CN110295232B - 用于结直肠癌的microRNA生物标记物 - Google Patents
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Abstract
本发明提供一种用于结直肠癌的microRNA生物标记物，包括：编码hsa‑miR‑423‑5p、编码hsa‑miR‑451a、编码hsa‑miR‑30b‑5p、编码hsa‑miR‑27b‑3p、编码hsa‑miR‑199a‑3p、编码hsa‑let‑7d‑3p、编码hsa‑miR‑423‑5p中的至少一种核酸分子，所述核酸分子均编码microRNA序列，其核苷酸序列如SEQ ID NO:1～7所示。通过混合样本筛选、少量样本单个验证和大量样本单个验证获得这些microRNA生物标记物。本发明还提供了用于诊断和预测早期结直肠癌的分子标记诊断试剂盒。
Description
技术领域
本发明属于分子诊断医学技术领域，具体涉及一种用于结直肠癌的microRNA生物标记物。
背景技术
结直肠癌(colorectal cancer,CRC)是世界上发病率高居第三、死亡率高居第二的肿瘤疾病。据美国癌症学会发表题名为“Global cancer statistics 2018:GLOBOCANestimates of incidence and mortality worldwide for 36cancers in 185countries”的报道，2018年将有180万新增结直肠癌患者和88.1万死亡人数，占据所有肿瘤患者和死亡人数的十分之一。如果在早期确诊，结直肠癌的死亡率将会大大降低，但是，不幸的是，结直肠癌早期患者一般没有明显症状。目前结直肠癌常用的筛查手段包括结肠镜、大便潜血试验和大便DNA检测等，这些检测手段要么不容易被患者接受，要么灵敏度有限。因此，更加方便，灵敏，无创的检测手段急需被开发。循环miRNA作为分子诊断标志物在肿瘤早期筛查中显示应用前景。其中，一些文献报道了结直肠癌循环miRNA生物标记物，例如miR-17-5p/miR-135b、miR-92a-3p/miR135b和miR-451a/miR-491-5p三个组合的比值可以区分结前期病变结直肠腺癌和健康组的ROC曲下面积AUC达到0.831，区分直肠癌(CRC)和健康组AUC达到0.735(Zhang,Jinhua,et al.,2018,Cancer,124(4):785-796)；miR-27a和miR-130a作为联合指标区分结直肠癌和健康组的AUC最高可达0.898(Wang,Shukui,et al.,2018,CancerEpidemiology Biomarkers&Prevention,27(7):746-754)；miR-17-3p和miR-92在结直肠癌组显著上调，区分健康组AUC可达0.885，灵敏度和特异性分别为89％和70％(Ng,Eko,etal.,2009,Gut,136.5:1375-1381.)。目前这些研究成果都还未应用于临床检测，其可能的原因一方面是miRNA生物标记物的灵敏度和稳定性不足，另一方面是这些miRNA都是基于提核酸的方法进行检测，费时费力，对操作人员要求较高，不适合临床应用推广。
发明内容
有鉴于此，有必要针对现有结直肠癌检测手段繁琐、灵敏度和稳定性有限等问题，提供一种用于结直肠癌的microRNA生物标记物。本发明的技术方案为：
第一个方面，本发明提供一种用于结直肠癌的microRNA生物标记物，包括：编码hsa-miR-423-5p(如SEQ ID NO:84所示)、编码hsa-miR-451a(如SEQ ID NO:92所示)、编码hsa-miR-30b-5p(如SEQ ID NO:56所示)、编码hsa-miR-27b-3p(如SEQ ID NO:51所示)、编码hsa-miR-199a-3p(如SEQ ID NO:38所示)、编码hsa-let-7d-3p(如SEQ ID NO:3所示)、编码hsa-miR-197-3p(如SEQ ID NO:37所示)中的至少一种核酸分子，所述核酸分子均编码microRNA序列。
第二个方面，本发明提供一种用于结直肠癌的microRNA生物标记物的筛选方法，包括以下步骤：
步骤一、混合样本筛选：首先在混合样本中确定候选miRNA范围，采用Direct S-Poly(T)Plus法检测所确定的候选miRNA，筛选出结直肠癌差异性表达的miRNA；
步骤二、少量样本单个验证：对步骤一中筛选出的结直肠癌差异性表达的miRNA作进一步验证；
步骤三、大量样本单个验证：确定结直肠癌早期诊断生物标记物。
进一步的，所述步骤一和所述步骤二中采用Direct S-Poly(T)Plus法检测miRNA，包括以下步骤：
(1)裂解离心：利用裂解用试剂将样本中的蛋白复合体充分裂解，使miRNA从样本的蛋白复合体中释放出来，并且降低样本中核糖核酸酶活性；将裂解后的混合物离心，得上清即为粗提RNA；
(2)加尾逆转录：将步骤(1)中的粗提RNA进行加Poly(A)尾及S-Poly(T)特异性逆转录；
(3)RT-qPCR定量检测：以步骤(2)中获得的逆转录产物cDNA为模板进行RT-qPCR定量检测。
进一步的，所述步骤(1)中裂解用试剂包括以下组分：2×lysis buffer和蛋白酶K，两者体积比20:1。
