CN111455052A

CN111455052A - 用于结直肠癌早期诊断的外泌体rna分子标记物组合及其应用

Info

Publication number: CN111455052A
Application number: CN202010316540.6A
Authority: CN
Inventors: 朱圣韬; 闵力; 孔关义; 刘翔; 张澍田
Original assignee: Beijing Friendship Hospital
Current assignee: Beijing Friendship Hospital
Priority date: 2020-04-21
Filing date: 2020-04-21
Publication date: 2020-07-28
Anticipated expiration: 2040-04-21
Also published as: CN111455052B

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RNA标记物及其应用。

Description

用于结直肠癌早期诊断的外泌体RNA分子标记物组合及其应用

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分子标志物组合及其应用。

背景技术

结直肠癌(Colorectal cancer，CRC)是常见的消化系统恶性肿瘤之一，在我国其发病率和病死率仅次于胃癌、食管癌和原发性肝癌。结直肠癌在疾病早期几乎无明显症状，随着病灶逐渐增大开始出现腹泻、便秘、腹痛甚至便血等症状，晚期则出现贫血、消瘦等全身症状。近年来，我国结直肠癌的发病率和死亡率呈持续上升趋势。有统计显示我国结直肠癌发病率、死亡率在全部恶性肿瘤中上升至第5位，其中新发病例近40万，死亡病例近20万。由于结直肠癌早期多无明显症状，多数患者发现时已属于中晚期，严重影响患者生存质量及预后。因此，结直肠癌的早期诊断、早期治疗显得尤为重要，已逐渐成为提高国家医疗水平和改善人民生活质量的重要问题。

在结直肠癌的筛查中，电子结肠镜检查仍然是金标准，研究显示电子结肠镜筛查能够显著降低结直肠癌死亡率。虽然结肠镜检查具有重要的临床意义，但由于检查前需要完备的术前准备，同时又是一项侵入性检查，因此在对每个人进行结肠镜检查仍具有挑战性。目前临床上粪便潜血检测(FOBT)及粪便DNA检测(FDT)是两种可行的无创性筛查手段，尽管这两种方法都有助于降低结直肠癌的死亡率，但较低的特异度和敏感度使其临床应用受到了一定的限制。血液septin9甲基化检测是基于血液的无创检测手段，但其检测的灵敏度并不高。因此开发一项基于血液的高灵敏度及特异度的无创检测手段至关重要。

细胞外囊泡(Extracellular Vesicles,EVs)是指由细胞分泌或从细胞膜上脱落的具有双层膜结构的囊泡状小体，其直径在30-1000nm左右，主要分为微囊泡(MicroVesicles,MVs)和外泌体(exosomes)两部分，其中外泌体功能复杂，具有多种生物活性，近年来逐渐成为了国内外研究热点。

目前研究显示，外泌体是由细胞内的内溶酶体微粒内陷形成多囊泡体后与细胞膜融合并向细胞外分泌的大小均一，直径在30～150nm的囊泡状结构，其外部主要由脂质双分子层构成，内部包含DNA、RNA及蛋白质等多种生物活性物质；近年来研究表明，外泌体广泛存在于血液、唾液、尿液、乳汁和胸腹水等部位，具有抗原呈递、细胞凋亡、炎症反应、肿瘤发展及转移等广泛的生物学功能。尤其在针对恶性肿瘤细胞的研究中发现，肿瘤细胞分泌的外泌体中含有miRNA、mRNA及lncRNA等分子，在肿瘤发生发展过程中起到不可忽视的作用，因此外泌体中富含的miRNA、mRNA及lncRNA分子可以作为肿瘤等多种疾病的无创诊断标志物，在结直肠癌早期诊断中具有巨大潜力。

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RNAlnc-MKRN2-42:1和lnc-MSI1-2:1、两个mRNA：TOP1和PPDF，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分子标志物组合，其包括miR-106b-3p、lnc-MKRN2-42:1、lnc-MSI1-2:1、TOP1基因转录本和PPDPF基因转录本。

在一个实施方案中，所述miR-106b-3p的序列如SEQ ID NO：17所示，具体为：CCGCACUGUGGGUACUUGCUGC(SEQ ID NO:17)；

所述lnc-MKRN2-42:1的序列如SEQ ID NO：18所示，具体为：

GCCGGGCACAGUGGCGCGUGCCUGUAGUCCCAGCUACUCGGGAGGCUGAGGUGGGAGGAUCGCUUGAGCCCAGGAGUUCUGGGCUGUAGUGCGCUAUGCCGAUCGGGUGUCCGCACUAAGUUCGGCAUCAAUAUGGUGACCUCCCGGGAGCGGGGGACCACCAGGUUGCCUAAGGAGGGGUGAACCGGCCCAGGUCGGAAACAGAGCAGGUCAAAACUCCCGUGCUGAUCAGGAGACAGAGUUUGUGAGCAGACAACUGGUCUGACCAAAAUUUAUGAGGUGGGAAUUUCCUCU(SEQ ID NO:18)；

