaga atctagaggt 15900
ggatgggatt cccgggatga cttcaattct ggtatcagta tttaaagtat caccacttaa 15960
tttttcctag gagccttgct gctgttctct gtttcttacg agtagcccgc ctggccggac 16020
tacttattta gttcctttaa caaatacaac tctcctgcag ggtgcccagg catgcaagta 16080
agcctctgtt agagcctgtg ggggcaggac ttgagcccca tgcaccttgt gtggtcacct 16140
ttgttagcct gcagctcctt ggatgtgtac gggcctgggt ccttgagctc gctcagcagt 16200
ctttctcaga ctctagccca gggaaactcc tctgtggttt tgtgtatggc accaagagaa 16260
gtactgttcc ttctggaaaa cataggatag aggtttgggt tcttacatgg tgaaaggtag 16320
tcctgatggc cctttttttt cttggaggat tgtggggagg tccagtccct tgatggtgga 16380
cctgatgcac ttcgccgttc tctttcttag ccaacccagg cagagcagct ggtggtatag 16440
cacaaagcat cagtccacac gccaggcctc tgccctgcag ggtgatttta agcattttta 16500
tcatctttac tttctgtttt taaattacaa aagtataatt ttttatatat gcacatgata 16560
aaaacttcag ttgtaaccca gaaaggtagg aagagaaaaa caaatttgtt ttctttcccg 16620
caaacttggg atgtgctgtc aatgctgtgc tgtatagaac gtcagcaccc tccccccatc 16680
aaaacaaaac cggtgtaaag tagtgggtga gtatgggagg gactggtgca cgctgtcttg 16740
tgagtctgag atttttaaaa actcagcttt ggctgaacag gtgacgacta cctgttgaat 16800
aatatagttc ttaaaagtcc tgtagccagt cagaaatgag cttattcata aaagtgcagt 16860
atggtgaagt caatctgtaa ttttatgtat aagctagtct ctgattgaaa catgcagcag 16920
ctgtcttctt agagccaacc agcagctcag ggaacggcct ttcccctgcc agtttggtat 16980
tcgcaggtgt tatagaagtt gcccacatac taacgtcttt ctacccttgg cttagcttgg 17040
ccactggcgc tcaagcagag acacagtgaa cacggccatg gcctggccgc caccagttct 17100
ggtgggaact gcagtgtttt ggtgacatct caaaccctat gtgggctcac tcgatggagt 17160
aatggctaat ttgatacttt atacgttatt tgcgagagta gatctttttt actttttaat 17220
aggatggtgc tatttttttt aatgatacct gctcatgtaa aaaacaaaat agagcaacag 17280
aaaagtacca agaaagcaaa aacatttcat catcagaaat agccagtgtt aaactaagct 17340
ttgtaaagaa aactctgaag attctcatga atagtttgct tcctagaaac ccaccaacag 17400
attttttgtt gttgtttttt ttgagacgga gtcttgctct gttgcccagg ctggagtgca 17460
gtggtgcgat ctcggctcac tgcaagctct gcctcctggg ttcatgccat tctcctgcct 17520
cagcctccca agtagctggg actacaggca cccaccacca cgcccggcta attttttgta 17580
tttttagcag agacggggtt tcatcatgtt agccaggatg gtctcgatct cctgacctcg 17640
tgatccacct gcctcggcct cccaaagtgc tgggattaca ggcatgagcc accgcgcccg 17700
gcccccacca acagatttga tgagagatcc ttgttcatct gcttcaccag cgtggtgtga 17760
gctatttgtt tcacgctaga cattggtggt cgaaatcatt taatctcata gtaaagggtg 17820
taaatttact ttgtactcac gtggaaaggt acacttcaca gtttagtttg ggggaaacgt 17880
gaagttaaat gttttaatac taactaggct tgttcactga actgtgtaat gtgggtaaaa 17940
tactttgtaa aggaccttat actcgactgt atgcctccca gtgtatttcc cctaaagcag 18000
tgctcttatt ggtctaaatc tgttcagact tgctttcatt acttggtttg ttaaaattta 18060
ttatttaact cagtctctga actgggtgtg agatcccttg gtttccaaaa cccttgccac 18120
ctttaaagaa tagaaatcgg cccccaccac aggtgatgca gtcagcgctt atcattggag 18180
gtggttctgt ggagttgcta gaacactgaa tgagcccgtg cggtgctgct cctgtgtgct 18240
acagatcaca acattttcgt cagctgatca tacctaacat tgctttatgt gacttttttt 18300
tttttctttt tttttttttt gagacagagg ctcactctgt tgcccaggct ggagtgcagt 18360
ggcatgattt cggttcactg caatctccgc ctccagggtt caagcgattc tcctgcctca 18420
gcctcccaag tagctgggat tacaggcgca caccaccaca cccagctaat ttttatattt 18480
ttagtagaga cggggtctct ccgtgttggt caggctggtc tcgaactccc agcctcaggt 18540
gatctgccca cctctgcctc ccagagtgct gggattacag gcgtgagcca ccacacccag 18600
cctatgtgac cttgatttta aagacgcctc atttaatgtt agtgttgatt catttaatag 18660
tgttaacatt gaactcaggg ccagctgtag taagtagtct gtggtgccat catttcagcc 18720
tgttagaaag ctcatcccac accggttttg tctgtaagac acctgacagc cagcctgcac 18780