进一步的，所述2×lysis buffer包括以下终浓度的组分：100mmol/LTris-HCl、300mmol/L NaCl、20mmol/L MgCl2；pH为8.0。
进一步的，所述蛋白酶K在所述裂解用试剂中的浓度为15U/mL。
进一步的，所述裂解条件为：50℃处理20分钟，然后95℃保持5分钟；
进一步的，所述步骤(2)中加尾逆转录的反应体系中所加入粗提RNA模板的体积百分比为5～75％，优选40％。
进一步的，所述步骤(2)中加尾逆转录的反应体系包括：1μL的0.05μM RT primer(逆转录引物)，1U的PolyA Polymerase(多聚腺苷酸聚合酶)，100U的MMLV(鼠白血病逆转录酶)，1.5μL的reaction buffer(反应缓冲液)，RNase-free Water(无RNA酶水)补足至10μL；加尾逆转录的反应条件为：37～40℃保温20～50min，42～～60℃保温20～50min，74～76℃保温3～7min以灭活酶，然后迅速置于冰上，静置2min以终止灭活。
进一步的，所述reaction buffer包含以下终浓度的组分：200mM Tris-HCl，600mMNaCl，40mM MgCl2，4mM ATP，2mM dNTP，pH 8.0。
进一步的，所述步骤(3)中以cDNA为模板进行real-time PCR定量检测的具体过程为：采用包含DNA聚合酶的热启动酶；real-time PCR反应体系为：4×qPCR reactionBuffer：5μL、1μmol/L Forward Primer 4μL、10μmol/L universal reverse primer0.4μL、10μmol/L universal Taqman probe0.5μL、100×ROX Rerference Dye0.2μL、hotstartAlpha Taq Polymerase0.0125μL、cDNA0.5μL、RNase-free Water加至20μL；反应条件为：预变性95℃5分钟，变性95℃10s，退火60℃40s，40个循环。
进一步的，所述包含DNA聚合酶的热启动酶的制备方法为：将DNA聚合酶和热启动抗体等体积混合，室温放置6小时。
进一步的，所述混合样本包括血清、血浆/血清、尿液、眼泪、乳汁、唾液、痰液或粪便抽提上清；优选混合样本为血浆。
第三个方面，本发明提供一种用于诊断和预测早期结直肠癌的分子标记诊断试剂盒，包括一种或多种核酸分子，每种核酸分子均编码microRNA序列，并且所述核酸分子的一种或多种在结直肠癌样本和对照样本中差异表达，所述核酸分子包括：编码hsa-miR-423-5p，如SEQ ID NO:84所示；编码hsa-miR-451a，如SEQ ID NO:92所示；编码hsa-miR-30b-5p，如SEQ ID NO:56所示；编码hsa-miR-27b-3p，如SEQ ID NO:51所示；编码hsa-miR-199a-3p，如SEQ ID NO:38所示；编码hsa-let-7d-3p，如SEQ ID NO:3所示；编码hsa-miR-197-3p，如SEQ ID NO:37所示。
进一步地，所述核酸分子在结直肠癌血浆和对照血浆中相比，编码hsa-miR-423-5p、hsa-miR-30b-5p、hsa-miR-27b-3p、hsa-miR-199a-3p、hsa-let-7d-3p、hsa-miR-197-3p的表达被上调；编码hsa-miR-451a的表达被下调。
进一步地，所述分子标记诊断试剂盒优选包括以下核酸分子的组合：编码hsa-miR-451a，如SEQ ID NO:92所示；编码hsa-miR-30b-5p，如SEQ ID NO:56所示；编码hsa-miR-27b-3p，如SEQ ID NO:51所示；编码hsa-miR-199a-3p，如SEQ ID NO:38所示；和编码hsa-let-7d-3p，如SEQ ID NO:3所示。
本发明的优势和有益效果是：
1.本发明提供的结直肠癌的miRNA生物标记物或组合通过检测体液中的循环miRNA的表达量，即可实现对结直肠癌进行早期诊断和预测，更快捷、准确，提前了结直肠癌的发现时机，有助于及时尽早的进行治疗，增加存活率。
2.本发明提供的结直肠癌的miRNA生物标记物或组合对结直肠癌的预测准确率较高，单个miRNA的ROC曲下面积AUC可达到0.785～0.940；miRNA生物标记物联合诊断结直肠癌个体与正常个体AUC可达到0.975，灵敏度和特异性分别为95.1％和90.3％。
3.本发明基于自主研发的无需提取核酸的miRNA检测方法，Direct S-Ploy(T)Plus方法，步骤简单，方法灵敏，20ul血浆可以完成近100个miRNA的检测，一组miRNA(≤48个miRNA)可在140分钟内完成检测。因此，本发明提供的结直肠癌miRNA生物标记物或组合非常适合于临床应用。
4.本发明筛选出的结直肠癌miRNA生物标记物基于以往研究成果，即从485个在血清/血浆中检测到的miRNA中筛选出3个结直肠癌内源性参照物(hsa-miR-93-5p，hsa-miR-25-3p和hsa-miR-106b-5p)，这三个内参在结直肠癌和对照组表达无显著差异，并且这三个miRNA位于同一个转录本，说明这个基因簇本身具有保守性。使用稳定内参作为归一化标准，可以避免样本差异对结果造成的影响，具有稳定性和可重复性。