所述lnc-MSI1-2:1的序列如SEQ ID NO：19所示，具体为：

GCUGGUAAUGGUCACACUAGGUGGCGUCCGCGUGAGGUGGAGGUGUCAGCGGCUCCUGUCUGCAGCUUCCUGGGGUCCUGGCGGCGCUCUUCUCCUGAGCCAGAAUCCAGCAGGAAGGACAGAAUGGGCGCUAGAGUGUGAAAAGAGGGCGAACUCACGUGGAUGUGACUCUCCGGGUCGCUCUUCCCUGUCACCCAGCUCCUCCUGCCGGUUUCCUGUGGGCAUCGCCUGCAUUGCUUUUAUCCCGGGGACGGAUUAGGGGGGUCCAGAGGAGGACUUGCUGGCACAAAUAACCCAAUGGAGGCUUUCGAGCACGGCUGGCUGGGGUUUGCGAUCCAACGGCCUUUUGCUGACCUGAAAGUCAACGCAAAGCUCAUCUUCAUUCUAGAGAAAAAGAAGCCUCAUAUCUGAGAUCAGGACCCCGCUUUCUCCUUGACAUUCCACAGCAAAACGUGAACAGCUGAACAAACACACAGUCCGGUUUCCCGAGACCAAAGGGUCAGGGGAAACCCCCGCUGAAAACGUGCAUCAGUGGAACAACUACUCUCUUCCAGGUCCCACCGAGGGCAAAGAGGAGAAAUAGAAUCCCUCACAAGAAAUCCACAACUUCUAGAAAUCCGCAGCACACACAGAACACACUUACCUCUCACAGCUCUACGGACACUUCACACCCCACAGUGGGACCGUCUGAUUGCAUUUGCAUUUUGGGACAUGUAGUCCAGAAAGCUUAAAUGAUGCGAGAAAAACCACAAGUCCCACAAUCCUCCGCGUCUCGCAAUUGACAGCCCCAGAUCAUCCAGGAACGCCCCCUGUCGGCUCUUUCUGGUCAGAGGUUCCCCCGUUAAUAUAAAAGCUUUUAAUCAUGGAUUUUUUUCUUUGUUUUCAAUUUCUGUGGGCACUUAGUAGGUGUAUAUAUUUAUAGUGUACAUAAGAUUUUUUUUCUUUUUUCUUUUUUUUUUUUAAGACGGAGUUGUUGUUUUUUUUUUUUGAAAAAACUCCAGUCUGGAGUGCAGUGGCACGAUCUCGGCUCACUGCAACCUCCCUCUCCCGGGUUCAACCGAUUCUCCUGCCUCAGCCUCCCGAGUAGCUGGGAUUAUUUUUGUAUUUUUAGUAGAGACGGGGGUUUCGCCAUGUUGGCCAGGCUCGUCUUGAACUCCUAACCUCAGGUGAUCCGCACGCCUUGACCUCCCAAAGUGCUGGGAUUAUAGGCGUGAGCCACUGCGCCCGGCCAUCUUUUCUCUUAUUUUUUGUCUUUUUUUCUUUUCUCUGUGCCUGUCUAUGCUUUACCCACAUGAGAUGUUUUGAUACAAACAUGCAAUGUGAAAUGAGCACGUCAUUAAUCAUGGAUCUAGAUUUGACUUUUUUUUAAAGUUUUUGUAUUUUUAGUAGAGACAGAGUUUCACCAUGUUGGCCAGGCUGGUCUCGAACUCCCGACCUCAGGUGAUCCGCCAACCUCGGCCCCCCAAAAUGCUGGGAUUACAGGCCUGAGCCACUGUUCCCGGCCUAGAUUUGACUCAUUCUAAACAUCAGAGUUUGUGAUCCUUUUCAUUCACAACGACAGUAUCAAGUUAUUUGCUAUUGGUAAAAUACCUUGAACUUGUGAAGAAAUGUGUUUAUACAUAUCUGAAAAAGUGAUAGUUUGUUGUUGUUGUUGUUGUUGUUUGUUUGUUUGUUUUUGAGACGGAGUCUCGCUCUGUUGCCCAGGCUGGAGUGCAAUGGUGCCAUUUCGGCUCACUGCAACCUCCGCCUCCCGGGUUCAAGCAAUUCUCCUGCCUCAGCCUCCUGAGUAGCAGGGAUUAUGGGCGCAUGCCACUACGCCCGGUUAAUUUUUUUAUUUUUAGUAGAGACAAGGUUUCACCAUGUUAGCCAGGAUGGUCUUGAUCUCCUGACCUCGUGAUCUGCCCACCCUAGCCUCCCAAAGUACUGGGAUUACAGGCGUAAGCCACUGUGCCCAGCCAAAAGUGAUGUUUUUACAGUUAAUAUGAAGCCAAAUAUAUUAUACCUGGACUAAUGUACUUAACCUGCUUGGCUUCUCCAAUUUUAUUUAUCAUAUUUUUUUUAUUAAGUUAAUCAAACAAGGCCUGCGUCUCACUCCCAGGGAUUCUGAUCUACAAAUUUCCUAAUUGUUCCUAAUGUGUAGACAACCAGAACUGCCGCUCUGCAGCAUCACCUAAAUCAAUAAAUGUAUAAGUGUCUUUGGACAUAAAGGUGGGCGGCCAGCAGGGACCUUGAAGACCACCUAAGGACUCCCUUCCCCCAUUUUAAGAUGAAUUGGGCUGAGUGCAGUGGUUCCCACCUGUAAUACCAACACUUUGGGAGGCCAAGGCAGGAGGAUCACUUGAGCCCAAAAGUUCAAGACCAGCUUGGGCAACAUAGUGAGACCCACUCACUACAAAAAAUAAACAAAAUUAGCGGGGUGUGGUGGUGUGUGCCUGUAGUCCCAGCUACUCAAGAGGCUCAGGUGGGAGGAUCACUUUGAGCCCAGGAGAUCGAGGCUGCAGUGAGCCAUAAUUGUGCGACUGCACUUCAGCCUGGGUGACAAAGUGAGAUCCCAUCUCUUCAAAAAAUAAUAAUAAUAAAUAAAGAUGAACUGAAGUGCAGAGAAGUAAUGUGGAAGCUUGAGAAAGAGUUUUUAGGUUUUUGGUUCUAUCAUUGUAUCUUUGGAGGUAAUAUUGGCAAAAUGACCAUUUAGUAGUUUGCCCAUUUCUGCUUUUUUUUUUUCUUUUUUUUGAGACAGGGUCUCGUUCUGUCACCCAGGCUGGAGUGCAGUUGUGACAUCACAGCUCACUGCAGUUUCUACUUCCCAGGCCCAAGGGAUCCUCCAACCUCAUCCUCUCGAGUAGCUGAGAUCACAGGCAUCCACCACCACGCCCAGCUAAAUUUUGUUAUUUUUUAUUUUUGUCGAGACACGGUCUCACUAUGUUGCUCAGGCUGGUCUCAAACUCCUGAACUCAAGGAAAGCUCUUGCUUUGGCCUCCCAAAGUGCUGGGAUUACAGGUGUGAGCCACCAUGCCGGGCCUGCUUAUUUCGACUUGGCAGCAUCGUUUCAUGAAGCCCCUGACUCUGUGACAACAUACAGCUCUCCAGAAAGAUGCUUUGAAGACAAAACAGAAUAGAGCACACAGCCCUCCAUGUCUCUUCCAAGUCACUAUAUUCAUUAAAAGAUAAAUGACUCUGGUCAUUGCCUUUUCCUGCACAAGAUAAUGUCUGAUGGGGUUAGUGAUGAUACCUCUGUAAUCCAUAACCAGAUAUACUCUUGCACCCAAACUUUGAUAUGAUUGUGCUUUAAUGUAACUUUGAAGCAAGUUUAAUGUGAUUUUGCAGGCGCCGUACACUCACCACUUGUAUAUGAGUGGCUGAAAUUCUGUGUUGGACCAGUCUGAUGGAACUGCUCCCACACUAUAGCCCUCGGUCUGUAAUCCUCAGUAAGACUUGUAAGUAAAACUAACUUUAAUUUUUUUUUUUUUUUUUUGAGAGAGUCUAGCUCUGUUGCCCAGGCUGGAGUGCAGUGGCGCGAUCUUCACUCACUGAAGCCUCUGCCUUCCCGGCUGAAGUGAUUCUGCUGCCUCGGCCUCCGGAGUAGCUAGGAUUACAGGCGCUCGCCACUGCGCCCGGCUAAUUUUUGUAUUUUUAGUAGAGAUGGGGUUUCGUCAUGUUGGCCAGGCUGGUUUUGAACUCGUGACCUCAGGUGACCCACCUGCCUCAGUCUCCUAAAAUGCUAGGAUUACAGGAGUGAGCCACUGCACCCCGCCACUUUUAUUAUUAUUAUUAUUGUAUUUAUUUAUUUAUUUAUUUAUUUUUGAGACGGAGUCUCGCUCUGUCGCCCAGGCUGGAGUGUAGUGGAGAGAUCUCGGCCCACUACAAGCUCCGCCUCCCGGGUUCACGCGAUUCUCCUGCCUCAGCCUCCGGACUACAGGCGCCCGCCACCACACCCGGCUAAUUUUUUGUAUUUUUUUUUUAGUAGAGACGGGGUUUCACCGUGUUAGCCAGGAUGGUCUCUAUCUCUUGACCUCGUGAUCCGCCCGCCUCAGCCUCCCAAAGUACUGGGAUUACAGGCGUGAGCCACCGCGCCCGGCUAUUUAUUUAUUUAUUUAUUUAUUUAUUUAUUUAUUUUUUGAGACGGAAUAUUGCUCUGUGCCCAGGCUGGAGUGCAGUGGCACGAUCUCAGAUCACUGCAACCUCCGCCUCCCGGGUUCAAGUGAUUCUCCUGCCUCAGCCUCCCGAGUAGCUGGGAUUACGGGUGCCCGCCACCAUGCCCGGCUAAUUUUUGUAUUUUUAGUAGAGACGGGGUUUCACCAUGCUUGUCAGCAUGGUCUCGAACUCCUGACCUCGCGAUCCGCCCGCCUCAGCCUCCCAAAGUGCUGGAAUUACAGGCAUGAGCCACUGCGCCCUGCCACUUUAAUUAUUUAAAAGCUUAUUUUUUUAUUUUGGGGACAUGGUCUCACUCUUUCACCCAGGCGGGAGUUCAGAGGCACGAUCACUGCGCACUACAGCCUCAACCUCUUGGACUCAAACCAUCCUCCCACCUCAGCCUCCUGAGUAGCUGGGACUACAGGUGGGCCACCAAGCCCAGCUAAUUUUUGUAUUUUUUUGUAGAGACGGGGUUUCACCAUGUUGCCCAGGCUGGUCUCGAACUCCUGGGCUCUAGGCUUUCCAAAGUGUUGUGAUUACGGGCGUGAGCUACUGCGCCUGGCCGAUUCUUUUUAACUUAGUACACAGGGAGUAGUAGCUCCAGCAACAGCGAAAGUCAGUAGCUGUAGGAUCUACUAGGCGGGGAAACUCAUAUGACAGCCCCCUCCUUUUUUUUUUUUUUUUUUUUUUUUUUGAGAUGGAGUUUCACUCUUGUUGACCAGGCUGGAGUGCAAUGGCGCCAUCUUGGCUCACCGCAACCUCCGCCUCCCGGGUUCAAGUGAUUCUCCUGCCUCAGCCUCCCCAGUAUCUGGGAUUACAGGUGUGAGCCACUGCGCCCGGCCACCCCCUUUUUUUUUCAAGGCUGCAGU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GUACUUGUUCGCAAACUCUCUAAAUGAGAGAUGGGGAAGUGUCUUACCAGCUGAUUCGCGAGAGCACCUGCAAGUUUACCUGUUUUAGUGAUUUGCGUUUAUGAAAGUUGGAGUUGCUGAUUUUGUGAUCUCUACUUUUAAGGUAAAAGCAGGGCGGUGUUGACUUUUCUUGCCAGUUGGCUGUCCUGUUUUCUUUCUCGCACUGUUUUCUUUUUGAGACGAAGUCUGUCGCUCAGGCUGGAGUGCACUGGCGUGAUCUCGGCUCGCUGCAAACUUUGCCGAACCCCUGUUUAGCGAUUUCCGUUUGUGAAAGUUGGAGUUGCUGAUUUUGUGAUCUCUACUUUUAAGGUAAAUGCAGGGCGGUGUUGACUUUUCUUGCCAGCUGGCUGUCCUGUUUUCUUUCUCUCGCACUGUUGUUUUCUUUUUGAGACGAAGUCUGUCGCUCAGGCUGGAGUGCACUGGCGCGAUCUCGGCUCGCUGCAAACUGCCGCCCGGGUUCAAGCGAUUCUCGUGCCUCAGCCUCCCGACUAGCUGGGAUUACAGGCGCCUGCCCACCGCACCCCGCUGAUUUUUUUGUAUUUUUAGUAGCGACCCCGUUUCACCAUCUUGUCCAGGCUGGUCUUGAACUCCUGACCUCGUGAUCCACCCGCCUCGGCCUCCCAGAGUGCUGGGAUUACAGGCGUGAGCCACCGCGCCCAGCCGUGUUCUCCAUUCAGAUUUUGUCACAGUGACGUCCUAAAAUUAUUCCCAACUCCCCCCACCCUGCUGUUUCCGCACCCUCGAGCACAGCUAGUCGUCCGUCUGCGCCCUCCAGGCUCGCUUUCCCACUGCUCACUCUCCUCGCUCAAAAUUCAAGUCUCAACCGAGUCAUCUCUGUGACGUCACGUUGAUUUGCAUAAGAUUCCCCAGCGUCCCAAGCGAAUAUUCUUAUGGUUUUCAAAACCUGAAUGUUUGACACGGGAUGUUCCAACAACAAGAAACCUCCUAUGCAGAUGGGCCUUAAAUACGGCUGGUGGAGUGGGAACACGUCGUAUACACGGACACACGGGCAGGCACUCACCCUCAAUGUAAUGGUAGUCAUCAUCCGUGGGGGAGCGGGGCGCGAACAGAACCUUUCCAGCUUUGCGCAGUGGCAGUAUCGUAGCCAAUGAGGUUUAUCCGAGGCGCGAUUAUUGCUAAUUGAAAACUUUUCCCAAUACCCCGCCAUGACGACUUGAAAUAUAGUCGGCAUUGGCAAUUUUUGACAGUCUCUACGGAGACUGAAUUUUUAAAAGUCUAAAGAAAAGGGUUUUAGGAAGUUGUAUUAUAGCAGGUUGUGUCUUAUGUUUGGUUUGAUAAGUUAUGAGCUGUUCCUAUGAUAGUUUUUUGGUUGUUGUUCAGGAGUAUUCCGUGAUUGAAUGUCCGGGGUUCUGUGCGGGUACUGUGAGUUGUUUUCCAAAAUGUAUGACACUAUGUUGGGAACUGGGUUCUAACUAAGACAGUUAUCGUUAAUCUUUUUUAUCCAAAUGCAGCCAGCGAGUGAGCUACUCCACUUCCUUUGUCACUGAAGGUCACAGAUUCCCUUAAAGGGAAGGGGAACUCUAAAACUAACACAUUAAUACAUCCAAAGUCUUCCUUUUUUUUUUUUUUUUUUUUUUUGAGACGGAGUUUUCGCUGUUUCCCAGGCUGGGGUACAGUGGCGCGAUCUCGGCUGACUGCAACCUCCACAUUCCGGGUUCAAGCGAUUCUCUGUCUCAGCCUCCGGAGUAGCUGGGAUUACGGGAGCCCAACAUCACGCCCGGCUCAUUUUUGUAUUUUUAGUGGAGACGGGGUUUCGCCUUCUUGGCCAGGCUGGUUUCAAACUCCUGACCUCAGGUGAUCCGCCUACCUCGGCCUUCCGAAGUGCUGGGAUUACAGGUGUGAACCACCGCGUUUUCUUCCUUUUAAAGCGACCAAGAGGGAAAAAGGCACAGAAGAUUGCUCAGGUCCUACUGUGGAGUCCUCGGCAGAGCUGGGGCUUAUCAGGUUCUCUUGCCCCUAGUGUGGUACUUGUUCCCACUACACCAGCCUCUUCCAGUCUUACCUUUUUACGAAGACCUGGGCUGUCCAUGGACAGAGGUCCUCAGGAGUCACAACUCUGGGCAUGGCGGAUCCAGCUGGACCUGCUUAGGACACCGCCCAUAAAAGACUCCUCCAGCCUGUAGCAAAAAGAGGUGGCAAAAAGGGUGGCAACAGUACCUACAAGGUCAAAGACGGUGCUGGGAAAAACAGCUCCAGUGGCAAAUUAAACUCAGUCCCCAGUUCAAGGACCUCACAGACUGGAGACGUGGAGGCGAAGGUAGCAGUGAGCUGAAAUUGCGCCACUGCACUCCAGCCUGGGUGACAGUGAGACUCUGUCUCAAAAAAAAAGAAAAAAGAAAGAAGAAGAAGCCGGGAGCGGUGGCCCACGCCUGUCAUCCCAGCACUUUGGGAGGCCAGGGCGGGCAGAUCACGAGGUCAGGAGUUCAAGACCAGCCUGGCCAACAUGGUGAAACCCUAUCUCUACUAAAAAUACAAAAAAAAAAAAAAAAAAAAAAAAUUAGCCAGCCAUGGUCGUGGGCGCCUGUAAUCCCAGCUACUCGGGAGGC(SEQ ID NO:19)；