ttaagaacag cagacagcac ttcacactgc acctggggcc acttcagaca gcgatgtcac 18840
cagaaaaagc ccaaagggca aaacacccga cactcatcgt gaaaagactt gacagctaat 18900
aactgaaaca ggcagagcat tatcgttttc tgcctcagct gggatcgtcg catctgtgca 18960
ggtccaggac tgactggaga aatgctgctg gtatctagct tgggattaca catgaatttt 19020
agcgactggg tggatttgca aatatgggat ctgtggataa tgaggatgac tgtatagtgc 19080
atactgcggg ctctgggggt tcttctcaaa gacaagctcc ctgaaagctt ggagccttgg 19140
caccactctc gcagagagta gcagtggcac cccctgagtg cgcaaccaac agcagcaagc 19200
tcatttgttt aattatggtt taacttcttt tcgccagttt tttttttttt tccttgagac 19260
agagtctcac ttcatcaccc aggctgaagt gtaatggcac gatcttggct cactgcaccc 19320
tctaccttcc aggttcgagc gattatcctg cctcagcctc atgagtagct ggaaccccag 19380
gcgtgcgcca ccatgcccag ctaatttttg tgttttttgt agagagtgag tctcactgtg 19440
ttgcccagtc tggtcctgaa ctcctatact cacctcccac ctcagcctcc caaagtgctg 19500
ggattacagg cgtgaaccag tctttttacc aaattttaac atcagtaggt tatacagtta 19560
aagtttagct ttcactgaag tacaagcctg aaggcccctt aagaccattg agaaaatact 19620
ctttcctcct acacaccagt gggaattcag catttctaaa tgagatgcag tttacttctg 19680
ccaagagccc acttagcagg cacacaaatg aatggggaaa gaacgctttg cccccacagg 19740
aggcgccggt gggggtgagg cgctgtgctc tctgctcatg cccctctgtg tagggcatgg 19800
ccaatttcgg ataactgctt tgtttaccac acaggtgctt tgcttgtaga atggcccaca 19860
cctaaaacgc agccaaaaca ggaaaattgg gactttgtgt ttttattttg tcttcatata 19920
gcgttttctt gtgatttttg tgttctgagt tgtaaatact tctaaatttc aggtttatct 19980
tttaaaatag ctgtatagat ttctattaga aaatagagtt caacctgctt tgaggctttc 20040
ttgttttgct taagccgtat taaaatacag tttcgtggta ttaatatttt tctacttttc 20100
atgcaagctg agcatgtaaa cagctcgttt gtttgctctc ctaagatctt gattcctttt 20160
tctttgggaa gtctgttagc gctgtcatta ctccacacat tggcatttgg acccactttt 20220
aacacacttt attttggaga gacttcagat agtggacaaa ctaaagaatt taaaatgaaa 20280
ctcaaacaat tcccagaaca ttattctata agcagcttgg agacgatagt ttgaaaagta 20340
attcagtgtc ctgtttcttc ctcaggcttc agggatgact tcttaggggg caggggaggt 20400
agtcgcccag gcgaccggcg aacaggcccc cccatgggca gccgcttcag agatggccct 20460
cccctccgtg gatccaacat ggatttcaga gaacccacag aaggtacggg ctcatgtgtc 20520
agtggagggc atcttgtcct gatgggatga tcatggccgg ttcacacccc gtggggactt 20580
ggcgttctac ggcacacaga gttgtcggca tagatcccca cgttctctgg aatagcaagt 20640
tcaccttggt cattagagca tctgctgttg gggtagcagg ccaggtgaat tttagaaagt 20700
gggctggttt gctgacagca acatcagcat ggtcgttttg ctggatatgg aatgggatcc 20760
acactctgac aaactctgct ttttctcccc cagaggaaag agcacagaga ccacgactcc 20820
agcttaaacc tcgaacagtc gcgacgcccc tcaatcaagt agccaatccc aactctgcta 20880
tcttcggggg tgccaggcct agagaggaag tcgttcaaaa ggagcaagaa tgagcctgcg 20940
gttgggaggg aatggggcgt ggggggttag agcaggacca cagcctggtg agtccccggg 21000
cagccgtcct gcagccgcca ctcctgcgcc tgccattggc ctcctcacag cggaaacaca 21060
gcttgtgagt gcatgtcagc tgttaacaag tggtttttag tacattctgg gctttgctgt 21120
atctatctag tgcctgtttg tgcgtttttt tctttcttcc gctgcttccc cattttcctt 21180
ctgtcctttt tctcctgctc cttgttttcc cagcagcaca tggggttcct cggaggagca 21240
gaggtggccg ccgtgggggg gcgtttgggc tgcggtgctg cgtcattttt cctttgcttt 21300
ctctttactt tagacactgg cccaactcca ggcgtttcct ttcattccct cagtgcttct 21360
cttctgacct gcatgttgag ttctgtattg ctggggcttc caacaaaaac cagagtcact 21420
gacagaggga acagcagaga ccttgttggt attcagctgt gatggatata gagaatcaga 21480
ggcaccttgt tttcacaact aggataaaaa tatctgcagg gtcctttcca ttcctattta 21540
gagggagtcc tggctccatg accccctccc gagtggactg tccaagcaga taggctcaca 21600
cgagaaacag tgaggctgaa agggggggct atggaagagc ggtagggagt ccacggagaa 21660
gatgcagtga atgcttgcat gcattcacac gtgtgtgtgt cccagctagt tcactccttt 21720
