CN113774137A - 检测生物标志物表达的试剂在制备鉴定白血病耐药和/或不良预后的试剂盒中的应用 - Google Patents
检测生物标志物表达的试剂在制备鉴定白血病耐药和/或不良预后的试剂盒中的应用 Download PDFInfo
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Abstract
本发明提供了检测生物标志物表达的试剂在制备鉴定白血病耐药和/或不良预后的试剂盒中的应用,涉及生物医药技术领域。本发明提供了miR‑25、miR‑93、miR‑106b和miR‑106b‑93‑25作为急性髓系白血病耐药和预后不良标志物的应用,其特异性引物对可用于制备检测急性髓系白血病诊断试剂或试剂盒,通过检测白血病干细胞中的miR‑25、miR‑93、miR‑106b表达水平,对急性髓系白血病的治疗进行用药指导,具备较好的临床应用价值和广阔的应用前景,同时本发明也指导通过凋亡发挥作用的小分子抑制剂在干预急性髓性白血病治疗过程中的应用。
Description
技术领域
本发明属于生物医药技术领域,具体涉及检测生物标志物表达的试剂在制备鉴定白血病耐药和/或不良预后的试剂盒中的应用。
背景技术
MicroRNA(miRNA)是真核生物中的一类内源性的具有调控功能的非编码短RNA,其长度一般为19-25个核苷酸。过去的研究表明miRNA参与了多重调节途径,包括发育、病毒防御、造血过程、器官形成、细胞增殖与死亡等。近年来,miRNA的丰度变化与肿瘤发生和发展的密切关系已经在科学界形成了共识并成为了目前的研究热点。
急性髓性白血病(Acute myeloidleukemia,AML)特征在于克隆性髓系前体细胞(clonal myeloidprecursor)的分化阻滞和恶性增殖,是一类在细胞遗传学上和分子上异质性很强的病症。具有中等风险和高风险的AML患者,大多选择化疗方法,但是经证实,长期化疗会产生耐药性和预后不良,导致药效降低,因此寻找AML的化疗耐药和预后不良生物标志物成为开发新型疗法的迫切需求。
发明内容
有鉴于此,本发明的目的在于提供检测生物标志物表达的试剂在制备鉴定白血病耐药和/或不良预后的试剂盒中的应用,证实白血病干细胞中miR-25、miR-93、miR-106b高表达与急性髓系白血病患者不良生存相关,为指导通过诱导凋亡发挥作用的小分子抑制剂在干预急性髓性白血病治疗过程中的应用提供了理论指导。
为了实现上述发明目的,本发明提供以下技术方案:
本发明提供了检测生物标志物表达的试剂在制备鉴定白血病耐药和/或不良预后的试剂盒中的应用,所述生物标志物选自以下至少一种:miR-25、miR-93、miR-106b和miR-106b-93-25基因簇。
优选的,所述检测生物标志物的表达的试剂包括检测所述生物标志物的靶基因的表达。
优选的,所述miR-93和miR-106b的直接靶点包括TP73,miR-25的直接靶点包括BAK1,miR-106b-93-25基因簇的直接靶点包括Caspase7。
优选的,所述试剂包括针对生物标志物的直接靶点设计的特异性引物,所述特异性引物包括:
针对TP73设计的引物对TP73-F和TP73-R,所述TP73-F的核苷酸序列如SEQ IDNO.1所示,所述TP73-R的核苷酸序列如SEQ ID NO.2所示;
针对BAK1设计的引物对BAK1-F和BAK1-R,所述BAK1-F的核苷酸序列如SEQ IDNO.3所示,所述BAK1-R的核苷酸序列如SEQ ID NO.4所示;
针对Caspase7设计的引物对Caspase7-F和Caspase7-R,所述Caspase7-F的核苷酸序列如SEQ ID NO.5所示,所述Caspase7-R的核苷酸序列如SEQ ID NO.6所示。
优选的,所述试剂盒中还包括内参基因GAPDH,针对GAPDH设计的引物对GAPDH-F和GAPDH-R,所述GAPDH-F的核苷酸序列如SEQ ID NO.7所示,所述GAPDH-R的核苷酸序列如SEQID NO.8所示。
优选的,所述白血病包括急性髓系白血病。
本发明还提供了一种鉴定白血病耐药和/或不良预后的试剂盒,所述试剂盒中包括:
针对miR-93和miR-106b的直接靶点TP73设计的引物对TP73-F和TP73-R,所述TP73-F的核苷酸序列如SEQ ID NO.1所示,所述TP73-R的核苷酸序列如SEQ ID NO.2所示;
针对miR-25的直接靶点BAK1设计的引物对BAK1-F和BAK1-R,所述BAK1-F的核苷酸序列如SEQ ID NO.3所示,所述BAK1-R的核苷酸序列如SEQ ID NO.4所示;