TOP1基因转录本的序列如SEQ ID NO：20所示，具体为：

CAAAUGCGAACUUAGGCUGUUACACAACUGCUGGGGUCUGUUCUCGCCGCCCGCCCGGCAGUCAGGCAGCGUCGCCGCCGUGGUAGCAGCCUCAGCCGUUUCUGGAGUCUCGGGCCCACAGUCACCGCCGCUUACCUGCGCCUCCUCGAGCCUCCGGAGUCCCCGUCCGCCCGCACAGGCCGGUUCGCCGUCUGCGUCUCCCCCACGCCGCCUCGCCUGCCGCCGCGCUCGUCCCUCCGGGCCGACAUGAGUGGGGACCACCUCCACAACGAUUCCCAGAUCGAAGCGGAUUUCCGAUUGAAUGAUUCUCAUAAACACAAAGAUAAACACAAAGAUCGAGAACACCGGCACAAAGAACACAAGAAGGAGAAGGACCGGGAAAAGUCCAAGCAUAGCAACAGUGAACAUAAAGAUUCUGAAAAGAAACACAAAGAGAAGGAGAAGACCAAACACAAAGAUGGAAGCUCAGAAAAGCAUAAAGACAAACAUAAAGACAGAGACAAGGAAAAACGAAAAGAGGAAAAGGUUCGAGCCUCUGGGGAUGCAAAAAUAAAGAAGGAGAAGGAAAAUGGCUUCUCUAGUCCACCACAAAUUAAAGAUGAACCUGAAGAUGAUGGCUAUUUUGUUCCUCCUAAAGAGGAUAUAAAGCCAUUAAAGAGACCUCGAGAUGAGGAUGAUGCUGAUUAUAAACCUAAGAAAAUUAAAACAGAAGAUACCAAGAAGGAGAAGAAAAGAAAACUAGAAGAAGAAGAGGAUGGUAAAUUGAAAAAACCCAAGAAUAAAGAUAAAGAUAAAAAAGUUCCUGAGCCAGAUAACAAGAAAAAGAAGCCGAAGAAAGAAGAGGAACAGAAGUGGAAAUGGUGGGAAGAAGAGCGCUAUCCUGAAGGCAUCAAGUGGAAAUUCCUAGAACAUAAAGGUCCAGUAUUUGCCCCACCAUAUGAGCCUCUUCCAGAGAAUGUCAAGUUUUAUUAUGAUGGUAAAGUCAUGAAGCUGAGCCCCAAAGCAGAGGAAGUAGCUACGUUCUUUGCAAAAAUGCUCGACCAUGAAUAUACUACCAAGGAAAUAUUUAGGAAAAAUUUCUUUAAAGACUGGAGAAAGGAAAUGACUAAUGAAGAGAAGAAUAUUAUCACCAACCUAAGCAAAUGUGAUUUUACCCAGAUGAGCCAGUAUUUCAAAGCCCAGACGGAAGCUCGGAAACAGAUGAGCAAGGAAGAGAAACUGAAAAUCAAAGAGGAGAAUGAAAAAUUACUGAAAGAAUAUGGAUUCUGUAUUAUGGAUAACCACAAAGAGAGGAUUGCUAACUUCAAGAUAGAGCCUCCUGGACUUUUCCGUGGCCGCGGCAACCACCCCAAGAUGGGCAUGCUGAAGAGACGAAUCAUGCCCGAGGAUAUAAUCAUCAACUGUAGCAAAGAUGCCAAGGUUCCUUCUCCUCCUCCAGGACAUAAGUGGAAAGAAGUCCGGCAUGAUAACAAGGUUACUUGGCUGGUUUCCUGGACAGAGAACAUCCAAGGUUCCAUUAAAUACAUCAUGCUUAACCCUAGUUCACGAAUCAAGGGUGAGAAGGACUGGCAGAAAUACGAGACUGCUCGGCGGCUGAAAAAAUGUGUGGACAAGAUCCGGAACCAGUAUCGAGAAGACUGGAAGUCCAAAGAGAUGAAAGUCCGGCAGAGAGCUGUAGCCCUGUACUUCAUCGACAAGCUUGCUCUGAGAGCAGGCAAUGAAAAGGAGGAAGGAGAAACAGCGGACACUGUGGGCUGCUGCUCACUUCGUGUGGAGCACAUCAAUCUACACCCAGAGUUGGAUGGUCAGGAAUAUGUGGUAGAGUUUGACUUCCUCGGGAAGGACUCCAUCAGAUACUAUAACAAGGUCCCUGUUGAGAAACGAGUUUUUAAGAACCUACAACUAUUUAUGGAGAACAAGCAGCCCGAGGAUGAUCUUUUUGAUAGACUCAAUACUGGUAUUCUGAAUAAGCAUCUUCAGGAUCUCAUGGAGGGCUUGACAGCCAAGGUAUUCCGUACAUACAAUGCCUCCAUCACGCUACAGCAGCAGCUAAAAGAACUGACAGCCCCGGAUGAGAACAUCCCAGCGAAGAUCCUUUCUUAUAACCGUGCCAAUCGAGCUGUUGCAAUUCUUUGUAACCAUCAGAGGGCACCACCAAAAACUUUUGAGAAGUCUAUGAUGAACUUGCAAACUAAGAUUGAUGCCAAGAAGGAACAGCUAGCAGAUGCCCGGAGAGACCUGAAAAGUGCUAAGGCUGAUGCCAAGGUCAUGAAGGAUGCAAAGACGAAGAAGGUAGUAGAGUCAAAGAAGAAGGCUGUUCAGAGACUGGAGGAACAGUUGAUGAAGCUGGAAGUUCAAGCCACAGACCGAGAGGAAAAUAAACAGAUUGCCCUGGGAACCUCCAAACUCAAUUAUCUGGACCCUAGGAUCACAGUGGCUUGGUGCAAGAAGUGGGGUGUCCCAAUUGAGAAGAUUUACAACAAAACCCAGCGGGAGAAGUUUGCCUGGGCCAUUGACAUGGCUGAUGAAGACUAUGAGUUUUAGCCAGUCUCAAGAGGCAGAGUUCUGUGAAGAGGAACAGUGUGGUUUGGGAAAGAUGGAUAAACUGAGCCUCACUUGCCCUCGUGCCUGGGGGAGAGAGGCAGCAAGUCUUAACAAACCAACAUCUUUGCGAAAAGAUAAACCUGGAGAUAUUAUAAGGGAGAGCUGAGCCAGUUGUCCUAUGGACAACUUAUUUAAAAAUAUUUCAGAUAUCAAAAUUCUAGCUGUAUGAUUUGUUUUGAAUUUUGUUUUUAUUUUCAAGAGGGCAAGUGGAUGGGAAUUUGUCAGCGUUCUACCAGGCAAAUUCACUGUUUCACUGAAAUGUUUGGAUUCUCUUAGCUACUGUAUGCAAAGUCCGAUUAUAUUGGUGCGUUUUUACAGUUAGGGUUUUGCAAUAACUUCUAUAUUUUAAUAGAAAUAAAUUCCUAAACUCCCUUCCCUCUCUCCCAUUUCAGGAAUUUAAAAUUAAGUAGAACAAAAAACCCAGCGCACCUGUUAGAGUCGUCACUCUCUAUUGUCAUGGGGAUCAAUUUUCAUUAAACUUGAAGCAGUCGUGGCUUUGGCAGUGUUUUGGUUCAGACACCUGUUCACAGAAAAAGCAUGAUGGGAAAAUAUUUCCUGACUUGAGUGUUCCUUUUUAAAUGUGAAUUUUUAUUUCUUUUUAAUUAUUUUAAAAUAUUUAAACCUUUUUCUUGAUCUUAAAGAUCGUGUAGAUUGGGGUUGGGGAGGGAUGAAGGGCGAGUGAAUCUAAGGAUAAUGAAAUAAUCAGUGACUGAAACCAUUUUCCCAUCAUCCUUUGUUCUGAGCAUUCGCUGUACCCUUUAAGAUAUCCAUCUUUUUCUUUUUAACCCUAAUCUUUCACUUGAAAGAUUUUAUUGUAUAAAAAGUUUCACAGGUCAAUAAACUUAGAGGAAAAUGAGUAUUUGGUCCAAAAAAAGGAAAAAUAAUCAAGAUUUUAGGGCUUUUAUUUUUUCUUUUGUAAUUGUGUAAAAAAUGGAAAAAAACAUAAAAAGCAGAAUUUUAAUGUGAAGACAUUUUUUGCUAUAAUCAUUAGUUUUAGAGGCAUUGUUAGUUUAGUGUGUGUGCAGAGUCCAUUUCCCACAUCUUUCCUCAAGUAUCUUCUAUUUUUAUCAUGAAUUCCCUUUUAAUCAACUGUAGGUUAUUUAAAAUAAAUUCCUACAACUUAAUGGAAA(SEQ ID NO:20)；