cgccgtgcgt ggtggaggct ggcctctctg gctgggtgca gtgaatggcc agcgggtttc 21780
ttttctgctg ggccaaggcg ctttgggggt ggagggggtg gtgctggtgc tgcactgggc 21840
tgactgcggc gctgacgcag cgtttccccc catccctgtt gcctgtgtgt tgtgtggatc 21900
tgttcctagt ataggcaaca taatgagata ctgtgcttcc cacctcccct tcagttcaga 21960
gccaaaatgg gtctagaatc tggcacttta ctcatttcct ttgataaatt gtactatgca 22020
gagctgtcag gaaccttcag atagcagtag aggactgcag ctgtctaggt ctgcggccac 22080
atcttgggga cacactggac tgttcccatg tgcagggttc agcagttatg tgggagtgct 22140
aggggttagg cttttgagct tgaacgcctg cgtgtgaaca gatgaaaaat ccttcagtac 22200
ccaagtccca gtctgtccta tggggagcag tttgggggcg gccggcagca ggagcctggg 22260
aaagaggccc tcgccaggtg atggcagggc cagggtggcc tggggcaccc agcggaatgt 22320
gcttagtatt tggtcaccag ccgtcatcct gggcttttcc tactgtgtct tgttacaagg 22380
cctcagcaat ccacagaact ctctctcctt ccttccacct gtcagcttct ctgcttctga 22440
gataagaacc atttgtgtaa caccaacact taacttcaga aagacatgca ttatgtggtg 22500
taatcaaacc cgatgctttc agatgaccta cttacatctt caatgtggat aagataaaga 22560
acaaaacaca tgcatctaaa ctgctgggca atccagttga cttttaaatg taagaatgga 22620
attccaaaca cttaacacat tcagctatat gacagaaagt aaatctatgg atatggtatt 22680
ttgtgaatga tcttttaaat aaaagaaaac cttacgtaat attt 22724
<210> 4
<211> 2546
<212> DNA
<213> Artificial
<400> 4
gacggcaaat ggcggacttc gacacctacg acgatcgggc ctacagcagc ttcggcggcg 60
gcagagggtc ccgcggcagt gctggtggcc atggttcccg tagccagaag gagttgccca 120
cagagccccc ctacacagca tacgtaggaa atctaccttt caatacggtt cagggcgaca 180
tagatgctat ctttaaggat ctcagcataa ggagtgtacg gctagtcaga gacaaagaca 240
cagataaatt taaaggattc tgctatgtag aattcgatga agtggattcc cttaaggaag 300
ccttgacata cgatggtgca ctgttgggcg atcggtcact tcgtgtggac attgcagaag 360
gcagaaaaca agataaaggt ggctttggat tcagaaaagg tggaccagat gacagaggaa 420
tgggtagctc tcgagaatct agaggtggat gggattcccg ggatgacttc aattctggct 480
tcagggatga cttcttaggg ggcaggggag gtagtcgccc aggcgaccgg cgaacaggcc 540
cccccatggg cagccgcttc agagatggcc ctcccctccg tggatccaac atggatttca 600
gagaacccac agaagaggaa agagcacaga gaccacgact ccagcttaaa cctcgaacag 660
tcgcgacgcc cctcaatcaa gtagccaatc ccaactctgc tatcttcggg ggtgccaggc 720
ctagagagga agtcgttcaa aaggagcaag aatgagcctg cggttgggag ggaatggggc 780
gtggggggtt agagcaggac cacagcctgg tgagtccccg ggcagccgtc ctgcagccgc 840
cactcctgcg cctgccattg gcctcctcac agcggaaaca cagcttgtga gtgcatgtca 900
gctgttaaca agtggttttt agtacattct gggctttgct gtatctatct agtgcctgtt 960
tgtgcgtttt tttctttctt ccgctgcttc cccattttcc ttctgtcctt tttctcctgc 1020
tccttgtttt cccagcagca catggggttc ctcggaggag cagaggtggc cgccgtgggg 1080
gggcgtttgg gctgcggtgc tgcgtcattt ttcctttgct ttctctttac tttagacact 1140
ggcccaactc caggcgtttc ctttcattcc ctcagtgctt ctcttctgac ctgcatgttg 1200
agttctgtat tgctggggct tccaacaaaa accagagtca ctgacagagg gaacagcaga 1260
gaccttgttg gtattcagct gtgatggata tagagaatca gaggcacctt gttttcacaa 1320
ctaggataaa aatatctgca gggtcctttc cattcctatt tagagggagt cctggctcca 1380
tgaccccctc ccgagtggac tgtccaagca gataggctca cacgagaaac agtgaggctg 1440
aaaggggggg ctatggaaga gcggtaggga gtccacggag aagatgcagt gaatgcttgc 1500
atgcattcac acgtgtgtgt gtcccagcta gttcactcct ttcgccgtgc gtggtggagg 1560
ctggcctctc tggctgggtg cagtgaatgg ccagcgggtt tcttttctgc tgggccaagg 1620
cgctttgggg gtggaggggg tggtgctggt gctgcactgg gctgactgcg gcgctgacgc 1680
agcgtttccc cccatccctg ttgcctgtgt gttgtgtgga tctgttccta gtataggcaa 1740
cataatgaga tactgtgctt cccacctccc cttcagttca gagccaaaat gggtctagaa 1800
tctggcactt tactcatttc ctttgataaa ttgtactatg cagagctgtc aggaaccttc 1860