针对miR-106b-93-25基因簇的直接靶点Caspase7设计的引物对Caspase7-F和Caspase7-R,所述Caspase7-F的核苷酸序列如SEQ ID NO.5所示,所述Caspase7-R的核苷酸序列如SEQ ID NO.6所示。
有益效果:本发明提供了miR-25、miR-93、miR-106b和miR-106b-93-25基因簇中的至少一种作为急性髓系白血病干细胞分子标志的应用,同时也作为蒽环类药物(如阿霉素)、酪氨酸激酶抑制剂(如伊马替尼)、和Bcl-2抑制剂(如ABT-737)耐药分子靶标预后不良分子标志,本发明检测miR-25、miR-93和miR-106b的特异性引物可用于制备检测急性髓系白血病诊断试剂或试剂盒,对急性髓系白血病的治疗过程中用药进行指导,同时本发明也指导通过诱导凋亡发挥作用的小分子抑制剂在干预急性髓性白血病治疗过程中的应用。在本发明实施例中,检测急性髓系白血病患者异常细胞群中白血病干细胞miR-25、miR-93、miR-106b的表达,发现白血病干细胞中miR-25、miR-93、miR-106b高表达与急性髓系白血病患者不良生存相关;同时还证实了miR-106b-93-25簇高表达与急性白血病治疗中的阿霉素、伊马替尼和ABT-737耐药和生存期缩短密切相关;通过细胞转染实验证实,miR-25、miR-93、miR-106b成功高表达的K562对阿霉素、伊马替尼和ABT-737耐药克隆形成能力明显增强,被凋亡诱导剂ABT-737(BCL-2抑制剂)诱导凋亡的比例减少。本发明实施例中还通过生物信息学和生物学的配合验证,过表达miR-25、miR-93、miR-106b和miR-106b-93-25的K562细胞凋亡相关基因表达明显降低,因此可通过检测白血病干细胞中的miR-25、miR-93、miR-106b表达水平,对急性髓系白血病的治疗进行用药指导,具备较好的临床应用价值和广阔的应用前景。
附图说明
图1表示miR-106b-93-25基因簇在耐药白血病细胞中上调,并与AML患者的不良预后相关;
图2表示miR-106b-93-25基因簇的过表达促进了髓系白血病细胞的细胞增殖;
图3表示miR-106b-93-25基因簇过表达促进了药物治疗后白血病细胞的存活;
图4表示miR-106b-93-25基因簇过表达抑制药物诱导的AML细胞系凋亡;
图5表示过表达miR-106b-93-25基因簇促进白血病细胞在体内的增殖和化疗药物耐受;
图6表示miR-106b、miR-93、miR-25在白血病细胞K562增殖和化疗耐受的影响;
图7表示凋亡通路上的关键调节基因被miR106b-93-25、miR-25、miR-93和miR-106b过表达诱导下调;
图8为miR106b-93-25、miR-25、miR-93和miR-106b的靶点图。
具体实施方式
本发明提供了检测生物标志物表达的试剂在制备鉴定白血病耐药和/或不良预后的试剂盒中的应用,所述生物标志物选自以下至少一种:miR-25、miR-93、miR-106b和miR-106b-93-25基因簇。
本发明实施例中以流式分选和实时荧光定量PCR技术为基础,以白血病患者的骨髓miR-106b-93-25基因簇(简称miR-106b-93-25簇)为检测对象,通过检测急性髓系白血病患者白血病干细胞亚群(CD34+细胞)中miR-25、miR-93、miR-106b的表达,发现白血病干细胞中miR-25、miR-93、miR-106b高表达与急性髓系白血病患者不良生存相关,可作为鉴定白血病不良预后的生物标志物。
本发明实施例以耐药实验和小鼠异种移植实验为基础,以耐药细胞为检测对象,发现在耐药的白血病细胞中的miR-106b-93-25基因簇中的单个microRNA:miR-25、miR-93、miR-106b呈现高表达;且转染了过表达miR-106b-93-25基因簇慢病毒载体的K562,HL-60细胞,miR-25、miR-93、miR-106b成功高表达,发现其阿霉素耐药曲线中的IC50明显升高,对阿霉素、伊马替尼和ABT-737耐药克隆形成能力明显增强。因此,miR-106b-93-25基因簇高表达与急性白血病治疗中的耐药和生存期缩短密切相关,可作为白血病耐药的生物标志物。
在本发明中,所述检测生物标志物的表达的试剂优选包括检测所述生物标志物的靶基因的表达,所述miR-93和miR-106b的直接靶点优选包括TP73,miR-25的直接靶点优选包括BAK1,miR-25、miR-93和miR-106b的直接靶点优选包括Caspase7。本发明所述试剂优选包括针对生物标志物的直接靶点设计的特异性引物,所述特异性引物优选包括:
针对TP73设计的引物对TP73-F和TP73-R,所述TP73-F的核苷酸序列如SEQ IDNO.1所示,所述TP73-R的核苷酸序列如SEQ ID NO.2所示;
针对BAK1设计的引物对BAK1-F和BAK1-R,所述BAK1-F的核苷酸序列如SEQ IDNO.3所示,所述BAK1-R的核苷酸序列如SEQ ID NO.4所示;
针对Caspase7设计的引物对Caspase7-F和Caspase7-R,所述Caspase7-F的核苷酸序列如SEQ ID NO.5所示,所述Caspase7-R的核苷酸序列如SEQ ID NO.6所示。
本发明所述试剂盒中优选还包括内参基因GAPDH,针对GAPDH设计的引物对GAPDH-F和GAPDH-R,所述GAPDH-F的核苷酸序列优选如SEQ ID NO.7所示,所述GAPDH-R的核苷酸序列优选如SEQ ID NO.8所示。
本发明所述白血病优选包括急性髓系白血病。
本发明还提供了一种鉴定白血病耐药和/或不良预后的试剂盒,所述试剂盒中包括:
针对miR-93和miR-106b的直接靶点TP73设计的引物对TP73-F和TP73-R,所述TP73-F的核苷酸序列如SEQ ID NO.1所示,所述TP73-R的核苷酸序列如SEQ ID NO.2所示;
针对miR-25的直接靶点BAK1设计的引物对BAK1-F和BAK1-R,所述BAK1-F的核苷酸序列如SEQ ID NO.3所示,所述BAK1-R的核苷酸序列如SEQ ID NO.4所示;
针对miR-106b-93-25基因簇的直接靶点Caspase7设计的引物对Caspase7-F和Caspase7-R,所述Caspase7-F的核苷酸序列如SEQ ID NO.5所示,所述Caspase7-R的核苷酸序列如SEQ ID NO.6所示。
本发明所述试剂盒优选与上述应用相同,在此不再赘述。
下面结合实施例对本发明提供的检测生物标志物的表达的试剂在制备鉴定白血病耐药和/或不良预后的试剂盒中的应用进行详细的说明,但是不能把它们理解为对本发明保护范围的限定。
实施例1
1、材料和方法
1.1患者样本
18例AML患者的骨髓(BM)样本从中国医学科学院血液病医院获得。所有患者均根据WHO分类为初诊AML。从这些样本中分离出BM单核细胞(MNC),并在知情同意的情况下进行冷冻。整个收集和分离程序符合1975年《赫尔辛基宣言》的指导原则,并经中国医学科学院血液病研究所伦理委员会批准。使用人CD34+微球从MNC中富集CD34+细胞(德国Miltenyi,130-046-702)。
1.2细胞系与细胞培养
K562、HL-60和THP-1细胞悬浮于含10%胎牛血清(FBS,Gibco)的RPMI 1640(Gibco,Carlsbad,USA)中,培养的同时补充2mM L-谷氨酰胺(Gibco)和1%青霉素/链霉素(Beyotime,上海,中国),在5%CO2培养箱中37℃条件下培养。将由阿霉素诱导的K562/A02和HL-60/ADR在含有1μg/ml阿霉素的RPMI 1640培养基中培养。
1.3慢病毒转导miRNA的异位表达
慢病毒载体SFFV-eGFP由苏州泓迅生物科技有限公司构建。将合成好的miR-25(SEQ ID NO.9)、miR-93(SEQ ID NO.10)、miR-106b(SEQ ID NO.11)或miR-106b-93-25(SEQID NO.12)前体序列与目的载体连接。含有miR-106b-93-25前体序列、miR-106b前体序列、miR-93前体序列、miR-25前体序列与eGFP荧光蛋白基因偶联的的慢病毒载体分别用于介导miR-106b-93-25簇、miR-106b、miR-93和miR-25过表达(SF-LV-GFP-miR-106b-93-25;SF-LV-GFP-miR-106b;SF-LV-GFP-miR-93;SF-LV-GFP-miR-25)。以同一骨架但是仅含有eGFP的载体用作对照(SF-LV-GFP-EV,简称EV)。
1.4实时定量PCR
使用hsa-miR-106b-5p(Invitrogen,000442)、hsa-miR-93-5p(Invitrogen,000432)和hsa-miR-25-3p(Invitrogen,000403)的Taqman MicroRNA分析来检测成熟miR-106b、miR-93和miR-25的表达。用于qPCR的其他引物列于补充表S2中。实验在生物学独立的重复样本中运行,使用2-ΔΔCT方法计算相对表达,并用U6作为内参基因(Invitrogen,001973)。
表1引物序列
Primer | Sequence(5’to3’) | SEQ ID NO |