PPDPF基因转录本的序列如SEQ ID NO：21所示，具体为：

ACCCCGCGCGCGCCUCUCUGUCGUGGCGCGGCUUCCCGCGGUCUUCUCUGCAAAUGGGCUCCGUGGCCUAGCGCCCCCGUCCCCGCCACCCGUGAUCGUGCGCCGAGGCCCGCGAGGGGUCGCCGCCCAGAUCCCACCAGCCAGCAAGCUAAAGCAUGGCGGCCAUCCCCUCCAGCGGCUCGCUCGUGGCCACCCACGACUACUACCGGCGCCGCCUGGGUUCCACUUCCAGCAACAGCUCCUGCAGCAGUACCGAGUGCCCCGGGGAAGCCAUUCCCCACCCCCCAGGUCUCCCCAAGGCUGACCCGGGUCAUUGGUGGGCCAGCUUCUUUUUCGGGAAGUCCACCCUCCCGUUCAUGGCCACGGUGUUGGAGUCCGCAGAGCACUCGGAACCUCCCCAGGCCUCCAGCAGCAUGACCGCCUGUGGCCUGGCUCGGGACGCCCCGAGGAAGCAGCCCGGCGGUCAGUCCAGCACAGCCAGCGCUGGGCCCCCGUCCUGACCUGAGCGGUUACCACCAGCCCCAGGCCUGCGGAGGCGCUAGUCCACCAGAGCCCCUCCCCGCCCCUCUCCCCACUCCGCAUCCCUCGCCCCCCUCCCCACCUCCCACCCCCCACCCUGUAAACUAGGCGGCUGCAGCAAGCAGACCUUCGCAUCAACACAGCAGACACCAAAAACCAGUGAGAGCCCCGCUCUCUACCGCCCGGCCCCAGCACUCGCUAGCUUUCCUGACACCUGGAACUGUGCACCUGGCACCAAGCGGAAAAUAAACUCCAAGCAGCCAGUAGCCCCGAUGGUGUGUGCCUGAGCUGUGUGGCCCGAGGUUCCA(SEQ ID NO:21)。

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的探针序列如SEQ ID NO:9所示；

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的探针序列如SEQ ID NO:12所示；和

检测miR-106b-3p的引物为：如SEQ ID NO:14所示的上游引物序列，以及如SEQ IDNO:16所示的下游引物序列；检测miR-106b-3p探针序列如SEQ ID NO:15所示。

在第三个方面中，本公开提供了上述第一个方面的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RNA分子标志物miR-106b-3p、lnc-MKRN2-42:1、lnc-MSI1-2:1、TOP1和PPDPF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，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。

在另一个实施方案何总，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，对早期结直肠癌种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miR-106b-3p、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，可以同时指代结肠癌、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，示例性的序列分别入SEQ ID NO：17和18所示；基因的转录本，具体为TOP1和PPDPF基因的转录本；以及miRNA，具体为miR-106b-3p，示例性的序列如SEQ ID NO：21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：19所示；PPDPF基因转录本即表示PPDPF基因转录得到的mRNA分子，示例性的序列如SEQ ID NO：20所示。

需要说明的是，在实际检测中的lncRNA、miRNA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，比较早期结直肠癌患者中相比于对照组具有差异表达的RNA，包括lncRNA、mRNA和miRNA，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，包括两个lncRNA、两个mRNA以及一个miRNA，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2miRNA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3miRNA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PrimeScript^TMRT reagent Kit(Perfect Real Time)和PremixEx Taq^TM(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；

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：13所示；上游引物具体为如SEQ ID NO:14所示的核苷酸序列，下游引物具体为如SEQ IDNO:16所示的核苷酸序列，探针为如SEQ ID NO:15所示的核苷酸序列。

所有上述引物和探针序列见表5：

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)按表7所列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和53例对照样本(健康人和良性病变)的血浆外泌体RNA进行检测，分别检测miR-106b-3p、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)。

标志物miR-106b-3p、lnc-MKRN2-42:1、lnc-MSI1-2:1、TOP1和PPDPF的联合检测

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

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分子标志物联合应用于检测结直肠癌的准确度。

如图1所示，联合应用miR-106b-3p、lnc-MKRN2-42:1、lnc-MSI1-2:1、TOP1和PPDPF时，基于表8和表9的结果，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。miR-106b-3p、lnc-MKRN2-42:1、lnc-MSI1-2:1、TOP1和PPDPF联合诊断早期结直肠癌的AUC为0.93，其阴性预测值为93.18％、灵敏度为93.61％、特异性为77.36％，具有极其优越的诊断性能，可以作为有效的临床诊断标志物。