附图说明
图1为本发明的用于结直肠癌的microRNA生物标记物的筛选流程。
图2为本发明的用于结直肠癌的microRNA生物标记物的筛选流程中104个miRNA在结直肠癌病人和健康对照血浆中表达谱分析。样本使用的是172例结直肠癌病人血浆和172例健康对照血浆混合样本(其信息如表1所示)。
图3为本发明的用于结直肠癌的microRNA生物标记物的筛选流程中11个miRNA少量单样本验证，其中11个miRNA在38例结直肠癌血浆和38例正常对照的相对表达量，数据为平均值±SE，ns表示差异不显著，*表示<0.05,***表示<0.001。
图4为本发明筛选出的7个结直肠癌潜在miRNA生物标记物在大量样本中的单个验证，其中样本共包含106例结直肠癌血浆样本和106例健康对照，**表示<0.01，***表示<0.001。
图5为本发明筛选出的7个结直肠癌潜在miRNA生物标记物ROC曲线图。
具体实施方式
在本发明的描述中，需要说明的是，实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品，其中引物、探针来自美国Integrated DNA Technologies(IDT)公司。
下面结合附图和具体的实施例对本发明做进一步详细说明，所述是对本发明的解释而不是限定。
本发明具体实施例中临床样本收集于深圳人民医院，共包含172例健康志愿者血浆和172例结直肠癌病人血浆，两组样本年龄性别基本匹配，具体信息如表1。
表1本发明具体实施例中使用的血浆样本信息
实施例1、从混合样本中筛选结直肠癌差异性表达的miRNA
本实施例提供一种从混合样本中筛选结直肠癌差异性表达的miRNA的方法，具体筛选流程如图1所示，初筛数据直接采用已发表的文献数据(Niu,Yanqin,et al.,2016,Scientific Reports 6:35611)，即485个miRNA在两个混合样本(结直肠癌和健康对照)中的表达谱数据，从中挑选出104个变化倍数大于1.5的miRNA。检测这104个miRNA的方法为Direct S-Poly(T)Plus，具体步骤详见专利“一种直接定量检测循环miRNA的RT-qPCR方法”，专利申请号为：201710442989。使用Direct S-Poly(T)Plus方法检测104个miRNA，20ul血浆可以完成近100个miRNA的检测，一组miRNA(≤48个miRNA，一个96孔板)可在140分钟内完成检测。
所述方法包含以下步骤：
(一)制备混合样本。
172例结直肠癌血浆样本和172例健康对照血浆样本分别混合均匀，制备成两个混合样本。
(二)制备粗提RNA，具体步骤为：
1)20uL血浆/血清与20uL 2×reaction buffer混合均匀，加入1uL蛋白酶K，50℃处理20分钟，然后95℃保持5分钟，置于冰上；
2)13,000rmp、4℃离心5分钟；吸取上清液(粗提RNA)转移至另一新的离心管中或直接用于步骤(三)；
(三)Direct S-Poly(T)Plus法检测miRNA，在本实施例中，在每一组的混合血浆样本中分别检测104个miRNA(所采用的引物序列如表2所示)，具体步骤如下：
1)加尾逆转录：miRNA加Poly(A)尾和逆转录(第一链cDNA的合成)在一个反应体系中进行，利用S-Poly(T)引物(如表2所示)进行miRNA的逆转录。
加尾逆转录的反应体系包含：4uL粗提RNA，1μL的0.05μM RT primer(逆转录引物)，1U的PolyA Polymerase(多聚腺苷酸聚合酶)，100U的MMLV(鼠白血病逆转录酶)，1.5μL的reaction buffer(反应缓冲液)，RNase-free Water(无RNA酶水)补足至10μL。所述reaction buffer包含以下终浓度的组分：200mM Tris-HCl，600mM NaCl，40mM MgCl2，4mMATP，2mM dNTP，pH 8.0。加尾逆转录的反应条件为：37℃保温30min，42℃保温30min，75℃保温5min以灭活酶，然后迅速置于冰上，静置2min以终止灭活。
所述S-Poly(T)引物由四部分组成，其序列从5’端到3’端依次为：14-20个碱基的PCR通用引物序列、14-20个碱基的通用探针序列、11个oligo(dT)和5-7个与miRNA 3’配对的特异性碱基(如表2所示)。更优选地，所述S-Poly(T)引物序列从5’端到3’端依次为：16个碱基的PCR通用引物序列、17个碱基的通用探针序列、11个oligo(dT)和6个与miRNA 3’配对的特异性碱基。
本实施例中所检测的miRNA的序列来自于miRBase，根据各自序列设计不同的S-Poly(T)引物、上游引物，检测不同miRNA的S-Poly(T)引物序列如表2所示。
表2 S-Poly(T)Plus法检测miRNA所用到的引物序列
2)PCR：以步骤S2中逆转录获得的第一链cDNA为模板，用miRNA特异上游引物和下游通用引物进行real-time PCR定量检测。所述miRNA特异上游引物是不含3’端3-8个碱基的miRNA特异序列，所述miRNA的下游引物来自于S-Poly(T)通用引物序列(Universalreverse primer，表2)。