GAPDH-F | GAAGGTGAAGGTCGGAGTC | 7 |
GAPDH-R | GAAGATGGTGATGGGATTTC | 8 |
TP73-F | CCACCACTTTGAGGTCACTTT | 1 |
TP73-R | CTTCAAGAGCGGGGAGTACG | 2 |
BAK1-F | CAAACAGGCTGGTGGCAATC | 3 |
BAK1-R | TCATCGGGGACGACATCAAC | 4 |
BAX-F | GCCGACGGCAACTTCAACTG | 13 |
BAX-R | CCAACCACCCTGGTCTTGGA | 14 |
BTG2-F | AGCGAGCAGAGGCTTAAGGT | 15 |
BTG2-R | CGGTAGGACACCTCATAGGG | 16 |
Caspase7-F | CGTTTGTACCGTCCCTCTTC | 5 |
Caspase7-R | GCCCAGCTTTTCAAAATTCA | 6 |
CDKN1A-F | AGGTGGACCTGGAGACTCTCAG | 17 |
CDKN1A-R | TCCTCTTGGAGAAGATCAGCCG | 18 |
1.5细胞增殖试验
对miR106b-25、miR-106b、miR-93、miR-25或EV载体转导的K562、HL-60和THP-1细胞手动计数,并以每孔1000个细胞的密度接种到96孔板中。每个细胞系进行三个平行孔重复。在不同的时间点(第0、1、2、3、4天)将细胞计数试剂盒-8(CCK-8)试剂(DojindoLaboratories,Japan)(10μL)加到每个孔中。使用微孔板读取器(Synergy H4,BioTek,USA)在4小时内测量450nm处的吸光度。通过比较第0天和各时间点的吸光度绘制细胞生长曲线。
1.6用细胞示踪法标记细胞
使用CFSE细胞增殖试剂盒(Life Technologies)按照使用说明书,培养过程中收集不同时间点(0小时、24小时、48小时、72小时、96小时、120小时)的细胞(3×105)PBS洗一次,2%PFA固定,待所有时间点取完后通过流式细胞术进行分析。使用ModFit软件(VeritySoftware House,USA)拟合分割曲线。
1.7细胞克隆形成(CFC)分析
用阿霉素(K562为0.05μM,HL-60为0.1μM)、伊马替尼(0.1μM)、ABT-737(10μM)在37℃下孵育24小时。然后将来自不同组的细胞以300或500个细胞/mL的浓度接种于0.5mL甲基纤维素培养基(MethoCult H4230,StemCell Technologies)中的24孔板中。在37℃下培养12~14天后计数菌落数。
1.8细胞凋亡分析
将过表达miR-25、miR-93、miR-106b、miR-106b-93-25或EV载体的K562细胞以8.9×104细胞/mL的密度接种到24孔板中,并与阿霉素(0.05μM)、伊马替尼(0.1μM)、ABT-737(10μM)一起培养72h以诱导细胞凋亡,然后收集细胞并用AnnexinV和DAPI标记。通过流式细胞仪分析细胞,以评估多种化疗药物诱导的凋亡细胞百分比。
1.9RNA测序和生物信息学分析
感染miR-25、miR-93、miR-106b、miR-106b-93-25或EV的K562细胞在Novogene(Beijing,China)公司进行RNA测序(RNA seq)实验。通过使用“limma”软件包的RNA-seq数据,差异表达基因(DEG)的p值小于0.05,log2 fold change(LFC)大于0.5。使用DAVIDBioinformatics Resources(6.8version)对GO富集和KEGG途径进行分析。基因集富集分析(GSEA)分析也用于确定先验定义的基因集是否具有统计学意义。
1.10荧光素酶报告试验
将含有miRNA靶位点的BAK1、TP73或CASP7的3’UTR或CDS片段克隆到pmirGLO双荧光素酶miRNA靶表达载体(Promega,USA)中,将载体命名为pmirGLO-TP73、pmirGLO-BAK1或pmirGLO-CASP7(Youbio Bio Biological Technology,China)。利用Youbio BiologicalTechnology将miR-25的靶向种子序列(5’-TGCAAT-3’)突变为5’-TAGACT-3’,并将miR-106b/93的种子序列(5’-CACTT-3’)突变为5’-AAAGGT-3’。根据制造商的说明(Promega,USA),通过测量萤火虫和海肾荧光素酶活性,使用双荧光素酶报告系统测定荧光素酶活性。
