CN111455053B - 用于结直肠腺瘤诊断的外泌体rna分子标记物组合及其应用 - Google Patents
用于结直肠腺瘤诊断的外泌体rna分子标记物组合及其应用 Download PDFInfo
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Abstract
本发明涉及生物检测领域,更具体涉及生物的分子检测,更具体涉及一组与结直肠腺瘤相关的外泌体RNA标记物及其应用。
Description
技术领域
本发明涉及生物检测领域,更具体涉及生物的分子检测,更具体涉及一组与结直肠腺瘤相关的外泌体RNA标记物及其应用。
背景技术
结直肠腺瘤是起源于结直肠黏膜腺上皮的良性肿瘤,但与结直肠癌的***密切,研究显示大约80%的结直肠癌由结直肠腺瘤发展而来,因此结直肠腺瘤被认为是结直肠癌的癌前病变。随着我国结直肠癌发病率及死亡率呈逐年上升趋势,研究显示及时发现和切除结直肠腺瘤可降低76%的结直肠癌发生率,因此研究结直肠腺瘤的诊断与治疗对于预防结直肠癌发生至关重要。
电子结肠镜检查有助于发现及观察结直肠粘膜病变,结合活组织检查能够提高检测结果的精确度及特异度,是结直肠腺瘤、结直肠癌等病变检出的重要手段,但病变检出率常常受检查医师经验、能力水平、电子结肠镜硬件设备、肠道清洁程度、受检者配合程度等多种因素影响,且常常因有创性操作而出现出血、穿孔等并发症。而目前临床上使用的无创性检测方法如粪便潜血检测、血液肿瘤标志物检测虽然应用广泛,但仍不具备良好的灵敏度及特异度,对于结直肠腺瘤的诊断只能作为一项参考指标。因此开发一种高灵敏度、特异度的无创结直肠腺瘤检测方法显得尤为重要。
作为近年来国内外的研究热点,细胞外囊泡(Extracellular Vesicles,EVs)开始逐渐受到人们重视,研究发现细胞外囊泡主要是由细胞分泌或由细胞膜脱落形成的的囊泡状小体,具有同细胞膜类似的双层膜结构,直径在30-1000nm不等。研究发现细胞外囊泡主要包含微囊泡(MicroVesicles,MVs)和外泌体(exosomes)两大部分,二者在经由细胞分泌后参与了体内多种多样的生物学功能,尤其是外泌体在疾病的发生发展中有着重要的意义。
外泌体本质上也属于细胞外囊泡,通常产生于细胞内多泡体,与细胞膜融合后被细胞分泌释放至细胞外,形成直径通常介于30~150nm之间的双层膜性小囊泡,研究显示外泌体可广泛分布于血液、唾液、尿液、乳汁和胸腹水等各种体液中,其内部富含DNA、RNA及蛋白质等多种活性物质,作为细胞间信息传递的重要媒介,可参与到人体内如抗原呈递、细胞凋亡、炎症反应、肿瘤发生发展及转移等多种病理生理过程。近年来的研究表明外泌体内部物质如miRNA、mRNA、LncRNA等分子与各***肿瘤及癌前病变的发生发展密切相关,在多种类型的肿瘤及其癌前病变的早期诊断中具有较大的潜力。
发明内容
发明人经过多年的研究,筛选获得了与结直肠腺瘤密切相关,能够用于临床诊断RNA分子标志物,其包括一个长链非编码RNAlnc-MKRN2-42:1两个基因的转录本(mRNA):TOP1和C19ORF43,以及一个miRNA:miR-106b-3p。所述RNA分子标志物的联合使用能够高灵敏度和特异性的指示结直肠腺瘤,具有优越的诊断性能。
本公开的第一个方面提供了一种用于结直肠腺瘤诊断、预后判断、疗效监测和/或复发监控的RNA分子标志物组合,其包括lnc-MKRN2-42:1、TOP1基因转录本、C19ORF43基因转录本和miR-106b-3p。
在一个实施方案中,所述lnc-MKRN2-42:1的序列如SEQ ID NO:14所示,具体为:
GCCGGGCACAGUGGCGCGUGCCUGUAGUCCCAGCUACUCGGGAGGCUGAGGUGGGAGGAUCGCUUGAGCCCAGGAGUUCUGGGCUGUAGUGCGCUAUGCCGAUCGGGUGUCCGCACUAAGUUCGGCAUCAAUAUGGUGACCUCCCGGGAGCGGGGGACCACCAGGUUGCCUAAGGAGGGGUGAACCGGCCCAGGUCGGAAACAGAGCAGGUCAAAACUCCCGUGCUGAUCAGGAGACAGAGUUUGUGAGCAGACAACUGGUCUGACCAAAAUUUAUGAGGUGGGAAUUUCCUCU(SEQ ID NO:14);
所述TOP1基因转录本的序列如SEQ ID NO:15所示,具体为:
CAAAUGCGAACUUAGGCUGUUACACAACUGCUGGGGUCUGUUCUCGCCGCCCGCCCGGCAGUCAGGCAGCGUCGCCGCCGUGGUAGCAGCCUCAGCCGUUUCUGGAGUCUCGGGCCCACAGUCACCGCCGCUUACCUGCGCCUCCUCGAGCCUCCGGAGUCCCCGUCCGCCCGCACAGGCCGGUUCGCCGUCUGCGUCUCCCCCACGCCGCCUCGCCUGCCGCCGCGCUCGUCCCUCCGGGCCGACAUGAGUGGGGACCACCUCCACAACGAUUCCCAGAUCGAAGCGGAUUUCCGAUUGAAUGAUUCUCAUAAACACAAAGAUAAACACAAAGAUCGAGAACACCGGCACAAAGAACACAAGAAGGAGAAGGACCGGGAAAAGUCCAAGCAUAGCAACAGUGAACAUAAAGAUUCUGAAAAGAAACACAAAGAGAAGGAGAAGACCAAACACAAAGAUGGAAGCUCAGAAAAGCAUAAAGACAAACAUAAAGACAGAGACAAGGAAAAACGAAAAGAGGAAAAGGUUCGAGCCUCUGGGGAUGCAAAAAUAAAGAAGGAGAAGGAAAAUGGCUUCUCUAGUCCACCACAAAUUAAAGAUGAACCUGAAGAUGAUGGCUAUUUUGUUCCUCCUAAAGAGGAUAUAAAGCCAUUAAAGAGACCUCGAGAUGAGGAUGAUGCUGAUUAUAAACCUAAGAAAAUUAAAACAGAAGAUACCAAGAAGGAGAAGAAAAGAAAACUAGAAGAAGAAGAGGAUGGUAAAUUGAAAAAACCCAAGAAUAAAGAUAAAGAUAAAAAAGUUCCUGAGCCAGAUAACAAGAAAAAGAAGCCGAAGAAAGAAGAGGAACAGAAGUGGAAAUGGUGGGAAGAAGAGCGCUAUCCUGAAGGCAUCAAGUGGAAAUUCCUAGAACAUAAAGGUCCAGUAUUUGCCCCACCAUAUGAGCCUCUUCCAGAGAAUGUCAAGUUUUAUUAUGAUGGUAAAGUCAUGAAGCUGAGCCCCAAAGCAGAGGAAGUAGCUACGUUCUUUGCAAAAAUGCUCGACCAUGAAUAUACUACCAAGGAAAUAUUUAGGAAAAAUUUCUUUAAAGACUGGAGAAAGGAAAUGACUAAUGAAGAGAAGAAUAUUAUCACCAACCUAAGCAAAUGUGAUUUUACCCAGAUGAGCCAGUAUUUCAAAGCCCAGACGGAAGCUCGGAAACAGAUGAGCAAGGAAGAGAAACUGAAAAUCAAAGAGGAGAAUGAAAAAUUACUGAAAGAAUAUGGAUUCUGUAUUAUGGAUAACCACAAAGAGAGGAUUGCUAACUUCAAGAUAGAGCCUCCUGGACUUUUCCGUGGCCGCGGCAACCACCCCAAGAUGGGCAUGCUGAAGAGACGAAUCAUGCCCGAGGAUAUAAUCAUCAACUGUAGCAAAGAUGCCAAGGUUCCUUCUCCUCCUCCAGGACAUAAGUGGAAAGAAGUCCGGCAUGAUAACAAGGUUACUUGGCUGGUUUCCUGGACAGAGAACAUCCAAGGUUCCAUUAAAUACAUCAUGCUUAACCCUAGUUCACGAAUCAAGGGUGAGAAGGACUGGCAGAAAUACGAGACUGCUCGGCGGCUGAAAAAAUGUGUGGACAAGAUCCGGAACCAGUAUCGAGAAGACUGGAAGUCCAAAGAGAUGAAAGUCCGGCAGAGAGCUGUAGCCCUGUACUUCAUCGACAAGCUUGCUCUGAGAGCAGGCAAUGAAAAGGAGGAAGGAGAAACAGCGGACACUGUGGGCUGCUGCUCACUUCGUGUGGAGCACAUCAAUCUACACCCAGAGUUGGAUGGUCAGGAAUAUGUGGUAGAGUUUGACUUCCUCGGGAAGGACUCCAUCAGAUACUAUAACAAGGUCCCUGUUGAGAAACGAGUUUUUAAGAACCUACAACUAUUUAUGGAGAACAAGCAGCCCGAGGAUGAUCUUUUUGAUAGACUCAAUACUGGUAUUCUGAAUAAGCAUCUUCAGGAUCUCAUGGAGGGCUUGACAGCCAAGGUAUUCCGUACAUACAAUGCCUCCAUCACGCUACAGCAGCAGCUAAAAGAACUGACAGCCCCGGAUGAGAACAUCCCAGCGAAGAUCCUUUCUUAUAACCGUGCCAAUCGAGCUGUUGCAAUUCUUUGUAACCAUCAGAGGGCACCACCAAAAACUUUUGAGAAGUCUAUGAUGAACUUGCAAACUAAGAUUGAUGCCAAGAAGGAACAGCUAGCAGAUGCCCGGAGAGACCUGAAAAGUGCUAAGGCUGAUGCCAAGGUCAUGAAGGAUGCAAAGACGAAGAAGGUAGUAGAGUCAAAGAAGAAGGCUGUUCAGAGACUGGAGGAACAGUUGAUGAAGCUGGAAGUUCAAGCCACAGACCGAGAGGAAAAUAAACAGAUUGCCCUGGGAACCUCCAAACUCAAUUAUCUGGACCCUAGGAUCACAGUGGCUUGGUGCAAGAAGUGGGGUGUCCCAAUUGAGAAGAUUUACAACAAAACCCAGCGGGAGAAGUUUGCCUGGGCCAUUGACAUGGCUGAUGAAGACUAUGAGUUUUAGCCAGUCUCAAGAGGCAGAGUUCUGUGAAGAGGAACAGUGUGGUUUGGGAAAGAUGGAUAAACUGAGCCUCACUUGCCCUCGUGCCUGGGGGAGAGAGGCAGCAAGUCUUAACAAACCAACAUCUUUGCGAAAAGAUAAACCUGGAGAUAUUAUAAGGGAGAGCUGAGCCAGUUGUCCUAUGGACAACUUAUUUAAAAAUAUUUCAGAUAUCAAAAUUCUAGCUGUAUGAUUUGUUUUGAAUUUUGUUUUUAUUUUCAAGAGGGCAAGUGGAUGGGAAUUUGUCAGCGUUCUACCAGGCAAAUUCACUGUUUCACUGAAAUGUUUGGAUUCUCUUAGCUACUGUAUGCAAAGUCCGAUUAUAUUGGUGCGUUUUUACAGUUAGGGUUUUGCAAUAACUUCUAUAUUUUAAUAGAAAUAAAUUCCUAAACUCCCUUCCCUCUCUCCCAUUUCAGGAAUUUAAAAUUAAGUAGAACAAAAAACCCAGCGCACCUGUUAGAGUCGUCACUCUCUAUUGUCAUGGGGAUCAAUUUUCAUUAAACUUGAAGCAGUCGUGGCUUUGGCAGUGUUUUGGUUCAGACACCUGUUCACAGAAAAAGCAUGAUGGGAAAAUAUUUCCUGACUUGAGUGUUCCUUUUUAAAUGUGAAUUUUUAUUUCUUUUUAAUUAUUUUAAAAUAUUUAAACCUUUUUCUUGAUCUUAAAGAUCGUGUAGAUUGGGGUUGGGGAGGGAUGAAGGGCGAGUGAAUCUAAGGAUAAUGAAAUAAUCAGUGACUGAAACCAUUUUCCCAUCAUCCUUUGUUCUGAGCAUUCGCUGUACCCUUUAAGAUAUCCAUCUUUUUCUUUUUAACCCUAAUCUUUCACUUGAAAGAUUUUAUUGUAUAAAAAGUUUCACAGGUCAAUAAACUUAGAGGAAAAUGAGUAUUUGGUCCAAAAAAAGGAAAAAUAAUCAAGAUUUUAGGGCUUUUAUUUUUUCUUUUGUAAUUGUGUAAAAAAUGGAAAAAAACAUAAAAAGCAGAAUUUUAAUGUGAAGACAUUUUUUGCUAUAAUCAUUAGUUUUAGAGGCAUUGUUAGUUUAGUGUGUGUGCAGAGUCCAUUUCCCACAUCUUUCCUCAAGUAUCUUCUAUUUUUAUCAUGAAUUCCCUUUUAAUCAACUGUAGGUUAUUUAAAAUAAAUUCCUACAACUUAAUGGAAA(SEQ ID NO:15);
所述C19ORF43基因转录本的序列如SEQ ID NO:16所示,具体为:
GUCCUUUGCGCGGCACCUGGCGACAAAAUGGCUGCCCGAGGGAGACGGGCGGAGCCUCAGGGCCGGGAGGCUCCGGGCCCCGCGGGCGGUGGCGGUGGCGGGAGCCGUUGGGCUGAGUCGGGAUCGGGGACGUCGCCCGAGAGCGGGGACGAGGAGGUGUCGGGCGCGGGUUCGAGCCCGGUGUCGGGCGGCGUGAACUUGUUCGCCAACGACGGCAGCUUCCUGGAGCUGUUCAAGCGGAAGAUGGAGGAGGAGCAGCGGCAGCGGCAGGAGGAGCCGCCCCCGGGUCCGCAGCGACCCGACCAGUCGGCCGCCGCCGCUGGCCCCGGGGAUCCGAAGAGGAAGGGCGGUCCGGGCUCCACACUUAGCUUCGUGGGCAAACGCAGAGGCGGGAACAAACUAGCCCUCAAGACGGGAAUAGUAGCCAAGAAGCAGAAGACGGAGGAUGAGGUAUUAACAAGUAAAGGUGACGCGUGGGCCAAGUACAUGGCAGAAGUGAAAAAGUACAAAGCUCACCAGUGCGGUGACGAUGAUAAAACUCGGCCCCUGGUGAAAUGACGCCCCUCCCCCACCUGCCCAUGGCCUGGGACUCUCUGCGAUGUACAUAACUAUUUAAUGCAGCGGCAGCGGCGACAGCCUUCCCUGAGAGGACUUAAAAGCAGAAGGAAACCGAGAUGCUUCCCGCAGCCGUGGACGAUUCUCCAGGACUCUUUUUUUACCUUGAGCACUUGCCUCGUGAGACUUCAUAGAACAGUGGUUUACUGUCCCCCCCUUCUCACCUCCUCAUUCUCUCUGGCUCUUUCUGUCUUCCUCUUCUCACCCUCCUCCCUCCCCUUAGCCAUCACUUCUGGGAAGUAAAGAACUUGACUUAGUGCCGGA(SEQ ID NO:16);和
所述miR-106b-3p的序列如SEQ ID NO:17所示,具体为:
CCGCACUGUGGGUACUUGCUGC(SEQ ID NO:17)。
在另一个实施方案中,所述RNA分子标志物来自于外泌体,优选地,所述外泌体来自于体液,例如血液、尿液、唾液或痰液。
本公开的第二个方面提供了一种用于检测上述第一个方面任一实施方案的RNA分子标志物的试剂,所述试剂例如引物和/或探针。
在一个实施方案中,检测lnc-MKRN2-42:1的引物为:如SEQ ID NO:1所示的上游引物序列,以及如SEQ ID NO:2所示的下游引物序列;检测lnc-MKRN2-42:1的探针序列如SEQID NO:3所示;
检测TOP1基因转录本的引物为:如SEQ ID NO:4示的上游引物序列,以及如SEQ IDNO:5示的下游引物序列;检测TOP1基因转录本的探针序列如SEQ ID NO:6所示;
检测C19ORF43基因转录本的引物为:如SEQ ID NO:7所示的上游引物序列,以及如SEQ ID NO:8所示的下游引物序列;检测C19ORF43基因转录本的探针序列如SEQ ID NO:9所示;和
检测miR-106b-3p的引物为:如SEQ ID NO:11所示的上游引物序列,以及如SEQ IDNO:13所示的下游引物序列;检测miR-106b-3p探针序列如SEQ ID NO:12所示。
本公开的第三个方面提供了上述第一个方面的RNA分子标志物或者第二个方面所述的试剂在制备用于结直肠腺瘤诊断、预后判断、疗效监测和/或复发监控的试剂盒中的应用。