Real-time PCR定量检测采用探针法或者SYBR荧光染料法。本实施例中采用探针法，所用探针为通用探针，其序列来自于S-Poly(T)引物上14-20个碱基的PCR通用引物序列。Real-time PCR的反应体系如表3所示。
表3 Real-time PCR的反应体系组成
PCR运行仪器为ABI StepOnePlus thermal cycler，反应条件为：预变性95℃5分钟，变性95℃10s，退火60℃40s，40个循环。每个PCR反应两个复孔。
归一化标准采用内源性参照物hsa-miR-93-5p。前期研究结果从485个在血清/血浆中检测到的miRNA中筛选出3个结直肠癌内源性参照物(hsa-miR-93-5p、hsa-miR-25-3p和hsa-miR-106b-5p)，这三个内参在结直肠癌和对照组表达无显著差异，并且这三个miRNA位于同一个转录本，说明这个基因簇本身具有保守性。使用稳定内参作为归一化标准，可以避免样本差异对结果造成的影响，具有稳定性和可重复性((Niu,Yanqin,et al.,2016,Scientific Reports 6:35611)。
用Direct S-Ploy(T)Plus的方法在两个混合样本中分别检测104个miRNA，检测结果如图2。挑选变化倍数大于1.5，无非特异性扩增，Ct值小于33的miRNA，共计11个，即hsa-miR-423-5p(如SEQ ID NO:84所示)、hsa-miR-451a(如SEQ ID NO:92所示)、hsa-miR-30b-5p(如SEQ ID NO:56所示)、hsa-miR-27b-3p(如SEQ ID NO:51所示)、hsa-miR-199a-3p(如SEQ ID NO:5所示)、hsa-let-7d-3p(如SEQ ID NO:6所示)、hsa-miR-197-3p(如SEQ ID NO:37所示)、hsa-miR-99a-5p(如SEQ ID NO:104所示)、hsa-miR-148a-3p(如SEQ ID NO:25所示)、hsa-miR-144-3p(如SEQ ID NO:22所示)和hsa-miR-22-3p(如SEQ ID NO:45所示)。其中hsa-miR-423-5p、hsa-miR-30b-5p、hsa-miR-27b-3p、hsa-miR-199a-3p、hsa-let-7d-3p、hsa-miR-197-3p和hsa-miR-22-3p显著上调，hsa-miR-451a显著下调，hsa-miR-99a-5p、hsa-miR-148a-3p和hsa-miR-144-3p无显著变化(图3)。
实施例2、少量单个样本验证差异性表达的miRNA
在本实施例中，具体实验操作过程均同实施例1。实施例1中筛选出的11个miRNA首先用少量随机样本，结直肠癌组和健康组分别随机挑选38例样本。验证结果其中有7个miRNA在结直肠癌组和健康组有显著性变化，即hsa-miR-423-5p、hsa-miR-451a、hsa-miR-30b-5p、hsa-miR-27b-3p、hsa-miR-199a-3p、hsa-let-7d-3p和hsa-miR-423-5p，序列号分别如SEQ ID NO:1～7所示。其中，hsa-miR-451a在结直肠癌组显著下调，其余6个miRNA在结直肠癌组显著上调。
实施例3、大量样本验证7个miRNA在结直肠癌/健康组中的表达量
实施例2中筛选出的11个潜在生物标记物在106例健康血浆(健康组)，106例结直肠癌血浆(结直肠癌组)样本中进行单样本验证。目标miRNA在健康组和结直肠癌组之间的表达量都有显著差异(P<0.001)(如图4所示)。并且7个miRNA均能显著区分健康组和结直肠癌早期(I期)病人，因此共确定有7个腺癌早期筛查生物标记物，分别为hsa-miR-423-5p、hsa-miR-451a、hsa-miR-30b-5p、hsa-miR-27b-3p、hsa-miR-199a-3p、hsa-let-7d-3p和hsa-miR-423-5p。其中hsa-miR-451a表达量显著下调，其余6个miRNA表达量显著上调。
实施例4 7个miRNA生物标记物在结直肠癌诊断中的价值
为了构建区分结直肠癌与正常人的miRNA诊断标准，我们评估了7个miRNA在结直肠癌患者和正常人血浆中的相对表达量。ROC曲线表明目标miRNA作为结直肠癌生物标记物AUC(曲下面积)分别达到0.785～0.940(p value均小于0.001)，表明这7个miRNA对结直肠癌具有较好的诊断和预测效果(图5)。利用Logistic回归分析，其中5个miRNA(hsa-miR-451a，hsa-miR-30b-5p，hsa-miR-27b-3p，hsa-miR-199a-3p和hsa-let-7d-3p)作为联合诊断指标能以AUC 0.975区分结直肠癌个体和正常个体(图5)，灵敏度和特异性分别为95.1％和90.3％。
综上，本发明提供的结直肠癌的miRNA生物标记物或组合是一种无创的理想分子标记物，只要通过检测体液中的循环miRNA的表达量，即可实现对结直肠癌进行早期诊断和预测，更快捷、准确，提前了结直肠癌的发现时机，有助于及时尽早的进行治疗，增加存活率。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对本发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。