1.11异体移植
所有动物实验都得到了该研究所动物研究委员会的批准。我们选择5周龄体重16~20g的雌性裸鼠提前6~24小时用240cGy照射。裸鼠皮下注射K562-miR-106b-25或K562-EV细胞(1×107)。移植后每3天测量一次肿瘤的长度和宽度,并用以下公式计算肿瘤体积。肿瘤体积=(A×B2)×(1/2),其中A为较长尺寸,B为较短尺寸。当肿瘤体积达到2000mm3时,处死小鼠并取出肿瘤,然后称重肿瘤质量。
接下来,进行二次皮下移植和阿霉素治疗。二次移植后第3天起,每隔一天通过腹腔注射以4mg/kg剂量的阿霉素对小鼠进行治疗,共12天。在整个研究过程中,每3天测量一次肿瘤大小。治疗12天后,对照组肿瘤基本消失,停止给药。继续正常喂养小鼠,观察小鼠存活情况。此外,根据第12天相对于第3天的肿瘤体积计算肿瘤抑制率,如下所示:肿瘤抑制率(%)=(第3天肿瘤体积-第12天肿瘤体积/第3天肿瘤体积)×100%。根据肿瘤体积绘制肿瘤生长曲线,根据生存天数绘制生存曲线。
1.12统计分析
GraphPad Prism 7.0用于统计分析。两组或两组以上数据的差异分别通过非配对t检验或单因素方差分析进行分析。使用Kaplan–Meier分析和对数秩检验评估生存率差异。实验数据以平均值±标准差表示,当p值小于0.05时,差异被认为具有统计学意义。
2、结果与分析
2.1将miR-106b-93-25作为急性髓系白血病的标志物和分子靶标,为白血病的预后评估提供新的分子标志,同时也为个体化治疗提供新的分子靶标。
本发明以流式分选和实时荧光定量PCR技术为基础,以白血病患者的骨髓miR-106b-93-25为检测对象,通过检测急性髓系白血病患者异常细胞群中白血病干细胞miR-25、miR-93、miR-106b的表达,发现白血病干细胞中miR-25、miR-93、miR-106b高表达与急性髓系白血病患者不良生存相关(图1)。
2.2本发明以耐药实验和小鼠异种移植实验为基础,说明miR-106b-93-25簇与耐药和预后不良密切相关。以K562/K562A02,HL60/HL60ADR为检测对象(K562/A02和HL-60/ADR为阿霉素诱导的,但是多种化疗药物耐药的多药耐药白血病细胞系),发现在耐药细胞中的miR-106b-93-25簇中的单个microRNA:miR-25、miR-93、miR-106b呈现高表达(图2)。
与转染空载的对照细胞相比,过表达miR-106b-93-25慢病毒载体的K562,HL-60细胞,miR-25、miR-93、miR-106b成功高表达。我们发现其阿霉素耐药曲线中的IC50明显升高,K562、HL-60和THP-1对阿霉素,imatinib和ABT-737、HL-60对阿霉素耐药克隆形成能力明显增强。同时进行miR-106b-93-25过表达的K562细胞进行小鼠皮下瘤移植体内实验,我们发现miR-106b-93-25组小鼠瘤块体积和重量大于对照组,接下来进行阿霉素治疗(图3和图4)。干预后,发现miR-106b-93-25组小鼠皮下瘤更能耐受阿霉素治疗,同时观察到该组小鼠生存期缩短。因此,该发明发现miR-106b-93-25簇高表达与急性白血病治疗中的阿霉素、imatinib和ABT-737耐药和生存期缩短密切相关(图5)。
2.3miR-25,miR-93,miR-106b通过抑制凋亡路径诱导阿霉素耐药
将转染miR-25、miR-93、miR-106b慢病毒的K562,与转染空载体的对照细胞相比,miR-25、miR-93、miR-106b成功高表达的K562对阿霉素耐药克隆形成能力明显增强,对凋亡诱导剂ABT-737(BCL-2抑制剂)凋亡比例减少(图6)。
2.4利用RNA-seq技术结合系统的生物信息学分析方法,同时进行生物学实验验证,分析出耐药机制可能是凋亡通路被抑制。
与转染空载体的对照K562细胞相比,过表达miR-25、miR-93、miR-106b和miR-106b-93-25的K562细胞差异基因GO和KEGG分析显示集中在与细胞增殖,对药物反应和凋亡相关通路(图7)。
检测到凋亡过程中关键基因BAK1、BAX、CASP7等基因明显减少,体外进行RT-qPCR进行证实,并且在ABT-737诱导后的细胞中基因表达进一步减少,与转染空载体的对照K562细胞相比,过表达miR-25、miR-93、miR-106b和miR-106b-93-25的K562细胞凋亡细胞比例明显降低。
2.5利用萤火虫荧光素酶报告系统,证实TP73是miR-93和miR-106b的直接靶点,BAK1是miR-25的直接靶点,CASP7是miR-25、miR-93和miR-106b的直接靶点。(图8)