本公开的四个方面提供了一种用于结直肠腺瘤诊断、预后判断、疗效监测和/或复发监控的试剂盒,其包括上述第一个方面所述的RNA分子标志物或者上述第二个方面所述的试剂,任选地,所述试剂盒还包括RNA提取试剂、反转录试剂、和/或PCR扩增试剂。
本公开的第五个方面提供了一种结直肠腺瘤诊断、预后判断、疗效监测和/或复发监控的方法,其包括下列步骤:
(1)采集待检测对象的体液样本,例如血液、尿液、痰液和唾液
(2)从上述体液样本中分离外泌体;
(3)提取外泌体RNA;
(4)检测包含长单链非编码RNA lnc-MKRN2-42:1、TOP1基因转录本、C19ORF43基因转录本和miR-106b-3p的RNA分子标志物组合的表达水平,优选进一步与健康对照的表达水平相比较,以判断所述对象是否患有结直肠腺瘤或患有结直肠腺瘤的风险大小、结直肠腺瘤的治疗效果和/或预后情况,和/或结直肠腺瘤是否复发或者复发风险的大小。
在一个实施方案中,所述步骤(4)的检测包括反转录和定量PCR的步骤,优选地,所述定量PCR所使用的试剂为前述第二个方面所述的试剂。
在另一个实施方案中,步骤(4)还包括利用外参基因(优选为cel-miR-39)对所述表达水平进行归一化的步骤;优选地,进一步将上述归一化后的分子标志物的表达水平值进行逻辑(logistic)回归处理得到输出值,并将所述输出值与判定阈值进行比较,其中当输出值高于判定阈值时,说明所述待检测对象患有结直肠腺瘤或者患有结直肠腺瘤的风险较高、结直肠腺瘤的治疗效果和/或预后情况不佳,和/或结直肠腺瘤在治疗后发生了复发或者复发风险较高。
本发明提供的上述肿瘤标志物、试剂盒和检测方法,从而实现了基于基于病人血浆的无创检测方法,就快速、高效的特点,病人依从性好,对早期结直肠腺瘤种具有极高的灵敏度和特异性,对结直肠腺瘤的诊断具有重要价值。
附图说明
图1显示了lnc-MKRN2-42:1、TOP1、C19ORF43和miR-106b-3p联合诊断早期结直肠腺瘤的性能。
具体实施方式
还可进一步通过实施例来理解本发明,然而,要理解的是,这些实施例不限制本发明。现在已知的或进一步开发的本发明的变化被认为落入本文中描述的和以下要求保护的本发明范围之内。
定义
术语“结直肠腺瘤”是指位于源于结直肠部位黏膜腺上皮组织的肿瘤性息肉。在本发明中“结直肠腺瘤”可以包括仅在结肠部位发生腺瘤(也可被称为“结肠腺瘤”)、仅在直肠部位发生腺瘤(也可称为“直肠腺瘤”)或者结肠直肠同时出现腺瘤的情形。
术语“RNA分子标志物”是与特定的疾病、适应症或者性状相关联,能够用于指示该特定疾病、适应症或性状的RNA分子。在本发明中,所述RNA分子标志物用于指示结直肠腺瘤,因此可作为结直肠腺瘤RNA分子标志物。
在本发明中,RNA分子标志物包括长链非编码RNA,具体为lnc-MKRN2-42:1,示例性的序列如SEQ ID NO:14所示;和mRNA,具体为TOP1和C19ORF43;以及miRNA,具体为miR-106b-3p,示例性的序列如SEQ ID NO:17所示。其中,本发明作为RNA分子标志物的TOP1基因转录本即表示TOP1基因转录得到的mRNA分子,示例性的序列如SEQ ID NO:15所示;C19ORF43基因转录本即表示C19ORF43基因转录得到的mRNA分子,示例性的序列如SEQ IDNO:16所示。
需要说明的是,在实际检测中的lncRNA、miRNA以及基因的转录本序列可以和示例性的序列不同,这是因为人体基因广泛的存在着序列多态性的现象,因此检测的序列可以是上述示例性序列的变体序列,例如包括一个或更多个核苷酸的改变、***或缺失。
术语“外泌体”,是指由细胞内多泡体与细胞膜融合后被细胞分泌释放至细胞外的小型细胞外囊泡,其具有双层膜结构。本发明中作为RNA分子标志物的RNA分子即存在于并分离自外泌体。
实施例
实施例1基于高通量测序筛选与结直肠腺瘤相关的外泌体RNA分子标志物
取来自于15例早期结直肠腺瘤患者和15例对照组健康受试者的血液不少于10mL,分离血浆后利用经典超速离心法(Evaluation of circulating small extracellularvesicles derived miRNAs as biomarkers of early colon cancer:a comparison withplasma total miRNAs,L Min,S Zhu,L Chen,X Liu,R Wei,L Zhao,Y Yang,Z Zhang,GKong,Journal of extracellular vesicles 8(1),1643670)分离血浆中的外泌体,采用QIAGEN miRNeasy mini kit提取RNA,对制备的的RNA进行微量链特异性RNA建库测序(Evaluation of circulating small extracellular vesicles derived miRNAs asbiomarkers of early colon cancer:a comparison with plasma total miRNAs,L Min,S Zhu,L Chen,X Liu,R Wei,L Zhao,Y Yang,Z Zhang,G Kong,Journal ofextracellular vesicles 8(1),1643670)。对测序结果进行生物信息学分析,比较早期结直肠腺瘤患者中相比于对照组具有差异表达的RNA,最终鉴定得到在两组中具有显著差异表达的RNA分子标志物,包括一个lncRNA,一个miRNA和两个基因的转录本(mRNA),具体如表1所示。
表1 RNA分子标志物列表
