CN111485021B - 用于结直肠腺瘤诊断的外泌体rna及其应用 - Google Patents
用于结直肠腺瘤诊断的外泌体rna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分子标志物及其应用。
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分子标志物及其应用。
背景技术
结直肠腺瘤起源于结直肠黏膜腺上皮组织,从组织学角度上属于肿瘤性息肉,文献报道其癌变率在2.9%-9.4%,尤其是个别病理类型如绒毛管状腺瘤癌变率更甚。有报道称大部分结直肠癌均由结直肠腺瘤发展而来,因此目前普遍认为结直肠腺瘤是结直肠癌的癌前病变,与结直肠癌的发生发展密切相关。而在我国,流行病学显示结直肠癌发病率及死亡率逐年上升,及时地发现和治疗结直肠腺瘤可降低结直肠癌发生率及死亡率,因此防治结直肠腺瘤在预防结直肠癌的发生发展中占据重要地位。
目前临床上主要通过结肠镜检查来明确结直肠病变,并通过对病变进行活组织检查来判断良恶性,进一步指导临床诊疗。但结直肠腺瘤直径通常较小,时常位于结肠粘膜皱襞间导致漏诊,而且结肠镜下的病变检出率高低取决于检查医师能力及经验、结肠镜配置高低、受试者肠道清洁程度、受检者配合程度等多种因素,且具有一定几率发生出血、穿孔等严重并发症,在所有人群中进行此检查受到一定的限制。目前临床上存在的无创性检测手段包括粪便潜血检测、血液肿瘤标志物检测等,虽然检测方法简单、受试者接受程度高、风险低,但在病变检出的灵敏度及特异度仍存在一定不足,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)的出现使人们更进一步加深了对细胞间相互作用的认识。外泌体(Exosome)是细胞外囊泡中最受关注的类型,其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,外泌体外层是与细胞膜相似的双层膜结构,直径通常介于30~150nm之间。多年来研究发现外泌体内部富含DNA、RNA及蛋白质等多种活性物质,被细胞释放后可广泛分布于血液、唾液、尿液、乳汁和胸腹水等各种体液中,研究发现外泌体作为细胞间信息传递的重要媒介,可参与到人体内多种病理生理过程,如抗原呈递、细胞凋亡、炎症反应、肿瘤发生发展及转移等。
由于外泌体内部携带的RNA被磷脂双分子层包裹,其对于血液环境中的蛋白酶与RNA酶降解有一定的抵御能力,其作为诊断检测靶标具有天然的优势。目前对于外泌体中较短的miRNA作为肿瘤标记物的研究较多,而对于长链RNA(包括mRNA、lncRNA)由于其丰度较低、更易于降解,对其实现检测具有更高技术难度,考虑到长链RNA的种类与多样性远高于miRNA,对其作为标记物的潜力进行挖掘与开发具有明显的商业意义与社会价值。
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以及三个基因的转录本:PPDPF、TOP1和C19ORF43。所述RNA分子标志物不论单独使用还是部分或者全部的联合使用均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,其包括选自lnc-MKRN2-42:1、PPDPF基因转录本、TOP1基因转录本和C19ORF43基因转录本中的一种或更多种(例如两种、三种或四种)RNA分子的组合。
在一个实施方案中,所述lnc-MKRN2-42:1的序列如SEQ ID NO:13所示,具体为;
GCCGGGCACAGUGGCGCGUGCCUGUAGUCCCAGCUACUCGGGAGGCUGAGGUGGGAGGAUCGCUUGAGCCCAGGAGUUCUGGGCUGUAGUGCGCUAUGCCGAUCGGGUGUCCGCACUAAGUUCGGCAUCAAUAUGGUGACCUCCCGGGAGCGGGGGACCACCAGGUUGCCUAAGGAGGGGUGAACCGGCCCAGGUCGGAAACAGAGCAGGUCAAAACUCCCGUGCUGAUCAGGAGACAGAGUUUGUGAGCAGACAACUGGUCUGACCAAAAUUUAUGAGGUGGGAAUUUCCUCU(SEQ ID NO:13);
PPDPF基因转录本的的序列如SEQ ID NO:14所示,具体为:
ACCCCGCGCGCGCCUCUCUGUCGUGGCGCGGCUUCCCGCGGUCUUCUCUGCAAAUGGGCUCCGUGGCCUAGCGCCCCCGUCCCCGCCACCCGUGAUCGUGCGCCGAGGCCCGCGAGGGGUCGCCGCCCAGAUCCCACCAGCCAGCAAGCUAAAGCAUGGCGGCCAUCCCCUCCAGCGGCUCGCUCGUGGCCACCCACGACUACUACCGGCGCCGCCUGGGUUCCACUUCCAGCAACAGCUCCUGCAGCAGUACCGAGUGCCCCGGGGAAGCCAUUCCCCACCCCCCAGGUCUCCCCAAGGCUGACCCGGGUCAUUGGUGGGCCAGCUUCUUUUUCGGGAAGUCCACCCUCCCGUUCAUGGCCACGGUGUUGGAGUCCGCAGAGCACUCGGAACCUCCCCAGGCCUCCAGCAGCAUGACCGCCUGUGGCCUGGCUCGGGACGCCCCGAGGAAGCAGCCCGGCGGUCAGUCCAGCACAGCCAGCGCUGGGCCCCCGUCCUGACCUGAGCGGUUACCACCAGCCCCAGGCCUGCGGAGGCGCUAGUCCACCAGAGCCCCUCCCCGCCCCUCUCCCCACUCCGCAUCCCUCGCCCCCCUCCCCACCUCCCACCCCCCACCCUGUAAACUAGGCGGCUGCAGCAAGCAGACCUUCGCAUCAACACAGCAGACACCAAAAACCAGUGAGAGCCCCGCUCUCUACCGCCCGGCCCCAGCACUCGCUAGCUUUCCUGACACCUGGAACUGUGCACCUGGCACCAAGCGGAAAAUAAACUCCAAGCAGCCAGUAGCCCCGAUGGUGUGUGCCUGAGCUGUGUGGCCCGAGGUUCCA(SEQ ID NO:14);
TOP1基因转录本的序列如SEQ ID NO:15所示,具体为:
CAAAUGCGAACUUAGGCUGUUACACAACUGCUGGGGUCUGUUCUCGCCGCCCGCCCGGCAGUCAGGCAGCGUCGCCGCCGUGGUAGCAGCCUCAGCCGUUUCUGGAGUCUCGGGCCCACAGUCACCGCCGCUUACCUGCGCCUCCUCGAGCCUCCGGAGUCCCCGUCCGCCCGCACAGGCCGGUUCGCCGUCUGCGUCUCCCCCACGCCGCCUCGCCUGCCGCCGCGCUCGUCCCUCCGGGCCGACAUGAGUGGGGACCACCUCCACAACGAUUCCCAGAUCGAAGCGGAUUUCCGAUUGAAUGAUUCUCAUAAACACAAAGAUAAACACAAAGAUCGAGAACACCGGCACAAAGAACACAAGAAGGAGAAGGACCGGGAAAAGUCCAAGCAUAGCAACAGUGAACAUAAAGAUUCUGAAAAGAAACACAAAGAGAAGGAGAAGACCAAACACAAAGAUGGAAGCUCAGAAAAGCAUAAAGACAAACAUAAAGACAGAGACAAGGAAAAACGAAAAGAGGAAAAGGUUCGAGCCUCUGGGGAUGCAAAAAUAAAGAAGGAGAAGGAAAAUGGCUUCUCUAGUCCACCACAAAUUAAAGAUGAACCUGAAGAUGAUGGCUAUUUUGUUCCUCCUAAAGAGGAUAUAAAGCCAUUAAAGAGACCUCGAGAUGAGGAUGAUGCUGAUUAUAAACCUAAGAAAAUUAAAACAGAAGAUACCAAGAAGGAGAAGAAAAGAAAACUAGAAGAAGAAGAGGAUGGUAAAUUGAAAAAACCCAAGAAUAAAGAUAAAGAUAAAAAAGUUCCUGAGCCAGAUAACAAGAAAAAGAAGCCGAAGAAAGAAGAGGAACAGAAGUGGAAAUGGUGGGAAGAAGAGCGCUAUCCUGAAGGCAUCAAGUGGAAAUUCCUAGAACAUAAAGGUCCAGUAUUUGCCCCACCAUAUGAGCCUCUUCCAGAGAAUGUCAAGUUUUAUUAUGAUGGUAAAGUCAUGAAGCUGAGCCCCAAAGCAGAGGAAGUAGCUACGUUCUUUGCAAAAAUGCUCGACCAUGAAUAUACUACCAAGGAAAUAUUUAGGAAAAAUUUCUUUAAAGACUGGAGAAAGGAAAUGACUAAUGAAGAGAAGAAUAUUAUCACCAACCUAAGCAAAUGUGAUUUUACCCAGAUGAGCCAGUAUUUCAAAGCCCAGACGGAAGCUCGGAAACAGAUGAGCAAGGAAGAGAAACUGAAAAUCAAAGAGGAGAAUGAAAAAUUACUGAAAGAAUAUGGAUUCUGUAUUAUGGAUAACCACAAAGAGAGGAUUGCUAACUUCAAGAUAGAGCCUCCUGGACUUUUCCGUGGCCGCGGCAACCACCCCAAGAUGGGCAUGCUGAAGAGACGAAUCAUGCCCGAGGAUAUAAUCAUCAACUGUAGCAAAGAUGCCAAGGUUCCUUCUCCUCCUCCAGGACAUAAGUGGAAAGAAGUCCGGCAUGAUAACAAGGUUACUUGGCUGGUUUCCUGGACAGAGAACAUCCAAGGUUCCAUUAAAUACAUCAUGCUUAACCCUAGUUCACGAAUCAAGGGUGAGAAGGACUGGCAGAAAUACGAGACUGCUCGGCGGCUGAAAAAAUGUGUGGACAAGAUCCGGAACCAGUAUCGAGAAGACUGGAAGUCCAAAGAGAUGAAAGUCCGGCAGAGAGCUGUAGCCCUGUACUUCAUCGACAAGCUUGCUCUGAGAGCAGGCAAUGAAAAGGAGGAAGGAGAAACAGCGGACACUGUGGGCUGCUGCUCACUUCGUGUGGAGCACAUCAAUCUACACCCAGAGUUGGAUGGUCAGGAAUAUGUGGUAGAGUUUGACUUCCUCGGGAAGGACUCCAUCAGAUACUAUAACAAGGUCCCUGUUGAGAAACGAGUUUUUAAGAACCUACAACUAUUUAUGGAGAACAAGCAGCCCGAGGAUGAUCUUUUUGAUAGACUCAAUACUGGUAUUCUGAAUAAGCAUCUUCAGGAUCUCAUGGAGGGCUUGACAGCCAAGGUAUUCCGUACAUACAAUGCCUCCAUCACGCUACAGCAGCAGCUAAAAGAACUGACAGCCCCGGAUGAGAACAUCCCAGCGAAGAUCCUUUCUUAUAACCGUGCCAAUCGAGCUGUUGCAAUUCUUUGUAACCAUCAGAGGGCACCACCAAAAACUUUUGAGAAGUCUAUGAUGAACUUGCAAACUAAGAUUGAUGCCAAGAAGGAACAGCUAGCAGAUGCCCGGAGAGACCUGAAAAGUGCUAAGGCUGAUGCCAAGGUCAUGAAGGAUGCAAAGACGAAGAAGGUAGUAGAGUCAAAGAAGAAGGCUGUUCAGAGACUGGAGGAACAGUUGAUGAAGCUGGAAGUUCAAGCCACAGACCGAGAGGAAAAUAAACAGAUUGCCCUGGGAACCUCCAAACUCAAUUAUCUGGACCCUAGGAUCACAGUGGCUUGGUGCAAGAAGUGGGGUGUCCCAAUUGAGAAGAUUUACAACAAAACCCAGCGGGAGAAGUUUGCCUGGGCCAUUGACAUGGCUGAUGAAGACUAUGAGUUUUAGCCAGUCUCAAGAGGCAGAGUUCUGUGAAGAGGAACAGUGUGGUUUGGGAAAGAUGGAUAAACUGAGCCUCACUUGCCCUCGUGCCUGGGGGAGAGAGGCAGCAAGUCUUAACAAACCAACAUCUUUGCGAAAAGAUAAACCUGGAGAUAUUAUAAGGGAGAGCUGAGCCAGUUGUCCUAUGGACAACUUAUUUAAAAAUAUUUCAGAUAUCAAAAUUCUAGCUGUAUGAUUUGUUUUGAAUUUUGUUUUUAUUUUCAAGAGGGCAAGUGGAUGGGAAUUUGUCAGCGUUCUACCAGGCAAAUUCACUGUUUCACUGAAAUGUUUGGAUUCUCUUAGCUACUGUAUGCAAAGUCCGAUUAUAUUGGUGCGUUUUUACAGUUAGGGUUUUGCAAUAACUUCUAUAUUUUAAUAGAAAUAAAUUCCUAAACUCCCUUCCCUCUCUCCCAUUUCAGGAAUUUAAAAUUAAGUAGAACAAAAAACCCAGCGCACCUGUUAGAGUCGUCACUCUCUAUUGUCAUGGGGAUCAAUUUUCAUUAAACUUGAAGCAGUCGUGGCUUUGGCAGUGUUUUGGUUCAGACACCUGUUCACAGAAAAAGCAUGAUGGGAAAAUAUUUCCUGACUUGAGUGUUCCUUUUUAAAUGUGAAUUUUUAUUUCUUUUUAAUUAUUUUAAAAUAUUUAAACCUUUUUCUUGAUCUUAAAGAUCGUGUAGAUUGGGGUUGGGGAGGGAUGAAGGGCGAGUGAAUCUAAGGAUAAUGAAAUAAUCAGUGACUGAAACCAUUUUCCCAUCAUCCUUUGUUCUGAGCAUUCGCUGUACCCUUUAAGAUAUCCAUCUUUUUCUUUUUAACCCUAAUCUUUCACUUGAAAGAUUUUAUUGUAUAAAAAGUUUCACAGGUCAAUAAACUUAGAGGAAAAUGAGUAUUUGGUCCAAAAAAAGGAAAAAUAAUCAAGAUUUUAGGGCUUUUAUUUUUUCUUUUGUAAUUGUGUAAAAAAUGGAAAAAAACAUAAAAAGCAGAAUUUUAAUGUGAAGACAUUUUUUGCUAUAAUCAUUAGUUUUAGAGGCAUUGUUAGUUUAGUGUGUGUGCAGAGUCCAUUUCCCACAUCUUUCCUCAAGUAUCUUCUAUUUUUAUCAUGAAUUCCCUUUUAAUCAACUGUAGGUUAUUUAAAAUAAAUUCCUACAACUUAAUGGAAA(SEQ ID NO:15);
C19ORF43基因转录本的序列如SEQ ID NO:16所示,具体为:
GUCCUUUGCGCGGCACCUGGCGACAAAAUGGCUGCCCGAGGGAGACGGGCGGAGCCUCAGGGCCGGGAGGCUCCGGGCCCCGCGGGCGGUGGCGGUGGCGGGAGCCGUUGGGCUGAGUCGGGAUCGGGGACGUCGCCCGAGAGCGGGGACGAGGAGGUGUCGGGCGCGGGUUCGAGCCCGGUGUCGGGCGGCGUGAACUUGUUCGCCAACGACGGCAGCUUCCUGGAGCUGUUCAAGCGGAAGAUGGAGGAGGAGCAGCGGCAGCGGCAGGAGGAGCCGCCCCCGGGUCCGCAGCGACCCGACCAGUCGGCCGCCGCCGCUGGCCCCGGGGAUCCGAAGAGGAAGGGCGGUCCGGGCUCCACACUUAGCUUCGUGGGCAAACGCAGAGGCGGGAACAAACUAGCCCUCAAGACGGGAAUAGUAGCCAAGAAGCAGAAGACGGAGGAUGAGGUAUUAACAAGUAAAGGUGACGCGUGGGCCAAGUACAUGGCAGAAGUGAAAAAGUACAAAGCUCACCAGUGCGGUGACGAUGAUAAAACUCGGCCCCUGGUGAAAUGACGCCCCUCCCCCACCUGCCCAUGGCCUGGGACUCUCUGCGAUGUACAUAACUAUUUAAUGCAGCGGCAGCGGCGACAGCCUUCCCUGAGAGGACUUAAAAGCAGAAGGAAACCGAGAUGCUUCCCGCAGCCGUGGACGAUUCUCCAGGACUCUUUUUUUACCUUGAGCACUUGCCUCGUGAGACUUCAUAGAACAGUGGUUUACUGUCCCCCCCUUCUCACCUCCUCAUUCUCUCUGGCUCUUUCUGUCUUCCUCUUCUCACCCUCCUCCCUCCCCUUAGCCAUCACUUCUGGGAAGUAAAGAACUUGACUUAGUGCCGGA(SEQ ID NO:16)。
在另一个实施方案中,其中所述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PPDPF基因转录本的引物为:如SEQ ID NO:4所示的上游引物序列,以及如SEQID NO:5所示的下游引物序列;检测PPDPF基因转录本的探针序列如SEQ ID NO:6所示;
检测TOP1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TOP1基因转录本的探针序列如SEQ ID NO:9所示;和/或
检测C19ORF43基因转录本的引物为:如SEQ ID NO:10所示的上游引物序列,以及如SEQ ID NO:11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: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。
本公开的第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)检测选自长单链非编码RNAlnc-MKRN2-42:1、和PPDPF基因转录本、TOP1基因转录本和C19ORF43基因转录本的一种或更多种(例如两种、三种或者四种)RNA分子标志物的表达水平,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。