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
序列表
<110> 中国医学科学院血液病医院(中国医学科学院血液学研究所)
<120> 检测生物标志物表达的试剂在制备鉴定白血病耐药和/或不良预后的试剂盒中的应用
<160> 18
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
<212> DNA
<213> 人工序列(artificial sequence)
<400> 1
ccaccacttt gaggtcactt t 21
<210> 2
<211> 20
<212> DNA
<213> 人工序列(artificial sequence)
<400> 2
cttcaagagc ggggagtacg 20
<210> 3
<211> 20
<212> DNA
<213> 人工序列(artificial sequence)
<400> 3
caaacaggct ggtggcaatc 20
<210> 4
<211> 20
<212> DNA
<213> 人工序列(artificial sequence)
<400> 4
tcatcgggga cgacatcaac 20
<210> 5
<211> 20
<212> DNA
<213> 人工序列(artificial sequence)
<400> 5
cgtttgtacc gtccctcttc 20
<210> 6
<211> 20
<212> DNA
<213> 人工序列(artificial sequence)
<400> 6
gcccagcttt tcaaaattca 20
<210> 7
<211> 19
<212> DNA
<213> 人工序列(artificial sequence)
<400> 7
gaaggtgaag gtcggagtc 19
<210> 8
<211> 20
<212> DNA
<213> 人工序列(artificial sequence)
<400> 8
gaagatggtg atgggatttc 20
<210> 9
<211> 383
<212> DNA/RNA
<213> 人工序列(artificial sequence)
<400> 9
ctccctcaca ggacagctga actccgggac tggccagtgt tgagaggcgg agacttgggc 60
aattgctgga cgctgccctg ggcattgcac ttgtctcggt ctgacagtgc cggcccaaca 120
ctgcggatgc tggggggagg ggggattcca ctcctgtttt gtgagtaggc gacccatggg 180
ctgcccagcc ttaaagccag aacaagggtg tcccctgacc tcgttccact gccctcctcc 240
cgttcccatc tttcccccct accttcccct taggcacgtc tgagaatggt ggatgtggtg 300
gagaaagaag atgtgaatga agccatcagg ctaatggaga tgtcaaagga ctctcttcta 360
ggagacaagg ggcagacagc tag 383
<210> 10
<211> 180
<212> DNA/RNA
<213> 人工序列(artificial sequence)
<400> 10
ctcagtcctg ggggctccaa agtgctgttc gtgcaggtag tgtgattacc caacctactg 60
ctgagctagc acttcccgag cccccgggac acgttctctc tgccaattgt cttcttggct 120
gagctcccca agctccatct gtcatgctgg ggagcccagt ggcgttcaaa agggtctggt 180
<210> 11
<211> 240
<212> DNA/RNA
<213> 人工序列(artificial sequence)
<400> 11
tccccacctc ccgctccagc cctgccgggg ctaaagtgct gacagtgcag atagtggtcc 60
tctccgtgct accgcactgt gggtacttgc tgctccagca gggcacgcac agcgtccgtg 120
gagggaaagg ccttttcccc acttcttaac cttcactgag agggtggttg gggtctgttt 180