实施例2基于荧光定量PCR平台的miRNA、mRNA及lncRNA检测体系
1.miRNA的检测体系
1)miRNA逆转录反应体系
miRNA反转录试剂、酶和oligdT从TAKARA采购,标准品从上海英潍捷基合成,特异性反转录的引物由苏州泓迅合成。采用20μL的反转录体系,如表2所示。
表2 miRNA的反转录体系
2)qPCR反应体系:
PCR反应混合液从TAKARA采购,上游引物、探针即通用下游引物由苏州泓迅合成、荧光定量PCR仪为ABI 7500。PCR反应体系如表3所示:
表3 miRNA的qPCR反应体系
2.mRNA及lncRNA检测体系
1)反转录体系
采用TAKARA公司的PrimeScriptTMRT reagent Kit(Perfect Real Time)和PremixEx TaqTM(Probe qPCR)试剂盒进行反转录和qPCR检测。
按表4所列组份配制反转录反应体系(反应液配制在冰上进行),然后放入PCR仪进行反应,反应条件为37℃60min,85℃5s,12℃∞,反转录结束后加入50μL DEPC-H2O稀释,取3uL作为模板,进行PCR反应。
表4长链RNA的反转录体系
2)qPCR体系
所述检测外泌体miRNA分子标志物的引物和探针包括:
用于检测lnc-MKRN2-42:1的引物和探针:所述lnc-MKRN2-42:1的上游引物具体为如SEQ ID NO:1所示的核苷酸序列,下游引物具体为如SEQ ID NO:2所示的核苷酸序列,探针为如SEQ ID NO:3所示的核苷酸序列;
用于检测TOP1基因转录本的引物和探针:所述TOP1的上游引物具体为如SEQ IDNO:4所示的核苷酸序列,下游引物具体为如SEQ ID NO:5所示的核苷酸序列,探针为如SEQID NO:6所示的核苷酸序列;
用于检测C19ORF43基因转录本的引物和探针:所述C19ORF43的上游引物具体为如SEQ ID NO:7所示的核苷酸序列,下游引物具体为如SEQ ID NO:8所示的核苷酸序列,探针为如SEQ ID NO:9所示的核苷酸序列。
用于检测miR-106b-3p的引物和探针:所述miR-106b-3p的逆转录引物为如SEQ IDNO.10所示;上游引物具体为如SEQ ID NO:11所示的核苷酸序列,下游引物具体为如SEQ IDNO:13所示的核苷酸序列,探针为如SEQ ID NO:12所示的核苷酸序列。
所有上述引物和探针序列如表5所示。
表5实施例所用引物和探针序列
按表6所列组分配制qPCR反应体系(反应液配制在冰上进行),设置无模板对照作为阴性对照。然后放入实时荧光PCR仪(ABI7500)按如下反应条件进行扩增检测。
表6长链RNA的qPCR反应体系
表7长链RNA的qPCR反应条件
实施例3结直肠腺瘤早期诊断检测试剂盒效果评估
1)样本采集
收集经医院确诊的早期(I期和II期)结直肠腺瘤患者(24例)、结直肠良性病人和健康人对照样本(合计53例)的血液10mL,分离血浆。
2)外泌体RNA提取
采用Qiagen公司商业化ExoEasy试剂盒或恩泽康泰公司的Exosupur进行血浆外泌体分离,分离后的外泌体用Qiagen miReasy mini kit试剂盒提取外泌体中的RNA,并利用Agilent 2100检测RNA浓度和质量,记录RNA浓度。
3)RNA两步法检测体系
采用实施例2中基于荧光定量PCR平台的mRNA及lncRNA检测体系,对步骤2)中提取的血浆外泌体RNA进行检测,检测目标RNA的Ct值,根据Ct值和相对定量公式计算相对表达量。
4)结果:
对早期结直肠腺瘤病人24例和53例对照样本(健康人和良性病变)的血浆外泌体,检测lnc-MKRN2-42:1、miR-106b-3p、TOP1和C19ORF43的Ct值,所有样本的检测结果如表8所示。进一步,根据目标基因Ct值及参考RNACel-39-3p的Ct值计算目标RNA相对参考基因的ΔCt(如表9所示)。
表8各样本中所有RNA标记物的检测Ct值
表9目标RNA相对参考基因的ΔCt值
标志物lnc-MKRN2-42:1、miR-106b-3p、TOP1、C19ORF43的联合检测
使用相对定量公式值2-ΔCt,计算每个RNA分子标志物相对表达量的倍数变化,即可知晓每个RNA分子标志物相对于参照基因的相对表达量。在此基础上,进一步采用逻辑回归进行训练(使用R语言glm函数),并使用ROC特征曲线和AUC(使用R语言roc函数)来评估上述RNA分子标志物联合应用于检测结直肠腺瘤的准确度。
如图1所示,基于此前表8和表9的检测结果,采用R语言进行t检测分析得到p<=0.05,说明联合标志物与早期结直肠腺瘤显著相关。结果显示,lnc-MKRN2-42:1、miR-106b-3p、TOP1和C19ORF43联合诊断早期结直肠腺瘤的AUC为0.83,其阴性预测值为94.87%、灵敏度为91.67%、特异性为69.81%。灵敏度和特异性均满足临床应用的要求,可以作为有效的临床诊断标志物。
序列表
<110> 首都医科大学附属北京友谊医院
<120> 用于结直肠腺瘤诊断的外泌体RNA分子标记物组合及其应用
<130> MP1928955
<160> 17
<170> PatentIn version 3.2
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<212> DNA
<213> 人工序列
<400> 8
gaggaagaca gaaagagcca gag 23
<210> 9
<211> 22
<212> DNA
<213> 人工序列
<400> 9
tcccgcagcc gtggacgatt ct 22
<210> 10
<211> 19