在又一个实施方案中,步骤(4)还包括利用外参基因(例如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癌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诊断早期结直肠腺瘤的性能。
图2显示了PPDPF诊断早期结直肠腺瘤的性能。
图3显示了TOP1诊断结直肠腺瘤的性能。
图4显示了C19ORF43诊断早期结直肠腺瘤的性能。
图5显示了lnc-MKRN2-42:1和C19ORF43联合诊断早期结直肠腺瘤的性能。
图6显示了lnc-MKRN2-42:1、C19ORF43和TOP1联合诊断早期结直肠腺瘤的性能。
图7显示了lnc-MKRN2-42:1、PPDPF、TOP1和C19ORF43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,其示例性的RNA序列如SEQ ID NO:13所示;和基因的转录本,所述基因包括PPDPF、TOP1和/或C19ORF43。进一步的,本发明作为RNA分子标志物的PPDPF基因转录本表示PPDPF基因经过转录得到的mRNA分子,示例性的mRNA序列如SEQ ID NO:14所示;TOP1基因转录本表示TOP1基因经过转录得到的mRNA分子,示例性的mRNA序列如SEQ ID NO:15所示;C19ORF43表示C19ORF43基因经过转录得到的mRNA分子,示例性的mRNA序列如SEQ ID NO:16所示。需要说明的是,在实际检测中的lncRNA和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。本发明中的RNA分子标志物即存在于外泌体中。
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进行微量链特异性长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,GKong,Journal of extracellularvesicles 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长链RNA,最终鉴定得到在两组中具有显著差异表达的mRNA及lncRNA各两个,具体如表1所示。
表1RNA分子标志物列表
实施例2基于荧光定量PCR平台的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2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2反转录反应体系
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的上游引物为具体如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PPDPF的引物和探针:所述PPDPF的上游引物具体为如SEQ ID NO: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SEQ ID NO:6所示的核苷酸序列;
用于检测TOP1的引物和探针:所述TOP1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C19ORF43的引物和探针:所述C19ORF43的上游引物具体为如SEQ ID NO:10所示的核苷酸序列,下游引物具体为如SEQ ID NO:11所示的核苷酸序列,探针为如SEQID NO:12所示的核苷酸序列。具体如表3所列。
表3实施例所用引物和探针序列
按表4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)按表5所列反应条件进行扩增检测。
表4qPCR反应体系
表5qPCR反应条件
实施例3利用RNA分子标志物进行结直肠腺瘤早期诊断检测
1)样本采集
收集包括经医院确诊的早期(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。
4)结果:
对24例早期结直肠腺瘤病人和53例对照样本(健康人和良性病变)的血浆外泌体,分别检测lnc-MKRN2-42:1、PPDPF、TOP1和C19ORF43的Ct值,所有样本的的检测结果如表6所示。进一步,根据目标基因Ct值及参考RNA Cel-39-3p的Ct值计算目标RNA相对参考基因的ΔCt(如表7所示)。
表6各样本中所有RNA标记物的检测Ct值
表7目标RNA相对参考基因的ΔCt值
接下来使用相对定量公式值2-ΔCt,计算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分子标志物单独使用或者联合应用用于检测结直肠腺瘤的准确度。