agatagcagt agaggactgc agctgtctag gtctgcggcc acatcttggg gacacactgg 1920
actgttccca tgtgcagggt tcagcagtta tgtgggagtg ctaggggtta ggcttttgag 1980
cttgaacgcc tgcgtgtgaa cagatgaaaa atccttcagt acccaagtcc cagtctgtcc 2040
tatggggagc agtttggggg cggccggcag caggagcctg ggaaagaggc cctcgccagg 2100
tgatggcagg gccagggtgg cctggggcac ccagcggaat gtgcttagta tttggtcacc 2160
agccgtcatc ctgggctttt cctactgtgt cttgttacaa ggcctcagca atccacagaa 2220
ctctctctcc ttccttccac ctgtcagctt ctctgcttct gagataagaa ccatttgtgt 2280
aacaccaaca cttaacttca gaaagacatg cattatgtgg tgtaatcaaa cccgatgctt 2340
tcagatgacc tacttacatc ttcaatgtgg ataagataaa gaacaaaaca catgcatcta 2400
aactgctggg caatccagtt gacttttaaa tgtaagaatg gaattccaaa cacttaacac 2460
attcagctat atgacagaaa gtaaatctat ggatatggta ttttgtgaat gatcttttaa 2520
ataaaagaaa accttacgta atattt 2546
<210> 5
<211> 2486
<212> DNA
<213> Artificial
<400> 5
gacggcaaat ggcggacttc gacacctacg acgatcgggc ctacagcagc ttcggcggcg 60
gcagagggtc ccgcggcagt gctggtggcc atggttcccg tagccagaag gagttgccca 120
cagagccccc ctacacagca tacgtaggaa atctaccttt caatacggtt cagggcgaca 180
tagatgctat ctttaaggat ctcagcataa ggagtgtacg gctagtcaga gacaaagaca 240
cagataaatt taaaggattc tgctatgtag aattcgatga agtggattcc cttaaggaag 300
ccttgacata cgatggtgca ctgttgggcg atcggtcact tcgtgtggac attgcagaag 360
gcagaaaaca agataaaggt ggctttggat tcagaaaagg tggaccagat gacagaggct 420
tcagggatga cttcttaggg ggcaggggag gtagtcgccc aggcgaccgg cgaacaggcc 480
cccccatggg cagccgcttc agagatggcc ctcccctccg tggatccaac atggatttca 540
gagaacccac agaagaggaa agagcacaga gaccacgact ccagcttaaa cctcgaacag 600
tcgcgacgcc cctcaatcaa gtagccaatc ccaactctgc tatcttcggg ggtgccaggc 660
ctagagagga agtcgttcaa aaggagcaag aatgagcctg cggttgggag ggaatggggc 720
gtggggggtt agagcaggac cacagcctgg tgagtccccg ggcagccgtc ctgcagccgc 780
cactcctgcg cctgccattg gcctcctcac agcggaaaca cagcttgtga gtgcatgtca 840
gctgttaaca agtggttttt agtacattct gggctttgct gtatctatct agtgcctgtt 900
tgtgcgtttt tttctttctt ccgctgcttc cccattttcc ttctgtcctt tttctcctgc 960
tccttgtttt cccagcagca catggggttc ctcggaggag cagaggtggc cgccgtgggg 1020
gggcgtttgg gctgcggtgc tgcgtcattt ttcctttgct ttctctttac tttagacact 1080
ggcccaactc caggcgtttc ctttcattcc ctcagtgctt ctcttctgac ctgcatgttg 1140
agttctgtat tgctggggct tccaacaaaa accagagtca ctgacagagg gaacagcaga 1200
gaccttgttg gtattcagct gtgatggata tagagaatca gaggcacctt gttttcacaa 1260
ctaggataaa aatatctgca gggtcctttc cattcctatt tagagggagt cctggctcca 1320
tgaccccctc ccgagtggac tgtccaagca gataggctca cacgagaaac agtgaggctg 1380
aaaggggggg ctatggaaga gcggtaggga gtccacggag aagatgcagt gaatgcttgc 1440
atgcattcac acgtgtgtgt gtcccagcta gttcactcct ttcgccgtgc gtggtggagg 1500
ctggcctctc tggctgggtg cagtgaatgg ccagcgggtt tcttttctgc tgggccaagg 1560
cgctttgggg gtggaggggg tggtgctggt gctgcactgg gctgactgcg gcgctgacgc 1620
agcgtttccc cccatccctg ttgcctgtgt gttgtgtgga tctgttccta gtataggcaa 1680
cataatgaga tactgtgctt cccacctccc cttcagttca gagccaaaat gggtctagaa 1740
tctggcactt tactcatttc ctttgataaa ttgtactatg cagagctgtc aggaaccttc 1800
agatagcagt agaggactgc agctgtctag gtctgcggcc acatcttggg gacacactgg 1860
actgttccca tgtgcagggt tcagcagtta tgtgggagtg ctaggggtta ggcttttgag 1920
cttgaacgcc tgcgtgtgaa cagatgaaaa atccttcagt acccaagtcc cagtctgtcc 1980
tatggggagc agtttggggg cggccggcag caggagcctg ggaaagaggc cctcgccagg 2040
tgatggcagg gccagggtgg cctggggcac ccagcggaat gtgcttagta tttggtcacc 2100
agccgtcatc ctgggctttt cctactgtgt cttgttacaa ggcctcagca atccacagaa 2160
ctctctctcc ttccttccac ctgtcagctt ctctgcttct gagataagaa ccatttgtgt 2220
