CN111455051A - 用于结直肠癌诊断及预测的外泌体rna及其应用 - Google Patents

用于结直肠癌诊断及预测的外泌体rna及其应用 Download PDF

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CN111455051A
CN111455051A CN202010316161.7A CN202010316161A CN111455051A CN 111455051 A CN111455051 A CN 111455051A CN 202010316161 A CN202010316161 A CN 202010316161A CN 111455051 A CN111455051 A CN 111455051A
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CN111455051B (zh
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闵力
孔关义
朱圣韬
郭俊峰
张澍田
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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癌相关的外泌体长链RNA标记物及其应用。

Description

用于结直肠癌诊断及预测的外泌体RNA及其应用
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癌相关的外泌体RNA分子标志物及其组合与应用。
背景技术
结直肠癌(colorectal cancer,CRC)是我国常见的恶性肿瘤之一。在西方国家其发病率居恶性肿瘤的第2~3位,随着我国人民生活水平的不断提高和饮食习惯的改变,我国结直肠癌的发病率也在逐年升高,已跃居第3~5位。结直肠癌的预后与早期诊断密切相关,多数早期结直肠癌可以治愈,5年生存率可达90%,而晚期则不足10%,因此提高国人早期结直肠癌及癌前病变的检出率,从而降低结直肠癌的发病率,提高结直肠癌的治愈率,延长患者的生存期显得尤为重要。
电子结肠镜是结直肠癌的重要诊疗手段,通过观察病变粘膜及活组织检查等手段能够实现早期诊断及治疗。但是电子结肠镜作为有创检查手段,不仅会给受检者带来痛苦,同时可能出现出血、穿孔等并发症,因此在一部分患者的筛查应用中受到限制。临床上也可通过采集患者血液进行肿瘤标记物检测等无创检查方法,但由于其灵敏度、特异度受限,只能作为一项参考指标。因此,开发一种高灵敏度的无创结直肠癌早期检测方法至关重要。
近年来细胞外囊泡(Extracellular Vesicles,EVs)逐渐成为国内外研究热点,它是由细胞分泌的或由细胞膜脱落的囊泡状小体,具有双层膜结构,直径在30-1000nm不等,细胞外囊泡主要包含微囊泡(MicroVesicles,MVs)和外泌体(exosomes)两大部分,近年来发现二者尤其是外泌体具有多种类型的生物学功能,其在疾病诊断中有着重要的意义。
外泌体是细胞内多泡体与细胞膜融合后被细胞分泌到细胞外的膜性小囊泡,其直径通常介于30~150nm之间,近年来发现主要参与在抗原呈递、细胞凋亡、炎症反应、肿瘤发生发展及转移等过程,是细胞间信息传递的重要媒介。外泌体可广泛分布于包括血液、唾液、尿液、乳汁和胸腹水在内的各种体液系统,其内部可携带DNA、RNA及蛋白质等多种活性物质,研究表明外泌体内部mRNA、lncRNA等分子在肿瘤的发生发展中起到至关重要的作用,可作为多种类型肿瘤的无创诊断标志物,因而外泌体mRNA、lncRNA的检测可作为结直肠癌早期诊断的手段。
发明内容
发明人经过多年的研究,筛选获得了与结直肠癌密切相关,能够用于临床诊断RNA分子标志物组合,其包括两个长链非编码RNA:lnc-MKRN2-42:1和lnc-MSI1-2:1,以及两个基因的转录本(mRNA):TOP1和PPDPF。所述RNA分子标志物不论是单独使用还是部分或者全部的联合使用能够高灵敏度和特异性的指示结直肠癌,具有优越的诊断性能。
在第一个方面中,本公开提供了一种用于结直肠癌诊断、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、lnc-MSI1-2:1、TOP1基因转录本和PPDPF基因转录本中一种或多种RNA分子的组合。
在一个实施方案中,所述所述lnc-MKRN2-42:1的序列如SEQ ID NO:13所示,具体为:
GCCGGGCACAGUGGCGCGUGCCUGUAGUCCCAGCUACUCGGGAGGCUGAGGUGGGAGGAUCGCUUGAGCCCAGGAGUUCUGGGCUGUAGUGCGCUAUGCCGAUCGGGUGUCCGCACUAAGUUCGGCAUCAAUAUGGUGACCUCCCGGGAGCGGGGGACCACCAGGUUGCCUAAGGAGGGGUGAACCGGCCCAGGUCGGAAACAGAGCAGGUCAAAACUCCCGUGCUGAUCAGGAGACAGAGUUUGUGAGCAGACAACUGGUCUGACCAAAAUUUAUGAGGUGGGAAUUUCCUCU(SEQ ID NO:13);
所述lnc-MSI1-2:1的序列如SEQ ID NO:14所示,具体为:
GCUGGUAAUGGUCACACUAGGUGGCGUCCGCGUGAGGUGGAGGUGUCAGCGGCUCCUGUCUGCAGCUUCCUGGGGUCCUGGCGGCGCUCUUCUCCUGAGCCAGAAUCCAGCAGGAAGGACAGAAUGGGCGCUAGAGUGUGAAAAGAGGGCGAACUCACGUGGAUGUGACUCUCCGGGUCGCUCUUCCCUGUCACCCAGCUCCUCCUGCCGGUUUCCUGUGGGCAUCGCCUGCAUUGCUUUUAUCCCGGGGACGGAUUAGGGGGGUCCAGAGGAGGACUUGCUGGCACAAAUAACCCAAUGGAGGCUUUCGAGCACGGCUGGCUGGGGUUUGCGAUCCAACGGCCUUUUGCUGACCUGAAAGUCAACGCAAAGCUCAUCUUCAUUCUAGAGAAAAAGAAGCCUCAUAUCUGAGAUCAGGACCCCGCUUUCUCCUUGACAUUCCACAGCAAAACGUGAACAGCUGAACAAACACACAGUCCGGUUUCCCGAGACCAAAGGGUCAGGGGAAACCCCCGCUGAAAACGUGCAUCAGUGGAACAACUACUCUCUUCCAGGUCCCACCGAGGGCAAAGAGGAGAAAUAGAAUCCCUCACAAGAAAUCCACAACUUCUAGAAAUCCGCAGCACACACAGAACACACUUACCUCUCACAGCUCUACGGACACUUCACACCCCACAGUGGGACCGUCUGAUUGCAUUUGCAUUUUGGGACAUGUAGUCCAGAAAGCUUAAAUGAUGCGAGAAAAACCACAAGUCCCACAAUCCUCCGCGUCUCGCAAUUGACAGCCCCAGAUCAUCCAGGAACGCCCCCUGUCGGCUCUUUCUGGUCAGAGGUUCCCCCGUUAAUAUAAAAGCUUUUAAUCAUGGAUUUUUUUCUUUGUUUUCAAUUUCUGUGGGCACUUAGUAGGUGUAUAUAUUUAUAGUGUACAUAAGAUUUUUUUUCUUUUUUCUUUUUUUUUUUUAAGACGGAGUUGUUGUUUUUUUUUUUUGAAAAAACUCCAGUCUGGAGUGCAGUGGCACGAUCUCGGCUCACUGCAACCUCCCUCUCCCGGGUUCAACCGAUUCUCCUGCCUCAGCCUCCCGAGUAGCUGGGAUUAUUUUUGUAUUUUUAGUAGAGACGGGGGUUUCGCCAUGUUGGCCAGGCUCGUCUUGAACUCCUAACCUCAGGUGAUCCGCACGCCUUGACCUCCCAAAGUGCUGGGAUUAUAGGCGUGAGCCACUGCGCCCGGCCAUCUUUUCUCUUAUUUUUUGUCUUUUUUUCUUUUCUCUGUGCCUGUCUAUGCUUUACCCACAUGAGAUGUUUUGAUACAAACAUGCAAUGUGAAAUGAGCACGUCAUUAAUCAUGGAUCUAGAUUUGACUUUUUUUUAAAGUUUUUGUAUUUUUAGUAGAGACAGAGUUUCACCAUGUUGGCCAGGCUGGUCUCGAACUCCCGACCUCAGGUGAUCCGCCAACCUCGGCCCCCCAAAAUGCUGGGAUUACAGGCCUGAGCCACUGUUCCCGGCCUAGAUUUGACUCAUUCUAAACAUCAGAGUUUGUGAUCCUUUUCAUUCACAACGACAGUAUCAAGUUAUUUGCUAUUGGUAAAAUACCUUGAACUUGUGAAGAAAUGUGUUUAUACAUAUCUGAAAAAGUGAUAGUUUGUUGUUGUUGUUGUUGUUGUUUGUUUGUUUGUUUUUGAGACGGAGUCUCGCUCUGUUGCCCAGGCUGGAGUGCAAUGGUGCCAUUUCGGCUCACUGCAACCUCCGCCUCCCGGGUUCAAGCAAUUCUCCUGCCUCAGCCUCCUGAGUAGCAGGGAUUAUGGGCGCAUGCCACUACGCCCGGUUAAUUUUUUUAUUUUUAGUAGAGACAAGGUUUCACCAUGUUAGCCAGGAUGGUCUUGAUCUCCUGACCUCGUGAUCUGCCCACCCUAGCCUCCCAAAGUACUGGGAUUACAGGCGUAAGCCACUGUGCCCAGCCAAAAGUGAUGUUUUUACAGUUAAUAUGAAGCCAAAUAUAUUAUACCUGGACUAAUGUACUUAACCUGCUUGGCUUCUCCAAUUUUAUUUAUCAUAUUUUUUUUAUUAAGUUAAUCAAACAAGGCCUGCGUCUCACUCCCAGGGAUUCUGAUCUACAAAUUUCCUAAUUGUUCCUAAUGUGUAGACAACCAGAACUGCCGCUCUGCAGCAUCACCUAAAUCAAUAAAUGUAUAAGUGUCUUUGGACAUAAAGGUGGGCGGCCAGCAGGGACCUUGAAGACCACCUAAGGACUCCCUUCCCCCAUUUUAAGAUGAAUUGGGCUGAGUGCAGUGGUUCCCACCUGUAAUACCAACACUUUGGGAGGCCAAGGCAGGAGGAUCACUUGAGCCCAAAAGUUCAAGACCAGCUUGGGCAACAUAGUGAGACCCACUCACUACAAAAAAUAAACAAAAUUAGCGGGGUGUGGUGGUGUGUGCCUGUAGUCCCAGCUACUCAAGAGGCUCAGGUGGGAGGAUCACUUUGAGCCCAGGAGAUCGAGGCUGCAGUGAGCCAUAAUUGUGCGACUGCACUUCAGCCUGGGUGACAAAGUGAGAUCCCAUCUCUUCAAAAAAUAAUAAUAAUAAAUAAAGAUGAACUGAAGUGCAGAGAAGUAAUGUGGAAGCUUGAGAAAGAGUUUUUAGGUUUUUGGUUCUAUCAUUGUAUCUUUGGAGGUAAUAUUGGCAAAAUGACCAUUUAGUAGUUUGCCCAUUUCUGCUUUUUUUUUUUCUUUUUUUUGAGACAGGGUCUCGUUCUGUCACCCAGGCUGGAGUGCAGUUGUGACAUCACAGCUCACUGCAGUUUCUACUUCCCAGGCCCAAGGGAUCCUCCAACCUCAUCCUCUCGAGUAGCUGAGAUCACAGGCAUCCACCACCACGCCCAGCUAAAUUUUGUUAUUUUUUAUUUUUGUCGAGACACGGUCUCACUAUGUUGCUCAGGCUGGUCUCAAACUCCUGAACUCAAGGAAAGCUCUUGCUUUGGCCUCCCAAAGUGCUGGGAUUACAGGUGUGAGCCACCAUGCCGGGCCUGCUUAUUUCGACUUGGCAGCAUCGUUUCAUGAAGCCCCUGACUCUGUGACAACAUACAGCUCUCCAGAAAGAUGCUUUGAAGACAAAACAGAAUAGAGCACACAGCCCUCCAUGUCUCUUCCAAGUCACUAUAUUCAUUAAAAGAUAAAUGACUCUGGUCAUUGCCUUUUCCUGCACAAGAUAAUGUCUGAUGGGGUUAGUGAUGAUACCUCUGUAAUCCAUAACCAGAUAUACUCUUGCACCCAAACUUUGAUAUGAUUGUGCUUUAAUGUAACUUUGAAGCAAGUUUAAUGUGAUUUUGCAGGCGCCGUACACUCACCACUUGUAUAUGAGUGGCUGAAAUUCUGUGUUGGACCAGUCUGAUGGAACUGCUCCCACACUAUAGCCCUCGGUCUGUAAUCCUCAGUAAGACUUGUAAGUAAAACUAACUUUAAUUUUUUUUUUUUUUUUUUGAGAGAGUCUAGCUCUGUUGCCCAGGCUGGAGUGCAGUGGCGCGAUCUUCACUCACUGAAGCCUCUGCCUUCCCGGCUGAAGUGAUUCUGCUGCCUCGGCCUCCGGAGUAGCUAGGAUUACAGGCGCUCGCCACUGCGCCCGGCUAAUUUUUGUAUUUUUAGUAGAGAUGGGGUUUCGUCAUGUUGGCCAGGCUGGUUUUGAACUCGUGACCUCAGGUGACCCACCUGCCUCAGUCUCCUAAAAUGCUAGGAUUACAGGAGUGAGCCACUGCACCCCGCCACUUUUAUUAUUAUUAUUAUUGUAUUUAUUUAUUUAUUUAUUUAUUUUUGAGACGGAGUCUCGCUCUGUCGCCCAGGCUGGAGUGUAGUGGAGAGAUCUCGGCCCACUACAAGCUCCGCCUCCCGGGUUCACGCGAUUCUCCUGCCUCAGCCUCCGGACUACAGGCGCCCGCCACCACACCCGGCUAAUUUUUUGUAUUUUUUUUUUAGUAGAGACGGGGUUUCACCGUGUUAGCCAGGAUGGUCUCUAUCUCUUGACCUCGUGAUCCGCCCGCCUCAGCCUCCCAAAGUACUGGGAUUACAGGCGUGAGCCACCGCGCCCGGCUAUUUAUUUAUUUAUUUAUUUAUUUAUUUAUUUAUUUUUUGAGACGGAAUAUUGCUCUGUGCCCAGGCUGGAGUGCAGUGGCACGAUCUCAGAUCACUGCAACCUCCGCCUCCCGGGUUCAAGUGAUUCUCCUGCCUCAGCCUCCCGAGUAGCUGGGAUUACGGGUGCCCGCCACCAUGCCCGGCUAAUUUUUGUAUUUUUAGUAGAGACGGGGUUUCACCAUGCUUGUCAGCAUGGUCUCGAACUCCUGACCUCGCGAUCCGCCCGCCUCAGCCUCCCAAAGUGCUGGAAUUACAGGCAUGAGCCACUGCGCCCUGCCACUUUAAUUAUUUAAAAGCUUAUUUUUUUAUUUUGGGGACAUGGUCUCACUCUUUCACCCAGGCGGGAGUUCAGAGGCACGAUCACUGCGCACUACAGCCUCAACCUCUUGGACUCAAACCAUCCUCCCACCUCAGCCUCCUGAGUAGCUGGGACUACAGGUGGGCCACCAAGCCCAGCUAAUUUUUGUAUUUUUUUGUAGAGACGGGGUUUCACCAUGUUGCCCAGGCUGGUCUCGAACUCCUGGGCUCUAGGCUUUCCAAAGUGUUGUGAUUACGGGCGUGAGCUACUGCGCCUGGCCGAUUCUUUUUAACUUAGUACACAGGGAGUAGUAGCUCCAGCAACAGCGAAAGUCAGUAGCUGUAGGAUCUACUAGGCGGGGAAACUCAUAUGACAGCCCCCUCCUUUUUUUUUUUUUUUUUUUUUUUUUUGAGAUGGAGUUUCACUCUUGUUGACCAGGCUGGAGUGCAAUGGCGCCAUCUUGGCUCACCGCAACCUCCGCCUCCCGGGUUCAAGUGAUUCUCCUGCCUCAGCCUCCCCAGUAUCUGGGAUUACAGGUGUGAGCCACUGCGCCCGGCCACCCCCUUUUUUUUUCAAGGCUGCAGUGAGCCAUGAUCCUGUGACUGCACUCCAGCCACUGACAGCCCUUUCUGCAUUAGUUAACCUGACCACAUCUGCUGAAAGAAAGCACCAGAAGCUAGACUGAAAUUUUCGUUGUCAAAAAAAUUAUAAUCCCCCCCAAAAUUUAAAAAUUAUAAUCCAGCACAUUUUCUGCACACAUUUUCUGGGUAUUUGAUCAUAACUUUUAAAAAUGUGAGAACAGUGACAUUCUCCUUCCCCAUUCACCAAUUUUACUAUGAAAAAGUUCAAACCUAGGUUAUUUUUUCAGUUCUCCAAGUGGAAUGAUCAUCACUAAUAGAGAUUGGUUGGGAAUUCUAUUGUGCUUCUAGGCCCCCAAAAAUCUCAUGCUCUGAGGAAAAGUUAGCUGAGUUUUCAAUUUUUCUCAUAGUGCUGGAAAAUCAUCCCUGGACGGUGAAGUCAUCCAAGUCUUUUUUGUUUUGUUUUGUUUUUUCUUUCUUUCUUUCUUUCUUUUUUAAUUUUUGUAUGCUUUGUUUCUAUUUCUUUUUUUUUUUGUCAUCCAAGUCAGCUGGUGAAACACACAAUUUCACUUGAUAGCUACUGAGAGAGUGUAAUGGGCCACCCAUUCUAGAAUUCUCAAGGGCUAGGAAGUUCCUUUGAUACCCUCAUGGGAUUAGGAUUAGGGCUUUAGUCUUUUUCUCUCUCUCUUUUUUUUUUUUUUUUUUUUUUUUUGAUUACAGGCGUGAGCCACCACACCAAGCCCCAUUAUUUAUUUAUUUUAUUUUAUGUAUGUAUGUAUGUAUGUAUGUAUGUAUUUAUUUAUUUUGAGACGGAGUCUCGCUCUAUCGCCCAGGCUGGAGUGCAGUGGCGCAAUCUAGGCUCACUGCAACCUCCGCCUACCGGGUUCAAGUGAUUCUCCUAUGUCAGCCUCCCAAGUAGCUGGGAUUAUAGGCGCCCGCCACCAUGCCCAGCUAAUUUUUGUAUUUUUAGUAGAGACGAGGUUUCACCAUCUUGGCCAGGCUGGUCUUGAACUCCUGACCUUGUGAUCCACCCGCCUCGGCCUCCCAAAGUGCUGGGAUUACAGGCGUGAGCCACCGUGCCUGGCCUAGUGUUUUUCUCACAACUCCACCCCAUCCAGAAUUAAUGACUAAAUCCUUGACUGGAUGAUCAAAGUAAAAGAAUCUAAAUUCACCACAUUGCUAGUCUUUUCUUUUUAUCCAGGCUGUGAAUUAAAUAAGAAAUCUGCUGCAGUGGCUCAUGCCUGUAAUCCCUCCCCUUGGGGAGGCCGAGGCGGGCGGAUCACUUGAGCCCAGGAGCUGGAGACCAGCAUGGGCAACAUGGUGAAACCCUGUAUCUACAAAAAAUACAGAAAAAUUAGCUGGGCAUGGUGGUGUGUGCCUGUAGUCCCAGCUACCUGGGAGGCUGAGGUGGGAAGAUCGCUUGAGCCCAGGAGAUUUAGGCUGCACUGAGCCGUGAUUGCACCCCUGCGCUCCAACCUAAGCGACUGAGAACAUGUCUCAAAAAAAAAAAAACAAAAAAAAAAACACAAAACAAGUUAGAAAUAAUCACAUAACACAUCAGCAUCAAACAGGCCUUUUAAAAUGUCGUUUUUGACCAUCCUGGCUAACACGGUGAAACCCCAUCUCUACUAAAAAUAGAAAAAAUUAGCCGGCCGUGGUGGCGGGCGCCUGUAGUCCCAGCUACUCGGGAGGCUGAGGCAGGAGAAUGGCGUGAACCCGGAAGGCGGAGCUCGCAGUGAGCCGAGAUUGCGCCACUGCACUCCAGCCUGGGCGACAGAGCGAGACUCCGUCUCAAAAAAAAAAAAUGUCAUUUUUGUAUAUGGAUGAUGAGUACAUGAAUGUUCACUGAGGAAUUCUUUGAACUUUUCGUGUUUACAAUUUUUUAUGAUAAAAUGUUGGGGGAAAUGUUACUUUCUUUUUUUUUUUUUUUUUUUUUUUUUUUGAGACGGAGUCUCGCUUUGUCACCCAGGCUGGAUUACAGUGGCAUGAUCUCAGCUCAUGGCAACCUCUACCUCCCAGGUUCAAGCGAUCCUCCUGCCUCAGCCCCCCUAGUAGCUGGGAUUACAGGCAAGCGCCACCACGCCCCGUUAAUUUUUGUAUUUUGUAUUUUUGUAAUUUUGUAUUUUUGUAUUUUGUAUUUACAGGGAUGUCGCCAUGUUACCAGGCUGAUCUUGAACUCCUGACCUCAGGUGAUCCACCUGCCUUGGCCUCCCAAAGCGUUGGGAUUACACGCUCGAGCCACCACACCUAGACCAGAUGUAGGUUUCUAAUAGCUUUCUACAUCCUGAAGGAAAACAGGCAGUGAGGGUGAAAGUUGCUUCAAAUUUUAGAAACUGUGAACAUUCUGUGGAAAUAAAUCCUUAUUAUUUGAGCCUCUUUUUGAAUUAAGGUGUCCAAACCCUUUUGAAUUAACAUAUGAUCCAGCAAUUUUACUUCUAGGUAUGUACACCAAAGAAUAGAAAACAGGGGCCAGGCGCGGUGGCUCACACCUGUAAUCCCAGCACUUUGGGAGGCCGAGGCAGGCAGAUCACUUGAGGUCAGGAGUUCAAGACCAGCCUGACCAACAUGGAGAAACCCCGUCUCUACUAAAAAAAAAAAAAAAAAAAAAAUUAUCUAUAUAUAUAUUUAUACAUAUAUAUAUAUAAAAUUAGCCAGGCAUGGUGGCGCAUACCUGUCAUCCCAGCUACUUGGGAGGCUGAGGCAGGAGAAUCACCUGAACCCAGGAGGCAGAGGUUGUGGUGAGCUGAGAUUGCGCCUUUGCAGUCCAGACUGCGCAACAAGAGCGAAACUCAGUCUCAAAAAAAAAAAAAAGGAAAACAGAAACUUAAGCUGAUAUUUGCACACCAAUAGCAGCAUUAUUCCCAUAGACAAAAGGUGGAAACCAGCAAUGACCAUCUAAAAAUAAAUGGAUAGGCCGGGCGCCGUGGCUCAGCACUUUGGGAGGCUGAGGCAGGAGGAUCACUUGAGGUCAGGAGCUUGAGACCAGCCUGGCCAACAUGGUGAAACCCUGUCUCUACUAAAAAUACAAAAAUUAGCCAGGAUUGGUGGCACGCACCUGUAAUCCCAGUUAGUUACUCGGGAGGCUGAGACAGGAGAACUGCUUGAACCCCAGAGGCGGAGGUUGCAUGGGCUGAGAUUGAGUCUAUGUACUCCAGCUUGGGCAACAGAGCGAGACUCCCCCUCAAAAAAAAAAAAAAAAAGGAUAAACAGUUGGUCCGGGCGGCAGCUCAUGCCUAUAGUCCCAGCACUUUGGGAGGCCAAGAUGGGAGGAUCACUUGGGCCCAGGAGUUCGAGACCAGCCUGGGCAACAUGGUGAGAUCCUGUCUGUACGAAAAAUACAGAAAGUAUCCAGCUGAAGUGGGAGGAGGAUCACUUGAGGCCUGGAAGCAGAGGUUGAAGUGAGCUGAGAUCGCGCCACUGCAUUCCAGCCUGGGCGACAGAGCCAAAAGCUGUCUCAAAAAAGAAUGAAUGGGACCGGGCGCGGUGGCUCACGGCUGUAAUCCCAGCACUUUGGGAAACCAAGGCGGGUGGAUCACCUGAGGCCAGGAGUUCAAGACCAGCCUGACCAACAUGGUGAAACCCCGUGUCUACUAAAAAUAAUCCCAGCUCCUCGGGAGGCUGAGGCAGGAGAAUCGCUUGAACCUGGGAGGCGGAGGUUGAUCUCGCCAUUGCGCUCCAGCCUGGGCAACAAAAGCAAAACUUCGUCUCAAAAAAAAAAAUAGACAAGAAUAAAUGGGCUGGGUGAAGUGGCUCACACCUGUAAUUCCAGCACUUUGGGAGGCCGAGGCAGGCAGAUCACGUGAGGUCAGGAGUUUGAGACCAGCCUGGCCAACAUGGUGAAAUCCUGUCUCUACUAAAAAUACAAAGAGUGGUGGCAUGCAUCUGUAAUCCCAGCUACUUGAGAGAUGGAGGCAGGAGAAUCGCUUGAAUCCGGGAGGCAGUGGUUGCAAUGAGCCGAGAUCAUGCCACUGCACUCCAGCCUGGGCAACAAGAGCAAAAACUCCAUCUCAAAAAAAAAAAAAGAUAACAUCUCAUACAGUGAAAUAUUAUUCAGCCAUAAAAUUAAUGAAUUCCUGAUAUAUACUACAACAUGGAUGAACCUUAAAAACAUUAUUCUGAAUGAAAUAAGACUGACCUCAAGGACAAAUAUUGUAUGAUUCCAUUUAUAUAUAUAUAAAGUACUCACAAAGUCAUAGAGAUAGAAAGUAGAAAAGAGGUUGCCACGGGGGCUGGAGGAGAAGGGAACAGGGAGUAAGGGUUUAAUGGGUACAGAGUCUCAGUUUCCACCCUUGGGCAUGAAAGAUAAAGUUCUGGAGGUAAGUAGUGAUGAUGGUUACACAACAAUGUGAACAUAUUCUAUGCCAGUGAAUUUACACUUAAGGUUAAAAUGGUAAAUUUUAUGUAUACUUUGGCAUAAUUUAAAAAAAAGUUUGAAAAAGAAAAAAGACAAAACAAUAAUCGUCUUUGGAGAAGUCUGUUACACUGAAUCUGGAGCCAAGUGAACAGAACCCUGAUUUUGAUCCCUUUUCUCUCAAAAGCCCUUCGCAGUCUCUGAAUUAAGUCUAUUAGCAUGUUCCUCCCAUAGUGCUUUGCUUCAUAUCAACAAAAACCUAGCUAAGUGAAAUCAGCAACGAUAUGCAGAAACCACCUACGCAGGUCACAAACAUCUUUCUAUGAUUGUAUAAUUUUCAAGCAAGCAAUAAGUGAAGAUUUUUCCAUAGGCCCUAAACUCACCUUUGCGAAAUAGGAAGCUGGUUUAUUGGGAGUGAUGAGCAGGGGGCGUAACAAAUUAGCUUUGCGCAGUGGCAGUAUCGUAGCCAAUGAGGUCUAUCCGAGGCGCGAUUAUUGCUAAUUGAAAACUUUUCCCAAUACCCCGCCGUGACGACUUGCAAUAUAGUCGGCACUGGCAAUUUUUGACAGUCUCUACGGAGACUGAAUUUUCUUGCAGUUGAACAACAGAGGCUUUUUUUGUGUGUGUGGGGGUGCUUGGUUUUGGGAGGUUGAAGAGUACUUGUUCGCAAACUCUCUAAAUGAGAGAUGGGGAAGUGUCUUACCAGCUGAUUCGCGAGAGCACCUGCAAGUUUACCUGUUUUAGUGAUUUGCGUUUAUGAAAGUUGGAGUUGCUGAUUUUGUGAUCUCUACUUUUAAGGUAAAAGCAGGGCGGUGUUGACUUUUCUUGCCAGUUGGCUGUCCUGUUUUCUUUCUCGCACUGUUUUCUUUUUGAGACGAAGUCUGUCGCUCAGGCUGGAGUGCACUGGCGUGAUCUCGGCUCGCUGCAAACUUUGCCGAACCCCUGUUUAGCGAUUUCCGUUUGUGAAAGUUGGAGUUGCUGAUUUUGUGAUCUCUACUUUUAAGGUAAAUGCAGGGCGGUGUUGACUUUUCUUGCCAGCUGGCUGUCCUGUUUUCUUUCUCUCGCACUGUUGUUUUCUUUUUGAGACGAAGUCUGUCGCUCAGGCUGGAGUGCACUGGCGCGAUCUCGGCUCGCUGCAAACUGCCGCCCGGGUUCAAGCGAUUCUCGUGCCUCAGCCUCCCGACUAGCUGGGAUUACAGGCGCCUGCCCACCGCACCCCGCUGAUUUUUUUGUAUUUUUAGUAGCGACCCCGUUUCACCAUCUUGUCCAGGCUGGUCUUGAACUCCUGACCUCGUGAUCCACCCGCCUCGGCCUCCCAGAGUGCUGGGAUUACAGGCGUGAGCCACCGCGCCCAGCCGUGUUCUCCAUUCAGAUUUUGUCACAGUGACGUCCUAAAAUUAUUCCCAACUCCCCCCACCCUGCUGUUUCCGCACCCUCGAGCACAGCUAGUCGUCCGUCUGCGCCCUCCAGGCUCGCUUUCCCACUGCUCACUCUCCUCGCUCAAAAUUCAAGUCUCAACCGAGUCAUCUCUGUGACGUCACGUUGAUUUGCAUAAGAUUCCCCAGCGUCCCAAGCGAAUAUUCUUAUGGUUUUCAAAACCUGAAUGUUUGACACGGGAUGUUCCAACAACAAGAAACCUCCUAUGCAGAUGGGCCUUAAAUACGGCUGGUGGAGUGGGAACACGUCGUAUACACGGACACACGGGCAGGCACUCACCCUCAAUGUAAUGGUAGUCAUCAUCCGUGGGGGAGCGGGGCGCGAACAGAACCUUUCCAGCUUUGCGCAGUGGCAGUAUCGUAGCCAAUGAGGUUUAUCCGAGGCGCGAUUAUUGCUAAUUGAAAACUUUUCCCAAUACCCCGCCAUGACGACUUGAAAUAUAGUCGGCAUUGGCAAUUUUUGACAGUCUCUACGGAGACUGAAUUUUUAAAAGUCUAAAGAAAAGGGUUUUAGGAAGUUGUAUUAUAGCAGGUUGUGUCUUAUGUUUGGUUUGAUAAGUUAUGAGCUGUUCCUAUGAUAGUUUUUUGGUUGUUGUUCAGGAGUAUUCCGUGAUUGAAUGUCCGGGGUUCUGUGCGGGUACUGUGAGUUGUUUUCCAAAAUGUAUGACACUAUGUUGGGAACUGGGUUCUAACUAAGACAGUUAUCGUUAAUCUUUUUUAUCCAAAUGCAGCCAGCGAGUGAGCUACUCCACUUCCUUUGUCACUGAAGGUCACAGAUUCCCUUAAAGGGAAGGGGAACUCUAAAACUAACACAUUAAUACAUCCAAAGUCUUCCUUUUUUUUUUUUUUUUUUUUUUUGAGACGGAGUUUUCGCUGUUUCCCAGGCUGGGGUACAGUGGCGCGAUCUCGGCUGACUGCAACCUCCACAUUCCGGGUUCAAGCGAUUCUCUGUCUCAGCCUCCGGAGUAGCUGGGAUUACGGGAGCCCAACAUCACGCCCGGCUCAUUUUUGUAUUUUUAGUGGAGACGGGGUUUCGCCUUCUUGGCCAGGCUGGUUUCAAACUCCUGACCUCAGGUGAUCCGCCUACCUCGGCCUUCCGAAGUGCUGGGAUUACAGGUGUGAACCACCGCGUUUUCUUCCUUUUAAAGCGACCAAGAGGGAAAAAGGCACAGAAGAUUGCUCAGGUCCUACUGUGGAGUCCUCGGCAGAGCUGGGGCUUAUCAGGUUCUCUUGCCCCUAGUGUGGUACUUGUUCCCACUACACCAGCCUCUUCCAGUCUUACCUUUUUACGAAGACCUGGGCUGUCCAUGGACAGAGGUCCUCAGGAGUCACAACUCUGGGCAUGGCGGAUCCAGCUGGACCUGCUUAGGACACCGCCCAUAAAAGACUCCUCCAGCCUGUAGCAAAAAGAGGUGGCAAAAAGGGUGGCAACAGUACCUACAAGGUCAAAGACGGUGCUGGGAAAAACAGCUCCAGUGGCAAAUUAAACUCAGUCCCCAGUUCAAGGACCUCACAGACUGGAGACGUGGAGGCGAAGGUAGCAGUGAGCUGAAAUUGCGCCACUGCACUCCAGCCUGGGUGACAGUGAGACUCUGUCUCAAAAAAAAAGAAAAAAGAAAGAAGAAGAAGCCGGGAGCGGUGGCCCACGCCUGUCAUCCCAGCACUUUGGGAGGCCAGGGCGGGCAGAUCACGAGGUCAGGAGUUCAAGACCAGCCUGGCCAACAUGGUGAAACCCUAUCUCUACUAAAAAUACAAAAAAAAAAAAAAAAAAAAAAAAUUAGCCAGCCAUGGUCGUGGGCGCCUGUAAUCCCAGCUACUCGGGAGGC(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);
以及所述PPDPF转录本的序列如SEQ ID NO:16所示,具体为:
ACCCCGCGCGCGCCUCUCUGUCGUGGCGCGGCUUCCCGCGGUCUUCUCUGCAAAUGGGCUCCGUGGCCUAGCGCCCCCGUCCCCGCCACCCGUGAUCGUGCGCCGAGGCCCGCGAGGGGUCGCCGCCCAGAUCCCACCAGCCAGCAAGCUAAAGCAUGGCGGCCAUCCCCUCCAGCGGCUCGCUCGUGGCCACCCACGACUACUACCGGCGCCGCCUGGGUUCCACUUCCAGCAACAGCUCCUGCAGCAGUACCGAGUGCCCCGGGGAAGCCAUUCCCCACCCCCCAGGUCUCCCCAAGGCUGACCCGGGUCAUUGGUGGGCCAGCUUCUUUUUCGGGAAGUCCACCCUCCCGUUCAUGGCCACGGUGUUGGAGUCCGCAGAGCACUCGGAACCUCCCCAGGCCUCCAGCAGCAUGACCGCCUGUGGCCUGGCUCGGGACGCCCCGAGGAAGCAGCCCGGCGGUCAGUCCAGCACAGCCAGCGCUGGGCCCCCGUCCUGACCUGAGCGGUUACCACCAGCCCCAGGCCUGCGGAGGCGCUAGUCCACCAGAGCCCCUCCCCGCCCCUCUCCCCACUCCGCAUCCCUCGCCCCCCUCCCCACCUCCCACCCCCCACCCUGUAAACUAGGCGGCUGCAGCAAGCAGACCUUCGCAUCAACACAGCAGACACCAAAAACCAGUGAGAGCCCCGCUCUCUACCGCCCGGCCCCAGCACUCGCUAGCUUUCCUGACACCUGGAACUGUGCACCUGGCACCAAGCGGAAAAUAAACUCCAAGCAGCCAGUAGCCCCGAUGGUGUGUGCCUGAGCUGUGUGGCCCGAGGUUCCA(SEQ ID NO:16)。
在另一个实施方案中,所述RNA分子标志物存在于外泌体中,优选地,所述外泌体来自于体液,例如血液、尿液、唾液或痰液。
在第二个方面中,本公开提供了一种用于检测上述第一个方面任意实施方案的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lnc-MSI1-2:1的引物为:如SEQ ID NO:4所示的上游引物序列,以及如SEQ IDNO:5所示的下游引物序列;检测lnc-MSI1-2:1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
检测PPDPF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PPDPF基因转录本的探针序列如SEQ ID NO:12所示。
在第三个方面中,本公开提供了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在制备用于结直肠癌诊断、预后判断、疗效监测和/或复发监控的试剂盒中的应用。
在第四个方面中,本公开提供了一种用于结直肠癌诊断、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。
在第五个方面中,本公开提供了一种结直肠癌诊断、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lnc-MKRN2-42:1、lnc-MSI1-2:1、TOP1基因转录本和PPDPF基因转录本中的一种或更多种的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癌或患有结直肠癌的风险大小、结直肠癌的治疗效果和/或预后情况,和/或结直肠癌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癌的风险较高、结直肠癌的治疗效果和/或预后情况不佳,和/或结直肠癌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,对早期结直肠癌具有极高的灵敏度和特异性,对结直肠癌的诊断具有重要价值。
附图说明
图1显示了lnc-MKRN2-42:1诊断早期结直肠癌的性能。
图2显示了lnc-MSI1-2:1诊断早期结直肠癌的性能。
图3显示了TOP1诊断结直肠癌的性能。