<212> DNA
<213> 人工序列
<400> 10
cgcctgggtt ccacttcca 19
<210> 11
<211> 18
<212> DNA
<213> 人工序列
<400> 11
caccgcactg tgggtact 18
<210> 12
<211> 23
<212> DNA
<213> 人工序列
<400> 12
attcgcactg gatacgacgc tgc 23
<210> 13
<211> 16
<212> DNA
<213> 人工序列
<400> 13
gtgcagggtc cgaggt 16
<210> 14
<211> 294
<212> RNA
<213> 人
<400> 14
gccgggcaca guggcgcgug ccuguagucc cagcuacucg ggaggcugag gugggaggau 60
cgcuugagcc caggaguucu gggcuguagu gcgcuaugcc gaucgggugu ccgcacuaag 120
uucggcauca auauggugac cucccgggag cgggggacca ccagguugcc uaaggagggg 180
ugaaccggcc caggucggaa acagagcagg ucaaaacucc cgugcugauc aggagacaga 240
guuugugagc agacaacugg ucugaccaaa auuuaugagg ugggaauuuc cucu 294
<210> 15
<211> 3734
<212> RNA
<213> 人
<400> 15
caaaugcgaa cuuaggcugu uacacaacug cuggggucug uucucgccgc ccgcccggca 60
gucaggcagc gucgccgccg ugguagcagc cucagccguu ucuggagucu cgggcccaca 120
gucaccgccg cuuaccugcg ccuccucgag ccuccggagu ccccguccgc ccgcacaggc 180
cgguucgccg ucugcgucuc ccccacgccg ccucgccugc cgccgcgcuc gucccuccgg 240
gccgacauga guggggacca ccuccacaac gauucccaga ucgaagcgga uuuccgauug 300
aaugauucuc auaaacacaa agauaaacac aaagaucgag aacaccggca caaagaacac 360
aagaaggaga aggaccggga aaaguccaag cauagcaaca gugaacauaa agauucugaa 420
aagaaacaca aagagaagga gaagaccaaa cacaaagaug gaagcucaga aaagcauaaa 480
gacaaacaua aagacagaga caaggaaaaa cgaaaagagg aaaagguucg agccucuggg 540
gaugcaaaaa uaaagaagga gaaggaaaau ggcuucucua guccaccaca aauuaaagau 600
gaaccugaag augauggcua uuuuguuccu ccuaaagagg auauaaagcc auuaaagaga 660
ccucgagaug aggaugaugc ugauuauaaa ccuaagaaaa uuaaaacaga agauaccaag 720
aaggagaaga aaagaaaacu agaagaagaa gaggauggua aauugaaaaa acccaagaau 780
aaagauaaag auaaaaaagu uccugagcca gauaacaaga aaaagaagcc gaagaaagaa 840
gaggaacaga aguggaaaug gugggaagaa gagcgcuauc cugaaggcau caaguggaaa 900
uuccuagaac auaaaggucc aguauuugcc ccaccauaug agccucuucc agagaauguc 960
aaguuuuauu augaugguaa agucaugaag cugagcccca aagcagagga aguagcuacg 1020
uucuuugcaa aaaugcucga ccaugaauau acuaccaagg aaauauuuag gaaaaauuuc 1080
uuuaaagacu ggagaaagga aaugacuaau gaagagaaga auauuaucac caaccuaagc 1140
aaaugugauu uuacccagau gagccaguau uucaaagccc agacggaagc ucggaaacag 1200
augagcaagg aagagaaacu gaaaaucaaa gaggagaaug aaaaauuacu gaaagaauau 1260
ggauucugua uuauggauaa ccacaaagag aggauugcua acuucaagau agagccuccu 1320
ggacuuuucc guggccgcgg caaccacccc aagaugggca ugcugaagag acgaaucaug 1380
cccgaggaua uaaucaucaa cuguagcaaa gaugccaagg uuccuucucc uccuccagga 1440