aacaccaaca cttaacttca gaaagacatg cattatgtgg tgtaatcaaa cccgatgctt 2280
tcagatgacc tacttacatc ttcaatgtgg ataagataaa gaacaaaaca catgcatcta 2340
aactgctggg caatccagtt gacttttaaa tgtaagaatg gaattccaaa cacttaacac 2400
attcagctat atgacagaaa gtaaatctat ggatatggta ttttgtgaat gatcttttaa 2460
ataaaagaaa accttacgta atattt 2486
<210> 6
<211> 19
<212> RNA
<213> Artificial
<400> 6
uggguagcuc ucgagaauc 19
<210> 7
<211> 19
<212> RNA
<213> Artificial
<400> 7
gauucucgag agcuaccca 19
<210> 8
<211> 19
<212> RNA
<213> Artificial
<400> 8
ggguagcucu cgagaaucu 19
<210> 9
<211> 19
<212> RNA
<213> Artificial
<400> 9
agauucucga gagcuaccc 19
<210> 10
<211> 19
<212> RNA
<213> Artificial
<400> 10
gguagcucuc gagaaucua 19
<210> 11
<211> 19
<212> RNA
<213> Artificial
<400> 11
uagauucucg agagcuacc 19
<210> 12
<211> 19
<212> RNA
<213> Artificial
<400> 12
guagcucucg agaaucuag 19
<210> 13
<211> 19
<212> RNA
<213> Artificial
<400> 13
cuagauucuc gagagcuac 19
<210> 14
<211> 19
<212> RNA
<213> Artificial
<400> 14
gcucucgaga aucuagagg 19
<210> 15
<211> 19
<212> RNA
<213> Artificial
<400> 15
ccucuagauu cucgagagc 19
<210> 16
<211> 19
<212> RNA
<213> Artificial
<400> 16
cgagaaucua gagguggau 19
<210> 17
<211> 19
<212> RNA
<213> Artificial
<400> 17
auccaccucu agauucucg 19
<210> 18
<211> 19
<212> RNA
<213> Artificial
<400> 18
gagaaucuag agguggaug 19
<210> 19
<211> 19
<212> RNA
<213> Artificial
<400> 19
cauccaccuc uagauucuc 19
<210> 20
<211> 19
<212> RNA
<213> Artificial
<400> 20
ucuagaggug gaugggauu 19
<210> 21
<211> 19
<212> RNA
<213> Artificial
<400> 21
aaucccaucc accucuaga 19
<210> 22
<211> 19
<212> RNA
<213> Artificial
<400> 22
uggaugggau ucccgggau 19
<210> 23
<211> 19
<212> RNA
<213> Artificial
<400> 23
aucccgggaa ucccaucca 19
<210> 24
<211> 19
<212> RNA
<213> Artificial
<400> 24
gaugacuuca auucuggcu 19
<210> 25
<211> 19
<212> RNA
<213> Artificial
<400> 25
agccagaauu gaagucauc 19
<210> 26
<211> 19
<212> RNA
<213> Artificial
<400> 26
cagaugacag aggcuucag 19
<210> 27
<211> 19
<212> RNA
<213> Artificial
<400> 27
cugaagccuc ugucaucug 19
<210> 28
<211> 19
<212> RNA
<213> Artificial
<400> 28
augacagagg cuucaggga 19
<210> 29
<211> 19
<212> RNA
<213> Artificial
<400> 29
ucccugaagc cucugucau 19
<210> 30
<211> 19
<212> RNA
<213> Artificial
<400> 30
ugacagaggc uucagggau 19
<210> 31
<211> 19
<212> RNA
<213> Artificial
<400> 31
aucccugaag ccucuguca 19
<210> 32
<211> 19
<212> RNA
<213> Artificial
<400> 32
gacagaggcu ucagggaug 19
<210> 33
<211> 19
<212> RNA
<213> Artificial
<400> 33
caucccugaa gccucuguc 19
<210> 34
<211> 22
<212> DNA
<213> Artificial
<400> 34
aggtcgtgtc attaccttgc tg 22
<210> 35
<211> 22
<212> DNA
<213> Artificial
<400> 35
gaggtcgtgt cattaccttg ct 22
<210> 36
<211> 22
<212> DNA
<213> Artificial
<400> 36
tgaggtcgtg tcattacctt gc 22
<210> 37
<211> 22
<212> DNA
<213> Artificial
<400> 37
ttgaggtcgt gtcattacct tg 22
<210> 38
<211> 22
<212> DNA
<213> Artificial
<400> 38
gttgaggtcg tgtcattacc tt 22
<210> 39
<211> 22
<212> DNA
<213> Artificial
<400> 39
agttgaggtc gtgtcattac ct 22
<210> 40
<211> 22
<212> DNA
<213> Artificial
<400> 40
aagttgaggt cgtgtcatta cc 22
<210> 41
<211> 22
<212> DNA
<213> Artificial
<400> 41
aaagttgagg tcgtgtcatt ac 22
<210> 42
<211> 22
<212> DNA
<213> Artificial
<400> 42
taaagttgag gtcgtgtcat ta 22
<210> 43
<211> 22
<212> DNA
<213> Artificial
<400> 43
ataaagttga ggtcgtgtca tt 22
<210> 44
<211> 22
<212> DNA
<213> Artificial
<400> 44
gataaagttg aggtcgtgtc at 22
<210> 45
<211> 22
<212> DNA
<213> Artificial
<400> 45
agataaagtt gaggtcgtgt ca 22
<210> 46
<211> 22
<212> DNA
<213> Artificial