图4显示了PPDPF诊断早期结直肠癌的性能。
图5显示了lnc-MKRN2-42:1和lnc-MSI1-2:1联合诊断早期结直肠癌的性能。
图6显示了lnc-MKRN2-42:1、lnc-MSI1-2:1和TOP1联合诊断早期结直肠癌的性能。
图7显示了lnc-MKRN2-42:1、lnc-MSI1-2:1、TOP1和PPDPF联合诊断早期结直肠癌的性能。
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“结直肠癌”在本发明中,可以指代结肠癌、直肠癌或者结直肠癌。
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癌,因此可作为结直肠癌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和lnc-MSI1-2:1,示例性的序列分别如SEQ ID NO:13和14所示;以及基因的转录本,具体为TOP1和PPDPF基因的转录本,其中本发明中所述的TOP1基因转录本和PPDPF基因转录本即表示由TOP1和PPDPF基因(人类的TOP1和PPDPF基因)所编码的mRNA,示例性的序列分别如SEQ IDNO:15和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,因此所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、插入或缺失。
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。本发明中的作为RNA分子标志物的RNA分子存在并分离自外泌体。
实施例
实施例1基于高通量测序筛选与结直肠癌相关的外泌体RNA分子标志物
取来自于15例早期结直肠癌患者和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,G Kong,Journal ofextracellular vesicles 8(1),1643670)。对测序结果进行生物信息学分析,比较早期结直肠癌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4个RNA分子标记物(包括特定基因的转录本和lncRNA各两个),具体如表1所示。
表1 RNA分子标志物列表
Figure BDA0002459663990000181
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
Figure BDA0002459663990000182
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lnc-MSI1-2:1的引物和探针:所述lnc-MSI1-2:1的上游引物具体具体为如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PPDPF的引物和探针:所述PPDPF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SEQ ID NO:12所示的核苷酸序列。
所有上述引物和探针序列见表3:
表3实施例所用引物和探针序列
序列编号 名称 序列(5’-3’) 备注
SEQ ID NO:1 MKRNF1 TCCGCACTAAGTTCGGCATC MKRN正向引物
SEQ ID NO:2 MKRNR1 TTTTGACCTGCTCTGTTTCCG MKRN反向引物
SEQ ID NO:3 MKRNFP TCCTTAGGCAACCTGGTGGTCCC MKRN探针
SEQ ID NO:4 MSI1F1 TGCCAATGCCGACTATATTTCAAG MSI1正向引物
SEQ ID NO:5 MSI1R1 GCAGTATCGTAGCCAATGAGGTTT MSI1反向引物
SEQ ID NO:6 MSI1FP CTTTTCCCAATACCCCGCCATGACGA MSI1探针
SEQ ID NO:7 top1F1 CTGTAGCCCTGTACTTCATCGAC top1正向引物
SEQ ID NO:8 top1R1 TCTACCACATATTCCTGACCATCC top1反向引物
SEQ ID NO:9 top1FP CCTTCCTCCTTTTCATTGCCTGCTCT top1探针
SEQ ID NO:10 PPDPFF1 CGCCTGGGTTCCACTTCCA PPDPF正向引物
SEQ ID NO:11 PPDPFR1 CTCTGCGGACTCCAACACC PPDPF反向引物
SEQ ID NO:12 PPDPFFP AAAAGAAGCTGGCCCACCAATGACCC PPDPF探针
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4 qPCR反应体系
Figure BDA0002459663990000201
表5 qPCR反应条件
Figure BDA0002459663990000202
实施例3利用RNA分子标志物进行结直肠癌早期诊断
1)样本采集
收集包括经医院确诊的早期(I期和II期)结直肠癌患者(47例)、结直肠良性病人和健康人对照样本(合计53例)共计100例对象的血液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47例早期结直肠癌病人样本和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、lnc-MSI1-2:1、TOP1和PPDPF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RNACel-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标志物的检测Ct值
Figure BDA0002459663990000211
Figure BDA0002459663990000221
Figure BDA0002459663990000231
Figure BDA0002459663990000241
表7目标RNA相对参考基因的ΔCt值
Figure BDA0002459663990000251
Figure BDA0002459663990000261
Figure BDA0002459663990000271
Figure BDA0002459663990000281
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癌的准确度。
(1)临床样本lnc-MKRN2-42:1、lnc-MSI1-2:1、TOP1和PPDPF分别单独检测结果
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RNA分子标志物的表达量均单独与早期结直肠癌显著相关。lnc-MKRN2-42:1、lnc-MSI1-2:1、TOP1和PPDPF诊断早期结直肠癌的AUC分别为0.71、0.68、0.64和0.62,具有良好的灵敏性和特异性。
(2)lnc-MKRN2-42:1和lnc-MSI1-2:1的联合检测
如图5所示,联合应用lnc-MKRN2-42:1和lnc-MSI1-2:1时,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lnc-MSI1-2:1的联合表达量与早期结直肠癌显著相关。lnc-MKRN2-42:1和lnc-MSI1-2:1联合诊断早期结直肠癌的AUC=0.84,其阴性预测值为81.48%、灵敏度为78.72%、特异性为83.02%,具有更加显著优秀的灵敏性和特异性。