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<110> 首都医科大学附属北京友谊医院

<120> 用于结直肠癌早期诊断的RNA分子标记物组合及其应用

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<211> 24

<212> DNA

<213> 人工序列

<400> 8

tctaccacat attcctgacc atcc 24

<210> 9

<211> 26

<212> DNA

<213> 人工序列

<400> 9

ccttcctcct tttcattgcc tgctct 26

<210> 10

<211> 19

<212> DNA

<213> 人工序列

<400> 10

cgcctgggtt ccacttcca 19

<210> 11

<211> 19

<212> DNA

<213> 人工序列

<400> 11

ctctgcggac tccaacacc 19

<210> 12

<211> 26

<212> DNA

<213> 人工序列

<400> 12

aaaagaagct ggcccaccaa tgaccc 26

<210> 13

<211> 19

<212> DNA

<213> 人工序列

<400> 13

cgcctgggtt ccacttcca 19

<210> 14

<211> 18

<212> DNA

<213> 人工序列

<400> 14

caccgcactg tgggtact 18

<210> 15

<211> 23

<212> DNA

<213> 人工序列

<400> 15

attcgcactg gatacgacgc tgc 23

<210> 16

<211> 16

<212> DNA

<213> 人工序列

<400> 16

gtgcagggtc cgaggt 16

<210> 17

<211> 22

<212> RNA

<213> 人

<400> 17

ccgcacugug gguacuugcu gc 22

<210> 18

<211> 294

<212> RNA

<213> 人

<400> 18

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> 19

<211> 12606

<212> RNA

<213> 人

<400> 19

gcugguaaug gucacacuag guggcguccg cgugaggugg aggugucagc ggcuccuguc 60

ugcagcuucc ugggguccug gcggcgcucu ucuccugagc cagaauccag caggaaggac 120

agaaugggcg cuagagugug aaaagagggc gaacucacgu ggaugugacu cuccgggucg 180

cucuucccug ucacccagcu ccuccugccg guuuccugug ggcaucgccu gcauugcuuu 240

uaucccgggg acggauuagg gggguccaga ggaggacuug cuggcacaaa uaacccaaug 300

gaggcuuucg agcacggcug gcugggguuu gcgauccaac ggccuuuugc ugaccugaaa 360

gucaacgcaa agcucaucuu cauucuagag aaaaagaagc cucauaucug agaucaggac 420

cccgcuuucu ccuugacauu ccacagcaaa acgugaacag cugaacaaac acacaguccg 480

guuucccgag accaaagggu caggggaaac ccccgcugaa aacgugcauc aguggaacaa 540

cuacucucuu ccagguccca ccgagggcaa agaggagaaa uagaaucccu cacaagaaau 600

ccacaacuuc uagaaauccg cagcacacac agaacacacu uaccucucac agcucuacgg 660

acacuucaca ccccacagug ggaccgucug auugcauuug cauuuuggga cauguagucc 720

agaaagcuua aaugaugcga gaaaaaccac aagucccaca auccuccgcg ucucgcaauu 780

gacagcccca gaucauccag gaacgccccc ugucggcucu uucuggucag agguuccccc 840

guuaauauaa aagcuuuuaa ucauggauuu uuuucuuugu uuucaauuuc ugugggcacu 900

uaguaggugu auauauuuau aguguacaua agauuuuuuu ucuuuuuucu uuuuuuuuuu 960

uaagacggag uuguuguuuu uuuuuuuuga aaaaacucca gucuggagug caguggcacg 1020

aucucggcuc acugcaaccu cccucucccg gguucaaccg auucuccugc cucagccucc 1080

cgaguagcug ggauuauuuu uguauuuuua guagagacgg ggguuucgcc auguuggcca 1140

ggcucgucuu gaacuccuaa ccucagguga uccgcacgcc uugaccuccc aaagugcugg 1200

gauuauaggc gugagccacu gcgcccggcc aucuuuucuc uuauuuuuug ucuuuuuuuc 1260

uuuucucugu gccugucuau gcuuuaccca caugagaugu uuugauacaa acaugcaaug 1320

ugaaaugagc acgucauuaa ucauggaucu agauuugacu uuuuuuuaaa guuuuuguau 1380

uuuuaguaga gacagaguuu caccauguug gccaggcugg ucucgaacuc ccgaccucag 1440

gugauccgcc aaccucggcc ccccaaaaug cugggauuac aggccugagc cacuguuccc 1500

ggccuagauu ugacucauuc uaaacaucag aguuugugau ccuuuucauu cacaacgaca 1560

guaucaaguu auuugcuauu gguaaaauac cuugaacuug ugaagaaaug uguuuauaca 1620

uaucugaaaa agugauaguu uguuguuguu guuguuguug uuuguuuguu uguuuuugag 1680

acggagucuc gcucuguugc ccaggcugga gugcaauggu gccauuucgg cucacugcaa 1740

ccuccgccuc ccggguucaa gcaauucucc ugccucagcc uccugaguag cagggauuau 1800

gggcgcaugc cacuacgccc gguuaauuuu uuuauuuuua guagagacaa gguuucacca 1860

uguuagccag gauggucuug aucuccugac cucgugaucu gcccacccua gccucccaaa 1920

guacugggau uacaggcgua agccacugug cccagccaaa agugauguuu uuacaguuaa 1980

uaugaagcca aauauauuau accuggacua auguacuuaa ccugcuuggc uucuccaauu 2040

uuauuuauca uauuuuuuuu auuaaguuaa ucaaacaagg ccugcgucuc acucccaggg 2100

auucugaucu acaaauuucc uaauuguucc uaauguguag acaaccagaa cugccgcucu 2160

gcagcaucac cuaaaucaau aaauguauaa gugucuuugg acauaaaggu gggcggccag 2220

cagggaccuu gaagaccacc uaaggacucc cuucccccau uuuaagauga auugggcuga 2280

gugcaguggu ucccaccugu aauaccaaca cuuugggagg ccaaggcagg aggaucacuu 2340

gagcccaaaa guucaagacc agcuugggca acauagugag acccacucac uacaaaaaau 2400

aaacaaaauu agcggggugu gguggugugu gccuguaguc ccagcuacuc aagaggcuca 2460

ggugggagga ucacuuugag cccaggagau cgaggcugca gugagccaua auugugcgac 2520

ugcacuucag ccugggugac aaagugagau cccaucucuu caaaaaauaa uaauaauaaa 2580

uaaagaugaa cugaagugca gagaaguaau guggaagcuu gagaaagagu uuuuagguuu 2640

uugguucuau cauuguaucu uuggagguaa uauuggcaaa augaccauuu aguaguuugc 2700

ccauuucugc uuuuuuuuuu ucuuuuuuuu gagacagggu cucguucugu cacccaggcu 2760

ggagugcagu ugugacauca cagcucacug caguuucuac uucccaggcc caagggaucc 2820

uccaaccuca uccucucgag uagcugagau cacaggcauc caccaccacg cccagcuaaa 2880

uuuuguuauu uuuuauuuuu gucgagacac ggucucacua uguugcucag gcuggucuca 2940

aacuccugaa cucaaggaaa gcucuugcuu uggccuccca aagugcuggg auuacaggug 3000

ugagccacca ugccgggccu gcuuauuucg acuuggcagc aucguuucau gaagccccug 3060