<400> 46
aagataaagt tgaggtcgtg tc 22
<210> 47
<211> 22
<212> DNA
<213> Artificial
<400> 47
aaagataaag ttgaggtcgt gt 22
<210> 48
<211> 22
<212> DNA
<213> Artificial
<400> 48
aaaagataaa gttgaggtcg tg 22
<210> 49
<211> 22
<212> DNA
<213> Artificial
<400> 49
aaaaagataa agttgaggtc gt 22
<210> 50
<211> 22
<212> DNA
<213> Artificial
<400> 50
aaaaaagata aagttgaggt cg 22
<210> 51
<211> 22
<212> DNA
<213> Artificial
<400> 51
caaaaaagat aaagttgagg tc 22
<210> 52
<211> 22
<212> DNA
<213> Artificial
<400> 52
ggatacacaa aaaagataaa gt 22
<210> 53
<211> 22
<212> DNA
<213> Artificial
<400> 53
ctgaggatac acaaaaaaga ta 22
<210> 54
<211> 22
<212> DNA
<213> Artificial
<400> 54
cctgaggata cacaaaaaag at 22
<210> 55
<211> 22
<212> DNA
<213> Artificial
<400> 55
tcctgaggat acacaaaaaa ga 22
<210> 56
<211> 22
<212> DNA
<213> Artificial
<400> 56
ttcctgagga tacacaaaaa ag 22
<210> 57
<211> 22
<212> DNA
<213> Artificial
<400> 57
attcctgagg atacacaaaa aa 22
<210> 58
<211> 22
<212> DNA
<213> Artificial
<400> 58
cattcctgag gatacacaaa aa 22
<210> 59
<211> 22
<212> DNA
<213> Artificial
<400> 59
ccattcctga ggatacacaa aa 22
<210> 60
<211> 22
<212> DNA
<213> Artificial
<400> 60
cccattcctg aggatacaca aa 22
<210> 61
<211> 22
<212> DNA
<213> Artificial
<400> 61
acccattcct gaggatacac aa 22
<210> 62
<211> 22
<212> DNA
<213> Artificial
<400> 62
tacccattcc tgaggataca ca 22
<210> 63
<211> 22
<212> DNA
<213> Artificial
<400> 63
ctacccattc ctgaggatac ac 22
<210> 64
<211> 22
<212> DNA
<213> Artificial
<400> 64
gctacccatt cctgaggata ca 22
<210> 65
<211> 22
<212> DNA
<213> Artificial
<400> 65
agctacccat tcctgaggat ac 22
<210> 66
<211> 22
<212> DNA
<213> Artificial
<400> 66
gagctaccca ttcctgagga ta 22
<210> 67
<211> 22
<212> DNA
<213> Artificial
<400> 67
agagctaccc attcctgagg at 22
<210> 68
<211> 22
<212> DNA
<213> Artificial
<400> 68
gagagctacc cattcctgag ga 22
<210> 69
<211> 22
<212> DNA
<213> Artificial
<400> 69
cgagagctac ccattcctga gg 22
<210> 70
<211> 22
<212> DNA
<213> Artificial
<400> 70
tcgagagcta cccattcctg ag 22
<210> 71
<211> 22
<212> DNA
<213> Artificial
<400> 71
ctcgagagct acccattcct ga 22
<210> 72
<211> 22
<212> DNA
<213> Artificial
<400> 72
tctcgagagc tacccattcc tg 22
<210> 73
<211> 22
<212> DNA
<213> Artificial
<400> 73
ttctcgagag ctacccattc ct 22
<210> 74
<211> 22
<212> DNA
<213> Artificial
<400> 74
attctcgaga gctacccatt cc 22
<210> 75
<211> 22
<212> DNA
<213> Artificial
<400> 75
gattctcgag agctacccat tc 22
<210> 76
<211> 22
<212> DNA
<213> Artificial
<400> 76
agattctcga gagctaccca tt 22
<210> 77
<211> 22
<212> DNA
<213> Artificial
<400> 77
tagattctcg agagctaccc at 22
<210> 78
<211> 22
<212> DNA
<213> Artificial
<400> 78
ctagattctc gagagctacc ca 22
<210> 79
<211> 22
<212> DNA
<213> Artificial
<400> 79
tctagattct cgagagctac cc 22
<210> 80
<211> 22
<212> DNA
<213> Artificial
<400> 80
ctctagattc tcgagagcta cc 22
<210> 81
<211> 22
<212> DNA
<213> Artificial
<400> 81
cctctagatt ctcgagagct ac 22
<210> 82
<211> 22
<212> DNA
<213> Artificial
<400> 82
acctctagat tctcgagagc ta 22
<210> 83
<211> 22
<212> DNA
<213> Artificial
<400> 83
cacctctaga ttctcgagag ct 22
<210> 84
<211> 22
<212> DNA
<213> Artificial
<400> 84
ccacctctag attctcgaga gc 22
<210> 85
<211> 22
<212> DNA
<213> Artificial
<400> 85
tccacctcta gattctcgag ag 22
<210> 86
<211> 22
<212> DNA
<213> Artificial
<400> 86
atccacctct agattctcga ga 22
<210> 87
<211> 22
<212> DNA
<213> Artificial
<400> 87
catccacctc tagattctcg ag 22
<210> 88
<211> 22
<212> DNA
<213> Artificial
<400> 88
ccatccacct ctagattctc ga 22
<210> 89