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<213> 人工序列(Artificial Sequence)
<400> 53
aaggagcuca cagucuauug ag 22
<210> 54
<211> 22
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 54
uaugugggau gguaaaccgc uu 22
<210> 55
<211> 23
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 55
cagugcaaua guauugucaa agc 23
<210> 56
<211> 22
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 56
uguaaacauc cuacacucag cu 22
<210> 57
<211> 23
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 57
uguaaacauc cuacacucuc agc 23
<210> 58
<211> 22
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 58
uguaaacauc cuugacugga ag 22
<210> 59
<211> 23
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 59
agaagaaggc ggucggucug cgg 23
<210> 60
<211> 22
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 60
aggugguccg uggcgcguuc gc 22
<210> 61
<211> 23
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 61
cgcauccccu agggcauugg ugu 23
<210> 62
<211> 22
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 62
uauugcacau uacuaaguug ca 22
<210> 63
<211> 22
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 63
cuggcccucu cugcccuucc gu 22
<210> 64
<211> 22
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 64
uccagcauca gugauuuugu ug 22
<210> 65
<211> 22
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 65
aacaauaucc uggugcugag ug 22
<210> 66
<211> 22
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 66
uuauaaagca augagacuga uu 22
<210> 67
<211> 22
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 67
gcugacuccu aguccagggc uc 22
<210> 68
<211> 23
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 68
ugucugcccg caugccugcc ucu 23
<210> 69
<211> 22
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 69
uuaucagaau cuccaggggu ac 22
<210> 70
<211> 22
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 70
aacacaccua uucaaggauu ca 22
<210> 71
<211> 22
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 71
uaaugccccu aaaaauccuu au 22
<210> 72
<211> 22
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 72
gccugcuggg guggaaccug gu 22
<210> 73
<211> 20
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 73
acucaaacug ugggggcacu 20