cauaagugga aagaaguccg gcaugauaac aagguuacuu ggcugguuuc cuggacagag 1500
aacauccaag guuccauuaa auacaucaug cuuaacccua guucacgaau caagggugag 1560
aaggacuggc agaaauacga gacugcucgg cggcugaaaa aaugugugga caagauccgg 1620
aaccaguauc gagaagacug gaaguccaaa gagaugaaag uccggcagag agcuguagcc 1680
cuguacuuca ucgacaagcu ugcucugaga gcaggcaaug aaaaggagga aggagaaaca 1740
gcggacacug ugggcugcug cucacuucgu guggagcaca ucaaucuaca cccagaguug 1800
gauggucagg aauauguggu agaguuugac uuccucggga aggacuccau cagauacuau 1860
aacaaggucc cuguugagaa acgaguuuuu aagaaccuac aacuauuuau ggagaacaag 1920
cagcccgagg augaucuuuu ugauagacuc aauacuggua uucugaauaa gcaucuucag 1980
gaucucaugg agggcuugac agccaaggua uuccguacau acaaugccuc caucacgcua 2040
cagcagcagc uaaaagaacu gacagccccg gaugagaaca ucccagcgaa gauccuuucu 2100
uauaaccgug ccaaucgagc uguugcaauu cuuuguaacc aucagagggc accaccaaaa 2160
acuuuugaga agucuaugau gaacuugcaa acuaagauug augccaagaa ggaacagcua 2220
gcagaugccc ggagagaccu gaaaagugcu aaggcugaug ccaaggucau gaaggaugca 2280
aagacgaaga agguaguaga gucaaagaag aaggcuguuc agagacugga ggaacaguug 2340
augaagcugg aaguucaagc cacagaccga gaggaaaaua aacagauugc ccugggaacc 2400
uccaaacuca auuaucugga cccuaggauc acaguggcuu ggugcaagaa gugggguguc 2460
ccaauugaga agauuuacaa caaaacccag cgggagaagu uugccugggc cauugacaug 2520
gcugaugaag acuaugaguu uuagccaguc ucaagaggca gaguucugug aagaggaaca 2580
gugugguuug ggaaagaugg auaaacugag ccucacuugc ccucgugccu gggggagaga 2640
ggcagcaagu cuuaacaaac caacaucuuu gcgaaaagau aaaccuggag auauuauaag 2700
ggagagcuga gccaguuguc cuauggacaa cuuauuuaaa aauauuucag auaucaaaau 2760
ucuagcugua ugauuuguuu ugaauuuugu uuuuauuuuc aagagggcaa guggauggga 2820
auuugucagc guucuaccag gcaaauucac uguuucacug aaauguuugg auucucuuag 2880
cuacuguaug caaaguccga uuauauuggu gcguuuuuac aguuaggguu uugcaauaac 2940
uucuauauuu uaauagaaau aaauuccuaa acucccuucc cucucuccca uuucaggaau 3000
uuaaaauuaa guagaacaaa aaacccagcg caccuguuag agucgucacu cucuauuguc 3060
auggggauca auuuucauua aacuugaagc agucguggcu uuggcagugu uuugguucag 3120
acaccuguuc acagaaaaag caugauggga aaauauuucc ugacuugagu guuccuuuuu 3180
aaaugugaau uuuuauuucu uuuuaauuau uuuaaaauau uuaaaccuuu uucuugaucu 3240
uaaagaucgu guagauuggg guuggggagg gaugaagggc gagugaaucu aaggauaaug 3300
aaauaaucag ugacugaaac cauuuuccca ucauccuuug uucugagcau ucgcuguacc 3360
cuuuaagaua uccaucuuuu ucuuuuuaac ccuaaucuuu cacuugaaag auuuuauugu 3420
auaaaaaguu ucacagguca auaaacuuag aggaaaauga guauuugguc caaaaaaagg 3480