(3)lnc-MKRN2-42:1、lnc-MSI1-2:1和TOP1的联合检测
如图6所示,联合应用lnc-MKRN2-42:1和lnc-MSI1-2:1和TOP1时,基于此前表6和表7的检测结果,对检测结果采用R语言进行t检测分析得到p<=0.05,说明lnc-MKRN2-42:1、lnc-MSI1-2:1和TOP1联合应用时,与早期结直肠癌显著相关,联合标志物诊断早期结直肠癌的AUC=0.89,其阴性预测值为92.5%、灵敏度为93.61%、特异性为69.81%,展现了极佳的灵敏度和特异性。
(4)lnc-MKRN2-42:1、lnc-MSI1-2:1、TOP1和PPDPF的联合检测
如图7所示,联合应用lnc-MKRN2-42:1和lnc-MSI1-2:1、TOP1和PPDPF时,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癌显著相关,四个标志物联合诊断早期结直肠癌的AUC达到了0.91,其阴性预测值为92.68%、灵敏度为93.61%、特异性为71.70%,展现了优秀的灵敏度和特异性。
以上结果表明,本公开提供的RNA分子标志物lnc-MKRN2-42:1、lnc-MSI1-2:1、TOP1和PPDPF,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。
序列表
<110> 首都医科大学附属北京友谊医院
<120> 用于结直肠癌诊断及预测的外泌体RNA及其应用
<130> MP1928957
<160> 16
<170> PatentIn version 3.2
<210> 1
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<212> DNA
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tccgcactaa gttcggcatc 20
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aaccuagcua agugaaauca gcaacgauau gcagaaacca ccuacgcagg ucacaaacau 9660
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aacucaccuu ugcgaaauag gaagcugguu uauugggagu gaugagcagg gggcguaaca 9780
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cacuguuuuc uuuuugagac gaagucuguc gcucaggcug gagugcacug gcgugaucuc 10260
ggcucgcugc aaacuuugcc gaaccccugu uuagcgauuu ccguuuguga aaguuggagu 10320
ugcugauuuu gugaucucua cuuuuaaggu aaaugcaggg cgguguugac uuuucuugcc 10380
agcuggcugu ccuguuuucu uucucucgca cuguuguuuu cuuuuugaga cgaagucugu 10440
cgcucaggcu ggagugcacu ggcgcgaucu cggcucgcug caaacugccg cccggguuca 10500
agcgauucuc gugccucagc cucccgacua gcugggauua caggcgccug cccaccgcac 10560
cccgcugauu uuuuuguauu uuuaguagcg accccguuuc accaucuugu ccaggcuggu 10620
cuugaacucc ugaccucgug auccacccgc cucggccucc cagagugcug ggauuacagg 10680
cgugagccac cgcgcccagc cguguucucc auucagauuu ugucacagug acguccuaaa 10740
auuauuccca acucccccca cccugcuguu uccgcacccu cgagcacagc uagucguccg 10800
ucugcgcccu ccaggcucgc uuucccacug cucacucucc ucgcucaaaa uucaagucuc 10860
aaccgaguca ucucugugac gucacguuga uuugcauaag auuccccagc gucccaagcg 10920
aauauucuua ugguuuucaa aaccugaaug uuugacacgg gauguuccaa caacaagaaa 10980
ccuccuaugc agaugggccu uaaauacggc ugguggagug ggaacacguc guauacacgg 11040
acacacgggc aggcacucac ccucaaugua augguaguca ucauccgugg gggagcgggg 11100
cgcgaacaga accuuuccag cuuugcgcag uggcaguauc guagccaaug agguuuaucc 11160
gaggcgcgau uauugcuaau ugaaaacuuu ucccaauacc ccgccaugac gacuugaaau 11220
auagucggca uuggcaauuu uugacagucu cuacggagac ugaauuuuua aaagucuaaa 11280
gaaaaggguu uuaggaaguu guauuauagc agguuguguc uuauguuugg uuugauaagu 11340
uaugagcugu uccuaugaua guuuuuuggu uguuguucag gaguauuccg ugauugaaug 11400
uccgggguuc ugugcgggua cugugaguug uuuuccaaaa uguaugacac uauguuggga 11460
acuggguucu aacuaagaca guuaucguua aucuuuuuua uccaaaugca gccagcgagu 11520
gagcuacucc acuuccuuug ucacugaagg ucacagauuc ccuuaaaggg aaggggaacu 11580
cuaaaacuaa cacauuaaua cauccaaagu cuuccuuuuu uuuuuuuuuu uuuuuuuuga 11640
gacggaguuu ucgcuguuuc ccaggcuggg guacaguggc gcgaucucgg cugacugcaa 11700
ccuccacauu ccggguucaa gcgauucucu gucucagccu ccggaguagc ugggauuacg 11760
ggagcccaac aucacgcccg gcucauuuuu guauuuuuag uggagacggg guuucgccuu 11820
cuuggccagg cugguuucaa acuccugacc ucaggugauc cgccuaccuc ggccuuccga 11880
agugcuggga uuacaggugu gaaccaccgc guuuucuucc uuuuaaagcg accaagaggg 11940
aaaaaggcac agaagauugc ucagguccua cuguggaguc cucggcagag cuggggcuua 12000
ucagguucuc uugccccuag ugugguacuu guucccacua caccagccuc uuccagucuu 12060