cactccatgt gtcctagatc ctgtgctaca gaccttcctt tctgtcctcc cgtcttggac 240
<210> 12
<211> 1043
<212> DNA/RNA
<213> 人工序列(artificial sequence)
<400> 12
atagccatgt gccgcgagaa gcagcccatg gtgccagagt ctctggctga ctacatcaca 60
gcagcatacg tggagatgag gcgagaggct tgggctagta aggatgccac ctatacttct 120
gcccggaccc tgctggctat cctgcgcctt tccactgctc tggtaagtgc ccaaattgct 180
ggagggccat ctgttttgac ccttaaaggg gtagctcctt accgtgctct cattgccgcc 240
tccccacctc ccgctccagc cctgccgggg ctaaagtgct gacagtgcag atagtggtcc 300
tctccgtgct accgcactgt gggtacttgc tgctccagca gggcacgcac agcgtccgtg 360
gagggaaagg ccttttcccc acttcttaac cttcactgag agggtggttg gggtctgttt 420
cactccatgt gtcctagatc ctgtgctaca gaccttcctt tctgtcctcc cgtcttggac 480
ctcagtcctg ggggctccaa agtgctgttc gtgcaggtag tgtgattacc caacctactg 540
ctgagctagc acttcccgag cccccgggac acgttctctc tgccaattgt cttcttggct 600
gagctcccca agctccatct gtcatgctgg ggagcccagt ggcgttcaaa agggtctggt 660
ctccctcaca ggacagctga actccgggac tggccagtgt tgagaggcgg agacttgggc 720
aattgctgga cgctgccctg ggcattgcac ttgtctcggt ctgacagtgc cggcccaaca 780
ctgcggatgc tggggggagg ggggattcca ctcctgtttt gtgagtaggc gacccatggg 840
ctgcccagcc ttaaagccag aacaagggtg tcccctgacc tcgttccact gccctcctcc 900
cgttcccatc tttcccccct accttcccct taggcacgtc tgagaatggt ggatgtggtg 960
gagaaagaag atgtgaatga agccatcagg ctaatggaga tgtcaaagga ctctcttcta 1020
ggagacaagg ggcagacagc tag 1043
<210> 13
<211> 20
<212> DNA
<213> 人工序列(artificial sequence)
<400> 13
gccgacggca acttcaactg 20
<210> 14
<211> 20
<212> DNA
<213> 人工序列(artificial sequence)
<400> 14
ccaaccaccc tggtcttgga 20
<210> 15
<211> 20
<212> DNA
<213> 人工序列(artificial sequence)
<400> 15
agcgagcaga ggcttaaggt 20
<210> 16
<211> 20
<212> DNA
<213> 人工序列(artificial sequence)
<400> 16
cggtaggaca cctcataggg 20
<210> 17
<211> 22
<212> DNA
<213> 人工序列(artificial sequence)
<400> 17
aggtggacct ggagactctc ag 22
<210> 18
<211> 22
<212> DNA
<213> 人工序列(artificial sequence)
<400> 18
tcctcttgga gaagatcagc cg 22
Claims (7)