<210> 74
<211> 22
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 74
uuauaauaca accugauaag ug 22
<210> 75
<211> 22
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 75
auauaauaca accugcuaag ug 22
<210> 76
<211> 22
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 76
acuggacuug gagucagaag gc 22
<210> 77
<211> 19
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 77
acuggacuug gaggcagaa 19
<210> 78
<211> 19
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 78
acuggacuug gagucagga 19
<210> 79
<211> 20
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 79
acugggcuug gagucagaag 20
<210> 80
<211> 21
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 80
acuggacuag gagucagaag g 21
<210> 81
<211> 22
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 81
uauacaaggg caagcucucu gu 22
<210> 82
<211> 22
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 82
uguguggauc cuggaggagg ca 22
<210> 83
<211> 23
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 83
agcucggucu gaggccccuc agu 23
<210> 84
<211> 23
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 84
ugaggggcag agagcgagac uuu 23
<210> 85
<211> 21
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 85
caaaacguga ggcgcugcua u 21
<210> 86
<211> 22
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 86
cagcagcaau ucauguuuug aa 22
<210> 87
<211> 22
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 87
aucgggaaug ucguguccgc cc 22
<210> 88
<211> 23
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 88
aaugacacga ucacucccgu uga 23
<210> 89
<211> 17
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 89
accccacucc ugguacc 17
<210> 90
<211> 19
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 90
gcauugugca gggcuauca 19
<210> 91
<211> 17
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 91
ccaguuuucc caggauu 17
<210> 92
<211> 22
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 92
aaaccguuac cauuacugag uu 22
<210> 93
<211> 22
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 93
aagacgggag gaaagaaggg ag 22
<210> 94
<211> 22
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 94
ucaggcucag uccccucccg au 22
<210> 95
<211> 22
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 95
cgucaacacu ugcugguuuc cu 22
<210> 96
<211> 22