acucugugac aacauacagc ucuccagaaa gaugcuuuga agacaaaaca gaauagagca 3120

cacagcccuc caugucucuu ccaagucacu auauucauua aaagauaaau gacucugguc 3180

auugccuuuu ccugcacaag auaaugucug augggguuag ugaugauacc ucuguaaucc 3240

auaaccagau auacucuugc acccaaacuu ugauaugauu gugcuuuaau guaacuuuga 3300

agcaaguuua augugauuuu gcaggcgccg uacacucacc acuuguauau gaguggcuga 3360

aauucugugu uggaccaguc ugauggaacu gcucccacac uauagcccuc ggucuguaau 3420

ccucaguaag acuuguaagu aaaacuaacu uuaauuuuuu uuuuuuuuuu uugagagagu 3480

cuagcucugu ugcccaggcu ggagugcagu ggcgcgaucu ucacucacug aagccucugc 3540

cuucccggcu gaagugauuc ugcugccucg gccuccggag uagcuaggau uacaggcgcu 3600

cgccacugcg cccggcuaau uuuuguauuu uuaguagaga ugggguuucg ucauguuggc 3660

caggcugguu uugaacucgu gaccucaggu gacccaccug ccucagucuc cuaaaaugcu 3720

aggauuacag gagugagcca cugcaccccg ccacuuuuau uauuauuauu auuguauuua 3780

uuuauuuauu uauuuauuuu ugagacggag ucucgcucug ucgcccaggc uggaguguag 3840

uggagagauc ucggcccacu acaagcuccg ccucccgggu ucacgcgauu cuccugccuc 3900

agccuccgga cuacaggcgc ccgccaccac acccggcuaa uuuuuuguau uuuuuuuuua 3960

guagagacgg gguuucaccg uguuagccag gauggucucu aucucuugac cucgugaucc 4020

gcccgccuca gccucccaaa guacugggau uacaggcgug agccaccgcg cccggcuauu 4080

uauuuauuua uuuauuuauu uauuuauuua uuuuuugaga cggaauauug cucugugccc 4140

aggcuggagu gcaguggcac gaucucagau cacugcaacc uccgccuccc ggguucaagu 4200

gauucuccug ccucagccuc ccgaguagcu gggauuacgg gugcccgcca ccaugcccgg 4260

cuaauuuuug uauuuuuagu agagacgggg uuucaccaug cuugucagca uggucucgaa 4320

cuccugaccu cgcgauccgc ccgccucagc cucccaaagu gcuggaauua caggcaugag 4380

ccacugcgcc cugccacuuu aauuauuuaa aagcuuauuu uuuuauuuug gggacauggu 4440

cucacucuuu cacccaggcg ggaguucaga ggcacgauca cugcgcacua cagccucaac 4500

cucuuggacu caaaccaucc ucccaccuca gccuccugag uagcugggac uacagguggg 4560

ccaccaagcc cagcuaauuu uuguauuuuu uuguagagac gggguuucac cauguugccc 4620

aggcuggucu cgaacuccug ggcucuaggc uuuccaaagu guugugauua cgggcgugag 4680

cuacugcgcc uggccgauuc uuuuuaacuu aguacacagg gaguaguagc uccagcaaca 4740

gcgaaaguca guagcuguag gaucuacuag gcggggaaac ucauaugaca gcccccuccu 4800

uuuuuuuuuu uuuuuuuuuu uuuuugagau ggaguuucac ucuuguugac caggcuggag 4860

ugcaauggcg ccaucuuggc ucaccgcaac cuccgccucc cggguucaag ugauucuccu 4920

gccucagccu ccccaguauc ugggauuaca ggugugagcc acugcgcccg gccacccccu 4980

uuuuuuuuca aggcugcagu gagccaugau ccugugacug cacuccagcc acugacagcc 5040

cuuucugcau uaguuaaccu gaccacaucu gcugaaagaa agcaccagaa gcuagacuga 5100

aauuuucguu gucaaaaaaa uuauaauccc ccccaaaauu uaaaaauuau aauccagcac 5160

auuuucugca cacauuuucu ggguauuuga ucauaacuuu uaaaaaugug agaacaguga 5220

cauucuccuu ccccauucac caauuuuacu augaaaaagu ucaaaccuag guuauuuuuu 5280

caguucucca aguggaauga ucaucacuaa uagagauugg uugggaauuc uauugugcuu 5340

cuaggccccc aaaaaucuca ugcucugagg aaaaguuagc ugaguuuuca auuuuucuca 5400

uagugcugga aaaucauccc uggacgguga agucauccaa gucuuuuuug uuuuguuuug 5460

uuuuuucuuu cuuucuuucu uucuuuuuua auuuuuguau gcuuuguuuc uauuucuuuu 5520

uuuuuuuguc auccaaguca gcuggugaaa cacacaauuu cacuugauag cuacugagag 5580

aguguaaugg gccacccauu cuagaauucu caagggcuag gaaguuccuu ugauacccuc 5640

augggauuag gauuagggcu uuagucuuuu ucucucucuc uuuuuuuuuu uuuuuuuuuu 5700

uuuuugauua caggcgugag ccaccacacc aagccccauu auuuauuuau uuuauuuuau 5760

guauguaugu auguauguau guauguauuu auuuauuuug agacggaguc ucgcucuauc 5820

gcccaggcug gagugcagug gcgcaaucua ggcucacugc aaccuccgcc uaccggguuc 5880

aagugauucu ccuaugucag ccucccaagu agcugggauu auaggcgccc gccaccaugc 5940

ccagcuaauu uuuguauuuu uaguagagac gagguuucac caucuuggcc aggcuggucu 6000

ugaacuccug accuugugau ccacccgccu cggccuccca aagugcuggg auuacaggcg 6060

ugagccaccg ugccuggccu aguguuuuuc ucacaacucc accccaucca gaauuaauga 6120

cuaaauccuu gacuggauga ucaaaguaaa agaaucuaaa uucaccacau ugcuagucuu 6180

uucuuuuuau ccaggcugug aauuaaauaa gaaaucugcu gcaguggcuc augccuguaa 6240

ucccuccccu uggggaggcc gaggcgggcg gaucacuuga gcccaggagc uggagaccag 6300

caugggcaac auggugaaac ccuguaucua caaaaaauac agaaaaauua gcugggcaug 6360

guggugugug ccuguagucc cagcuaccug ggaggcugag gugggaagau cgcuugagcc 6420

caggagauuu aggcugcacu gagccgugau ugcaccccug cgcuccaacc uaagcgacug 6480

agaacauguc ucaaaaaaaa aaaaacaaaa aaaaaaacac aaaacaaguu agaaauaauc 6540

acauaacaca ucagcaucaa acaggccuuu uaaaaugucg uuuuugacca uccuggcuaa 6600

cacggugaaa ccccaucucu acuaaaaaua gaaaaaauua gccggccgug guggcgggcg 6660

ccuguagucc cagcuacucg ggaggcugag gcaggagaau ggcgugaacc cggaaggcgg 6720

agcucgcagu gagccgagau ugcgccacug cacuccagcc ugggcgacag agcgagacuc 6780

cgucucaaaa aaaaaaaaug ucauuuuugu auauggauga ugaguacaug aauguucacu 6840

gaggaauucu uugaacuuuu cguguuuaca auuuuuuaug auaaaauguu gggggaaaug 6900

uuacuuucuu uuuuuuuuuu uuuuuuuuuu uuuuugagac ggagucucgc uuugucaccc 6960

aggcuggauu acaguggcau gaucucagcu cauggcaacc ucuaccuccc agguucaagc 7020