(1)标志物lnc-MKRN2-42:1、PPDPF、TOP1和C19ORF43单独检测
如图1-4所示,单独应用每个RNA分子标志物时,基于表6和表7的对检测结果,采用R语言进行t检测分析得到p<=0.05,表明其中每一个标志物的表达量变化均单独与早期结直肠腺瘤显著相关。lnc-MKRN2-42:1、PPDPF、TOP1和C19ORF43诊断早期结直肠腺瘤的AUC分别为0.69、0.68、0.62和0.60,良好的灵敏性和特异性。
(2)标志物lnc-MKRN2-42:1和C19ORF43的联合检测
如图5所示,联合应用lnc-MKRN2-42:1和C19ORF43,基于此前表6和表7的检测结果,采用R语言进行t检测分析得到p<=0.05,说明lnc-MKRN2-42:1和C19ORF43的联合应用与早期结直肠腺瘤显著相关。lnc-MKRN2-42:1和PPDPF联合诊断早期结直肠腺瘤的AUC=0.78,其阴性预测值为85.11%、灵敏度为70.83%、特异性为75.47%,具有显著的诊断能力。
(3)标志物lnc-MKRN2-42:1、C19ORF43和TOP1的联合检测
如图6所示,联合应用lnc-MKRN2-42:1、C19ORF43和TOP1时,基于表6和表7的检测结果,采用R语言进行t检测分析得到p<=0.05,说明lnc-MKRN2-42:1、C19ORF43和TOP1联合应用时,与早期结直肠腺瘤显著相关。lnc-MKRN2-42:1、PPDPF和TOP1联合诊断早期结直肠腺瘤的AUC=0.81,其阴性预测值为92.31%、灵敏度为87.5%、特异性为67.92%,展现了极佳的灵敏度和特异性。
(4)标志物lnc-MKRN2-42:1、PPDPF、TOP1和C19ORF43的联合检测
如图7所示,联合应用lnc-MKRN2-42:1、PPDPF、TOP1和C19ORF43,基于此前表6和表7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。lnc-MKRN2-42:1、PPDPF、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91.11%、灵敏度为83.33%、特异性为77.36%。
以上结果表明,本公开提供的RNA分子标志物lnc-MKRN2-42:1、PPDPF、TOP1和C19ORF43,不论是单独使用还是部分或者全部的联合使用,均能够有效的作为临床结直肠癌诊断的标志物。
SEQUENCE LISTING
<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及其应用
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Claims (3)
1.一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,其包括检测lnc-MKRN2-42:1、PPDPF基因转录本、TOP1基因转录本和C19ORF43基因转录本的引物和/或探针,其中
所述检测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SEQID NO: 2所示的下游引物序列;所述检测lnc-MKRN2-42:1的探针序列如SEQ ID NO: 3所示;
所述检测PPDPF基因转录本的引物为:如SEQ ID NO: 4所示的上游引物序列,以及如SEQ ID NO: 5所示的下游引物序列;所述检测PPDPF基因转录本的探针序列如SEQ ID NO:6所示;
所述检测TOP1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;所述检测TOP1基因转录本的探针序列如SEQ ID NO: 9所示;和
所述检测C19ORF43基因转录本的引物为:如SEQ ID NO: 10所示的上游引物序列,以及如SEQ ID NO: 11所示的下游引物序列;所述检测C19ORF43基因转录本的探针序列如SEQID NO: 12所示。
2.根据权利要求1所述的试剂盒,所述lnc-MKRN2-42:1的序列如SEQ ID NO:13所示、所述PPDPF基因转录本的序列如SEQ ID NO:14所示、所述TOP1基因转录本的序列如SEQ IDNO:15所示和所述C19ORF43基因转录本的序列如SEQ ID NO:16所示。
3.根据权利要求1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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