accuuuuuac gaagaccugg gcuguccaug gacagagguc cucaggaguc acaacucugg 12120
gcauggcgga uccagcugga ccugcuuagg acaccgccca uaaaagacuc cuccagccug 12180
uagcaaaaag agguggcaaa aaggguggca acaguaccua caaggucaaa gacggugcug 12240
ggaaaaacag cuccaguggc aaauuaaacu caguccccag uucaaggacc ucacagacug 12300
gagacgugga ggcgaaggua gcagugagcu gaaauugcgc cacugcacuc cagccugggu 12360
gacagugaga cucugucuca aaaaaaaaga aaaaagaaag aagaagaagc cgggagcggu 12420
ggcccacgcc ugucauccca gcacuuuggg aggccagggc gggcagauca cgaggucagg 12480
aguucaagac cagccuggcc aacaugguga aacccuaucu cuacuaaaaa uacaaaaaaa 12540
aaaaaaaaaa aaaaaaauua gccagccaug gucgugggcg ccuguaaucc cagcuacucg 12600
ggaggc 12606
<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> 827
<212> RNA
<213> 人
<400> 16
accccgcgcg cgccucucug ucguggcgcg gcuucccgcg gucuucucug caaaugggcu 60
ccguggccua gcgcccccgu ccccgccacc cgugaucgug cgccgaggcc cgcgaggggu 120
cgccgcccag aucccaccag ccagcaagcu aaagcauggc ggccaucccc uccagcggcu 180
cgcucguggc cacccacgac uacuaccggc gccgccuggg uuccacuucc agcaacagcu 240
ccugcagcag uaccgagugc cccggggaag ccauucccca ccccccaggu cuccccaagg 300
cugacccggg ucauuggugg gccagcuucu uuuucgggaa guccacccuc ccguucaugg 360
ccacgguguu ggaguccgca gagcacucgg aaccucccca ggccuccagc agcaugaccg 420
ccuguggccu ggcucgggac gccccgagga agcagcccgg cggucagucc agcacagcca 480
gcgcugggcc cccguccuga ccugagcggu uaccaccagc cccaggccug cggaggcgcu 540
aguccaccag agccccuccc cgccccucuc cccacuccgc aucccucgcc ccccucccca 600
ccucccaccc cccacccugu aaacuaggcg gcugcagcaa gcagaccuuc gcaucaacac 660
agcagacacc aaaaaccagu gagagccccg cucucuaccg cccggcccca gcacucgcua 720
gcuuuccuga caccuggaac ugugcaccug gcaccaagcg gaaaauaaac uccaagcagc 780
caguagcccc gauggugugu gccugagcug uguggcccga gguucca 827

Claims (13)

1.一种用于结直肠癌诊断、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、lnc-MSI1-2:1、TOP1基因转录本和PPDPF基因转录本中一种或更多种的RNA分子的组合。
2.根据权利要求1所示的RNA分子标志物,所述lnc-MKRN2-42:1的序列如SEQ ID NO:13所示,所述lnc-MSI1-2:1的序列如SEQ ID NO:14所示,TOP1转录本的序列如SEQ ID NO:15所示,和/或所述PPDPF转录本的序列如SEQ ID NO:16所示。
3.根据权利要求1或2所述的RNA分子标志物,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,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至3中任一项所述RNA分子标志物的试剂,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lnc-MSI1-2:1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;检测lnc-MSI1-2:1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SEQ IDNO: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PPDPF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 IDNO:11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:12所示。
6.根据权利要求1至4中任一项所述的RNA分子标志物或者权利要求5所述的试剂在制备用于结直肠癌诊断、预后判断、疗效监测和/或复发监控的试剂盒中的应用。
7.一种用于结直肠癌诊断、预后判断、疗效监测和/或复发监控的试剂盒,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。
9.一种结直肠癌诊断、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;
(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lnc-MKRN2-42:1、lnc-MSI1-2:1、TOP1基因转录本和PPDPF基因转录本中的一种或更多种(例如两种、三种或者四种)的RNA分子标志物的表达水平,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癌或患有结直肠癌的风险大小、结直肠癌的治疗效果和/或预后情况,和/或结直肠癌是否复发或者复发风险的大小。
10.根据权利要求9所述的方法,其中所述步骤(4)的检测包括反转录和定量PCR的步骤,所述定量PCR所使用的试剂为权利要求5所述的试剂。
11.根据权利要求9所述的方法,其中步骤(4)还包括利用外参基因对所述表达水平进行归一化的步骤。
12.根据权利要求11所述的方法,其中步骤(4)所述的外参基因是cel-miR-39。
13.根据权利要求11所述的方法,其中步骤(4)的归一化步骤之后,将上述归一化后的分子标志物的表达水平值进行逻辑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癌的风险较高、结直肠癌的治疗效果和/或预后情况不佳,和/或结直肠癌在治疗后发生了复发或者复发风险较高。
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