1.检测生物标志物表达的试剂在制备鉴定白血病耐药和/或不良预后的试剂盒中的应用,所述生物标志物选自以下至少一种:miR-25、miR-93、miR-106b和miR-106b-93-25基因簇。
2.根据权利要求1所述应用,其特征在于,所述检测生物标志物的表达的试剂包括检测所述生物标志物的靶基因的表达。
3.根据权利要求2所述应用,其特征在于,所述miR-93和miR-106b的直接靶点包括TP73,miR-25的直接靶点包括BAK1,miR-106b-93-25基因簇的直接靶点包括Caspase7。
4.根据权利要求3所述应用,其特征在于,所述试剂包括针对生物标志物的直接靶点设计的特异性引物,所述特异性引物包括:
针对TP73设计的引物对TP73-F和TP73-R,所述TP73-F的核苷酸序列如SEQ ID NO.1所示,所述TP73-R的核苷酸序列如SEQ ID NO.2所示;
针对BAK1设计的引物对BAK1-F和BAK1-R,所述BAK1-F的核苷酸序列如SEQ ID NO.3所示,所述BAK1-R的核苷酸序列如SEQ ID NO.4所示;
针对Caspase7设计的引物对Caspase7-F和Caspase7-R,所述Caspase7-F的核苷酸序列如SEQ ID NO.5所示,所述Caspase7-R的核苷酸序列如SEQ ID NO.6所示。
5.根据权利要求4所述应用,其特征在于,所述试剂盒中还包括内参基因GAPDH,针对GAPDH设计的引物对GAPDH-F和GAPDH-R,所述GAPDH-F的核苷酸序列如SEQ ID NO.7所示,所述GAPDH-R的核苷酸序列如SEQ ID NO.8所示。
6.根据权利要求1所述应用,其特征在于,所述白血病包括急性髓系白血病。
7.一种鉴定白血病耐药和/或不良预后的试剂盒,其特征在于,所述试剂盒中包括:
针对miR-93和miR-106b的直接靶点TP73设计的引物对TP73-F和TP73-R,所述TP73-F的核苷酸序列如SEQ ID NO.1所示,所述TP73-R的核苷酸序列如SEQ ID NO.2所示;
针对miR-25的直接靶点BAK1设计的引物对BAK1-F和BAK1-R,所述BAK1-F的核苷酸序列如SEQ ID NO.3所示,所述BAK1-R的核苷酸序列如SEQ ID NO.4所示;
针对miR-106b-93-25基因簇的直接靶点Caspase7设计的引物对Caspase7-F和Caspase7-R,所述Caspase7-F的核苷酸序列如SEQ ID NO.5所示,所述Caspase7-R的核苷酸序列如SEQ ID NO.6所示。
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US20110021609A1 (en) * | 2008-02-28 | 2011-01-27 | The Ohio State University Research Foundation | MicroRNA Signatures Associated with Cytogenetics and Prognosis in Acute Myeloid Leukemia (AML) and Uses Thereof |
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US20110021609A1 (en) * | 2008-02-28 | 2011-01-27 | The Ohio State University Research Foundation | MicroRNA Signatures Associated with Cytogenetics and Prognosis in Acute Myeloid Leukemia (AML) and Uses Thereof |
CN102007408A (zh) * | 2008-02-28 | 2011-04-06 | 俄亥俄州立大学研究基金会 | 与急性髓性白血病(aml)中的细胞遗传学和预后相关的微rna特征及其用途 |
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LONNEKE J. VERBOON: "MicroRNA-106b~25 cluster is upregulated in relapsed MLL-rearranged pediatric acute myeloid leukemia", 《ONCOTARGET》, vol. 7, no. 30, pages 48413 * |
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