gauccuccug ccucagcccc ccuaguagcu gggauuacag gcaagcgcca ccacgccccg 7080

uuaauuuuug uauuuuguau uuuuguaauu uuguauuuuu guauuuugua uuuacaggga 7140

ugucgccaug uuaccaggcu gaucuugaac uccugaccuc aggugaucca ccugccuugg 7200

ccucccaaag cguugggauu acacgcucga gccaccacac cuagaccaga uguagguuuc 7260

uaauagcuuu cuacauccug aaggaaaaca ggcagugagg gugaaaguug cuucaaauuu 7320

uagaaacugu gaacauucug uggaaauaaa uccuuauuau uugagccucu uuuugaauua 7380

agguguccaa acccuuuuga auuaacauau gauccagcaa uuuuacuucu agguauguac 7440

accaaagaau agaaaacagg ggccaggcgc gguggcucac accuguaauc ccagcacuuu 7500

gggaggccga ggcaggcaga ucacuugagg ucaggaguuc aagaccagcc ugaccaacau 7560

ggagaaaccc cgucucuacu aaaaaaaaaa aaaaaaaaaa aauuaucuau auauauauuu 7620

auacauauau auauauaaaa uuagccaggc augguggcgc auaccuguca ucccagcuac 7680

uugggaggcu gaggcaggag aaucaccuga acccaggagg cagagguugu ggugagcuga 7740

gauugcgccu uugcagucca gacugcgcaa caagagcgaa acucagucuc aaaaaaaaaa 7800

aaaaggaaaa cagaaacuua agcugauauu ugcacaccaa uagcagcauu auucccauag 7860

acaaaaggug gaaaccagca augaccaucu aaaaauaaau ggauaggccg ggcgccgugg 7920

cucagcacuu ugggaggcug aggcaggagg aucacuugag gucaggagcu ugagaccagc 7980

cuggccaaca uggugaaacc cugucucuac uaaaaauaca aaaauuagcc aggauuggug 8040

gcacgcaccu guaaucccag uuaguuacuc gggaggcuga gacaggagaa cugcuugaac 8100

cccagaggcg gagguugcau gggcugagau ugagucuaug uacuccagcu ugggcaacag 8160

agcgagacuc ccccucaaaa aaaaaaaaaa aaaggauaaa caguuggucc gggcggcagc 8220

ucaugccuau agucccagca cuuugggagg ccaagauggg aggaucacuu gggcccagga 8280

guucgagacc agccugggca acauggugag auccugucug uacgaaaaau acagaaagua 8340

uccagcugaa gugggaggag gaucacuuga ggccuggaag cagagguuga agugagcuga 8400

gaucgcgcca cugcauucca gccugggcga cagagccaaa agcugucuca aaaaagaaug 8460

aaugggaccg ggcgcggugg cucacggcug uaaucccagc acuuugggaa accaaggcgg 8520

guggaucacc ugaggccagg aguucaagac cagccugacc aacaugguga aaccccgugu 8580

cuacuaaaaa uaaucccagc uccucgggag gcugaggcag gagaaucgcu ugaaccuggg 8640

aggcggaggu ugaucucgcc auugcgcucc agccugggca acaaaagcaa aacuucgucu 8700

caaaaaaaaa aauagacaag aauaaauggg cugggugaag uggcucacac cuguaauucc 8760

agcacuuugg gaggccgagg caggcagauc acgugagguc aggaguuuga gaccagccug 8820

gccaacaugg ugaaauccug ucucuacuaa aaauacaaag agugguggca ugcaucugua 8880

aucccagcua cuugagagau ggaggcagga gaaucgcuug aauccgggag gcagugguug 8940

caaugagccg agaucaugcc acugcacucc agccugggca acaagagcaa aaacuccauc 9000

ucaaaaaaaa aaaaagauaa caucucauac agugaaauau uauucagcca uaaaauuaau 9060

gaauuccuga uauauacuac aacauggaug aaccuuaaaa acauuauucu gaaugaaaua 9120

agacugaccu caaggacaaa uauuguauga uuccauuuau auauauauaa aguacucaca 9180

aagucauaga gauagaaagu agaaaagagg uugccacggg ggcuggagga gaagggaaca 9240

gggaguaagg guuuaauggg uacagagucu caguuuccac ccuugggcau gaaagauaaa 9300

guucuggagg uaaguaguga ugaugguuac acaacaaugu gaacauauuc uaugccagug 9360

aauuuacacu uaagguuaaa augguaaauu uuauguauac uuuggcauaa uuuaaaaaaa 9420

aguuugaaaa agaaaaaaga caaaacaaua aucgucuuug gagaagucug uuacacugaa 9480

ucuggagcca agugaacaga acccugauuu ugaucccuuu ucucucaaaa gcccuucgca 9540

gucucugaau uaagucuauu agcauguucc ucccauagug cuuugcuuca uaucaacaaa 9600

aaccuagcua agugaaauca gcaacgauau gcagaaacca ccuacgcagg ucacaaacau 9660

cuuucuauga uuguauaauu uucaagcaag caauaaguga agauuuuucc auaggcccua 9720

aacucaccuu ugcgaaauag gaagcugguu uauugggagu gaugagcagg gggcguaaca 9780

aauuagcuuu gcgcaguggc aguaucguag ccaaugaggu cuauccgagg cgcgauuauu 9840

gcuaauugaa aacuuuuccc aauaccccgc cgugacgacu ugcaauauag ucggcacugg 9900

caauuuuuga cagucucuac ggagacugaa uuuucuugca guugaacaac agaggcuuuu 9960

uuugugugug ugggggugcu ugguuuuggg agguugaaga guacuuguuc gcaaacucuc 10020

uaaaugagag auggggaagu gucuuaccag cugauucgcg agagcaccug caaguuuacc 10080

uguuuuagug auuugcguuu augaaaguug gaguugcuga uuuugugauc ucuacuuuua 10140

agguaaaagc agggcggugu ugacuuuucu ugccaguugg cuguccuguu uucuuucucg 10200

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> 20

<211> 3734

<212> RNA

<213> 人

<400> 20

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> 21

<211> 827

<212> RNA

<213> 人

<400> 21

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

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分子标志物组合，其包括miR-106b-3p、lnc-MKRN2-42:1、lnc-MSI1-2:1、TOP1基因转录本和PPDPF基因转录本。

2.根据权利要求1所述的RNA分子标志物，所述miR-106b-3p的序列如SEQ ID NO：17所示；所述lnc-MKRN2-42:1的序列如SEQ ID NO：18所示、所述lnc-MSI1-2:1的序列如SEQ IDNO：19所示；TOP1基因转录本的序列如SEQ ID NO：20所示、和/或所述PPDF基因转录本的序列如SEQ ID NO：21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4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；

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；和

检测miR-106b-3p的引物为：如SEQ ID NO:14所示的上游引物序列，以及如SEQ ID NO:16所示的下游引物序列；检测miR-106b-3p探针序列如SEQ ID NO:15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RNA分子标志物miR-106b-3p、lnc-MKRN2-42:1、lnc-MSI1-2:1、TOP1和PPDPF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。