<211> 22
<212> DNA
<213> Artificial
<400> 89
atcccgggaa tcccatccac ct 22
<210> 90
<211> 22
<212> DNA
<213> Artificial
<400> 90
catcccggga atcccatcca cc 22
<210> 91
<211> 22
<212> DNA
<213> Artificial
<400> 91
tcatcccggg aatcccatcc ac 22
<210> 92
<211> 22
<212> DNA
<213> Artificial
<400> 92
gtcatcccgg gaatcccatc ca 22
<210> 93
<211> 22
<212> DNA
<213> Artificial
<400> 93
agtcatcccg ggaatcccat cc 22
<210> 94
<211> 22
<212> DNA
<213> Artificial
<400> 94
aagtcatccc gggaatccca tc 22
<210> 95
<211> 22
<212> DNA
<213> Artificial
<400> 95
gaagtcatcc cgggaatccc at 22
<210> 96
<211> 22
<212> DNA
<213> Artificial
<400> 96
tgaagtcatc ccgggaatcc ca 22
<210> 97
<211> 22
<212> DNA
<213> Artificial
<400> 97
ttgaagtcat cccgggaatc cc 22
<210> 98
<211> 22
<212> DNA
<213> Artificial
<400> 98
attgaagtca tcccgggaat cc 22
<210> 99
<211> 22
<212> DNA
<213> Artificial
<400> 99
aattgaagtc atcccgggaa tc 22
<210> 100
<211> 22
<212> DNA
<213> Artificial
<400> 100
gaattgaagt catcccggga at 22
<210> 101
<211> 22
<212> DNA
<213> Artificial
<400> 101
agaattgaag tcatcccggg aa 22
<210> 102
<211> 22
<212> DNA
<213> Artificial
<400> 102
cagaattgaa gtcatcccgg ga 22
<210> 103
<211> 22
<212> DNA
<213> Artificial
<400> 103
ccagaattga agtcatcccg gg 22
<210> 104
<211> 22
<212> DNA
<213> Artificial
<400> 104
accagaattg aagtcatccc gg 22
<210> 105
<211> 22
<212> DNA
<213> Artificial
<400> 105
taccagaatt gaagtcatcc cg 22
<210> 106
<211> 22
<212> DNA
<213> Artificial
<400> 106
ataccagaat tgaagtcatc cc 22
<210> 107
<211> 22
<212> DNA
<213> Artificial
<400> 107
gataccagaa ttgaagtcat cc 22
<210> 108
<211> 22
<212> DNA
<213> Artificial
<400> 108
tgataccaga attgaagtca tc 22
<210> 109
<211> 22
<212> DNA
<213> Artificial
<400> 109
ctgataccag aattgaagtc at 22
<210> 110
<211> 22
<212> DNA
<213> Artificial
<400> 110
actgatacca gaattgaagt ca 22
<210> 111
<211> 22
<212> DNA
<213> Artificial
<400> 111
tactgatacc agaattgaag tc 22
<210> 112
<211> 22
<212> DNA
<213> Artificial
<400> 112
atactgatac cagaattgaa gt 22
<210> 113
<211> 22
<212> DNA
<213> Artificial
<400> 113
aatactgata ccagaattga ag 22
<210> 114
<211> 22
<212> DNA
<213> Artificial
<400> 114
aaatactgat accagaattg aa 22
<210> 115
<211> 22
<212> DNA
<213> Artificial
<400> 115
taaatactga taccagaatt ga 22
<210> 116
<211> 22
<212> DNA
<213> Artificial
<400> 116
ttaaatactg ataccagaat tg 22
<210> 117
<211> 22
<212> DNA
<213> Artificial
<400> 117
tttaaatact gataccagaa tt 22
<210> 118
<211> 22
<212> DNA
<213> Artificial
<400> 118
ctttaaatac tgataccaga at 22
<210> 119
<211> 22
<212> DNA
<213> Artificial
<400> 119
actttaaata ctgataccag aa 22
<210> 120
<211> 22
<212> DNA
<213> Artificial
<400> 120
tactttaaat actgatacca ga 22
<210> 121
<211> 22
<212> DNA
<213> Artificial
<400> 121
atactttaaa tactgatacc ag 22
<210> 122
<211> 22
<212> DNA
<213> Artificial
<400> 122
gatactttaa atactgatac ca 22
<210> 123
<211> 22
<212> DNA
<213> Artificial
<400> 123
tgatacttta aatactgata cc 22
<210> 124
<211> 22
<212> DNA
<213> Artificial
<400> 124
gtgatacttt aaatactgat ac 22
<210> 125
<211> 22
<212> DNA
<213> Artificial
<400> 125
ggtgatactt taaatactga ta 22
<210> 126
<211> 22
<212> DNA
<213> Artificial
<400> 126
tggtgatact ttaaatactg at 22
<210> 127
<211> 22
<212> DNA
<213> Artificial
<400> 127
gtggtgatac tttaaatact ga 22
<210> 128
<211> 22
<212> DNA
<213> Artificial
<400> 128
agtggtgata ctttaaatac tg 22
<210> 129
<211> 22
<212> DNA
<213> Artificial
<400> 129
aagtggtgat actttaaata ct 22
<210> 130
<211> 22
<212> DNA
<213> Artificial
<400> 130
taagtggtga tactttaaat ac 22