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 96
ugugacagau ugauaacuga aa 22
<210> 97
<211> 23
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 97
ucggggauca ucaugucacg aga 23
<210> 98
<211> 22
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 98
cacgcucaug cacacaccca ca 22
<210> 99
<211> 22
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 99
gugagucucu aagaaaagag ga 22
<210> 100
<211> 21
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 100
uggguuuacg uugggagaac u 21
<210> 101
<211> 22
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 101
uccauuacac uacccugccu cu 22
<210> 102
<211> 22
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 102
acugcugagc uagcacuucc cg 22
<210> 103
<211> 22
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 103
ugagguagua aguuguauug uu 22
<210> 104
<211> 22
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 104
aacccguaga uccgaucuug ug 22
Claims (2)
1.一种microRNA生物标记物组合在制备用于诊断结直肠癌的产品中的用途，其特征在于：所述microRNA生物标记物组合由：hsa-miR-451a、hsa-miR-30b-5p、hsa-miR-27b-3p、hsa-miR-199a-3p和hsa-let-7d-3p组成；所述hsa-miR-451a的核酸序列如SEQ ID NO:92所示，所述hsa-miR-30b-5p的核酸序列如SEQ ID NO:56所示，所述hsa-miR-27b-3p的核酸序列如SEQ ID NO:51所示，所述hsa-miR-199a-3p的核酸序列如SEQ ID NO:38所示，所述hsa-let-7d-3p的核酸序列如SEQ ID NO:3所示。
2.一种microRNA生物标记物组合在制备用于诊断和预测早期结直肠癌的诊断试剂盒中的用途，其特征在于：所述microRNA生物标记物组合由：hsa-miR-451a、hsa-miR-30b-5p、hsa-miR-27b-3p、hsa-miR-199a-3p和hsa-let-7d-3p组成；所述hsa-miR-451a的核酸序列如SEQ ID NO:92所示，所述hsa-miR-30b-5p的核酸序列如SEQ ID NO:56所示，所述hsa-miR-27b-3p的核酸序列如SEQ ID NO:51所示，所述hsa-miR-199a-3p的核酸序列如SEQ IDNO:38所示，所述hsa-let-7d-3p的核酸序列如SEQ ID NO:3所示。
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Non-Patent Citations (4)
| Title |
|---|
| Identification of reference genes for circulating microRNA analysis in colorectal cancer;Yanqin Niu等;《SCIENTIFIC REPORTS》;20161019;第6卷;摘要、第2页倒数第2段、第7-8页、supplemental table 1 * |
| S-poly（T）PCR筛选结直肠癌血浆miRNA作为诊断标记物的研究;谭彦;《中国优秀硕士学位论文全文数据库 医药卫生科技辑》;20170715;1.2.3-1.2.4、1.3.1-1.3.3、表8 * |
| Yanqin Niu等.Identification of reference genes for circulating microRNA analysis in colorectal cancer.《SCIENTIFIC REPORTS》.2016,第6卷摘要、第2页倒数第2段、第7-8页、supplemental table 1. * |
| 谭彦.S-poly（T）PCR筛选结直肠癌血浆miRNA作为诊断标记物的研究.《中国优秀硕士学位论文全文数据库 医药卫生科技辑》.2017,1.2.3-1.2.4、1.3.1-1.3.3、表8. * |
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