aaaaauaauc aagauuuuag ggcuuuuauu uuuucuuuug uaauugugua aaaaauggaa 3540
aaaaacauaa aaagcagaau uuuaauguga agacauuuuu ugcuauaauc auuaguuuua 3600
gaggcauugu uaguuuagug ugugugcaga guccauuucc cacaucuuuc cucaaguauc 3660
uucuauuuuu aucaugaauu cccuuuuaau caacuguagg uuauuuaaaa uaaauuccua 3720
caacuuaaug gaaa 3734
<210> 16
<211> 879
<212> RNA
<213> 人
<400> 16
guccuuugcg cggcaccugg cgacaaaaug gcugcccgag ggagacgggc ggagccucag 60
ggccgggagg cuccgggccc cgcgggcggu ggcgguggcg ggagccguug ggcugagucg 120
ggaucgggga cgucgcccga gagcggggac gaggaggugu cgggcgcggg uucgagcccg 180
gugucgggcg gcgugaacuu guucgccaac gacggcagcu uccuggagcu guucaagcgg 240
aagauggagg aggagcagcg gcagcggcag gaggagccgc ccccgggucc gcagcgaccc 300
gaccagucgg ccgccgccgc uggccccggg gauccgaaga ggaagggcgg uccgggcucc 360
acacuuagcu ucgugggcaa acgcagaggc gggaacaaac uagcccucaa gacgggaaua 420
guagccaaga agcagaagac ggaggaugag guauuaacaa guaaagguga cgcgugggcc 480
aaguacaugg cagaagugaa aaaguacaaa gcucaccagu gcggugacga ugauaaaacu 540
cggccccugg ugaaaugacg ccccuccccc accugcccau ggccugggac ucucugcgau 600
guacauaacu auuuaaugca gcggcagcgg cgacagccuu cccugagagg acuuaaaagc 660
agaaggaaac cgagaugcuu cccgcagccg uggacgauuc uccaggacuc uuuuuuuacc 720
uugagcacuu gccucgugag acuucauaga acagugguuu acuguccccc ccuucucacc 780
uccucauucu cucuggcucu uucugucuuc cucuucucac ccuccucccu ccccuuagcc 840
aucacuucug ggaaguaaag aacuugacuu agugccgga 879
<210> 17
<211> 22
<212> RNA
<213> 人
<400> 17
ccgcacugug gguacuugcu gc 22
Claims (8)
1.一种用于结直肠腺瘤诊断的RNA分子标志物组合,其包括lnc-MKRN2-42:1、TOP1基因转录本、C19ORF43基因转录本和miR-106b-3p,
其中,所述TOP1基因转录本的序列如SEQ ID NO:15所示,所述C19ORF43基因转录本的序列如SEQ ID NO:16所示。
2.根据权利要求1所述的RNA分子标志物组合,所述lnc-MKRN2-42:1的序列如SEQ IDNO:14所示以及miR-106b-3p的序列如SEQ ID NO:17所示。
3.根据权利要求1所述的RNA分子标志物组合,其中所述RNA分子标志物存在于外泌体,所述外泌体来自体液。
4.根据权利要求3所述的RNA分子标志物组合,其中,所述体液为血液、尿液、唾液或痰液。
5.一种用于检测权利要求1所述RNA分子标志物组合的试剂,所述试剂包括引物和/或探针,其中
检测lnc-MKRN2-42:1的引物为:如SEQ ID NO:1所示的上游引物序列,以及如SEQ IDNO:2所示的下游引物序列;检测lnc-MKRN2-42:1的探针序列如SEQ ID NO:3所示;
检测TOP1基因转录本的引物为:如SEQ ID NO:4示的上游引物序列,以及如SEQ ID NO:5示的下游引物序列;检测TOP1的探针序列如SEQ ID NO:6所示;
检测C19ORF43基因转录本的引物为:如SEQ ID NO:7所示的上游引物序列,以及如SEQID NO:8所示的下游引物序列;检测C19ORF43基因转录本的探针序列如SEQ ID NO:9所示;和
检测miR-106b-3p的引物为:如SEQ ID NO:11所示的上游引物序列,以及如SEQ ID NO:13所示的下游引物序列;检测miR-106b-3p探针序列如SEQ ID NO:12所示。
6.根据权利要求1至4中任一项所述的RNA分子标志物组合或者权利要求5所述的试剂在制备用于结直肠腺瘤诊断的试剂盒中的应用。
7.一种用于结直肠腺瘤诊断的试剂盒,其包括权利要求1至4中任一项所述的RNA分子标志物组合或者权利要求5所述的试剂。
8.根据权利要求7所述的试剂盒,所述试剂盒还包括RNA提取试剂、反转录试剂、和/或PCR扩增试剂。
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