<210> 131
<211> 22
<212> DNA
<213> Artificial
<400> 131
ttaagtggtg atactttaaa ta 22
<210> 132
<211> 22
<212> DNA
<213> Artificial
<400> 132
attaagtggt gatactttaa at 22
<210> 133
<211> 22
<212> DNA
<213> Artificial
<400> 133
aattaagtgg tgatacttta aa 22
<210> 134
<211> 22
<212> DNA
<213> Artificial
<400> 134
aaattaagtg gtgatacttt aa 22
<210> 135
<211> 22
<212> DNA
<213> Artificial
<400> 135
aaaattaagt ggtgatactt ta 22
<210> 136
<211> 22
<212> DNA
<213> Artificial
<400> 136
aaaaattaag tggtgatact tt 22
<210> 137
<211> 22
<212> DNA
<213> Artificial
<400> 137
gaaaaattaa gtggtgatac tt 22
<210> 138
<211> 22
<212> DNA
<213> Artificial
<400> 138
ggaaaaatta agtggtgata ct 22
<210> 139
<211> 22
<212> DNA
<213> Artificial
<400> 139
aggaaaaatt aagtggtgat ac 22
<210> 140
<211> 22
<212> DNA
<213> Artificial
<400> 140
taggaaaaat taagtggtga ta 22
<210> 141
<211> 22
<212> DNA
<213> Artificial
<400> 141
ctaggaaaaa ttaagtggtg at 22
<210> 142
<211> 22
<212> DNA
<213> Artificial
<400> 142
cctaggaaaa attaagtggt ga 22
<210> 143
<211> 22
<212> DNA
<213> Artificial
<400> 143
tcctaggaaa aattaagtgg tg 22
<210> 144
<211> 22
<212> DNA
<213> Artificial
<400> 144
ctcctaggaa aaattaagtg gt 22
<210> 145
<211> 22
<212> DNA
<213> Artificial
<400> 145
gctcctagga aaaattaagt gg 22
<210> 146
<211> 22
<212> DNA
<213> Artificial
<400> 146
ggctcctagg aaaaattaag tg 22
<210> 147
<211> 22
<212> DNA
<213> Artificial
<400> 147
aggctcctag gaaaaattaa gt 22
<210> 148
<211> 22
<212> DNA
<213> Artificial
<400> 148
aaggctccta ggaaaaatta ag 22
<210> 149
<211> 22
<212> DNA
<213> Artificial
<400> 149
caaggctcct aggaaaaatt aa 22
<210> 150
<211> 22
<212> DNA
<213> Artificial
<400> 150
gcaaggctcc taggaaaaat ta 22
<210> 151
<211> 22
<212> DNA
<213> Artificial
<400> 151
agcaaggctc ctaggaaaaa tt 22
<210> 152
<211> 22
<212> DNA
<213> Artificial
<400> 152
cagcaaggct cctaggaaaa at 22

Claims (7)

1.一种寡聚核苷酸，其特征在于，该寡聚核苷酸具有干预EIF4H剪接亚型表达的活性，其序列是下列中的任意一项：
(1)SEQ ID NO 6-152中任意一项，或与SEQ ID NO 6-152中任意一项的序列重复超过5个核苷酸的干扰小RNA或者反义寡聚核苷酸；
(2)上述(1)中核苷酸的各种化学修饰形式；
(3)上述(1)中核苷酸带有连接物的衍生核苷酸序列。
2.如权利要求1所述的寡聚核苷酸，其特征在于，所述的干扰小RNA是双链或单链干扰小RNA、微小RNA、双链RNA、短发卡RNA或者其化学修饰形式。
3.如权利要求1所述的寡聚核苷酸，其特征在于，所述的反义寡聚核苷酸是反义寡聚核苷酸及其类似物或其化学修饰形式。
4.一种检测EIF4H不同剪接亚型表达的方法，其特征在于，通过不同剪接亚型对应的RNA及蛋白大小区分EIF4H不同剪接亚型，其包括：
采用反转录-聚合酶链反应检测EIF4H不同剪接亚型RNA表达；
或，
通过RNA测序检测EIF4H不同剪接亚型RNA表达；
或，
使用针对eIF4H的抗体检测蛋白表达产物。
5.权利要求1-3所述的寡聚核苷酸在制备诊断试剂或者治疗药物中的应用。
6.如权利要求5所述的应用，其特征在于，所述的药物通过抑制EIF4H特定剪接亚型表达治疗恶性肿瘤。
7.权利要求4所述的检测EIF4H不同剪接亚型表达的方法在制备评估、鉴定诊断试剂或者治疗药物标准系统中的应用。
CN201811569563.7A 2018-12-19 2018-12-19 干预eif4h剪接亚型的寡聚核苷酸及其应用 Pending CN111471679A (zh)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007119692A1 (ja) * 2006-04-12 2007-10-25 National University Corporation Chiba University eIF4H発現抑制による癌細胞の増殖阻害方法
CN103421792A (zh) * 2013-06-21 2013-12-04 浙江理工大学 真核生物翻译起始因子4H（eIF4H）基因沉默抑制肿瘤细胞生长的方法及其应用

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007119692A1 (ja) * 2006-04-12 2007-10-25 National University Corporation Chiba University eIF4H発現抑制による癌細胞の増殖阻害方法
CN103421792A (zh) * 2013-06-21 2013-12-04 浙江理工大学 真核生物翻译起始因子4H（eIF4H）基因沉默抑制肿瘤细胞生长的方法及其应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
TAKESHI TOMONAGA 等: "Identification of altered protein expression and post-translational modifications in primary colorectal cancer by using agarose two-dimensional gel electrophoresis", 《CLINICAL CANCER RESEARCH》, vol. 10, no. 6, 15 March 2004 (2004-03-15), XP002340980, DOI: 10.1158/1078-0432.CCR-03-0321 *

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