CN114182021A - 肾癌诊断用尿液miRNA标志物、诊断试剂及试剂盒 - Google Patents
肾癌诊断用尿液miRNA标志物、诊断试剂及试剂盒 Download PDFInfo
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Abstract
本发明公开了一种泌尿系统癌证诊断用尿液miRNA标志物、试剂盒及诊断试剂，所述尿液miRNA标志物选自hsa‑miR‑375、hsa‑miR‑520d‑5p、hsa‑miR‑199b‑5p、hsa‑miR‑518e‑5p、hsa‑miR‑31‑3p、hsa‑miR‑4306、hsa‑miR‑143‑3p、hsa‑miR‑183‑5p、hsa‑miR‑21‑3p、hsa‑miR‑219a‑5p、hsa‑miR‑1‑3p、hsa‑miR‑18a‑3p、hsa‑miR‑671‑3p、hsa‑miR‑153‑3p、hsa‑miR‑331‑5p、hsa‑miR‑885‑5p中的一种或多种的组合。本发明具备有更高的人群特异性；相较于其他miRNA分子标志物更可靠。
Description
技术领域
本发明涉及分子诊断技术领域，特别涉及一种肾癌诊断用尿液miRNA标志物、诊断试剂及试剂盒。
背景技术
肾细胞癌(renal cell carcinoma，RCC，简称肾癌)是起源于肾小管上皮的恶性肿瘤，占肾脏恶性肿瘤的80％～90％。肾癌发病率仅次于前列腺癌及膀胱癌，占泌尿系统肿瘤第三位。肾癌的组织病理类型最常见的为透明细胞癌，占所有RCC案件的75％至80％，每年全球约90000死亡病例。在过去的几十年里，RCC的发病率逐渐增加。尽管RCC的死亡率在高水平维持，但是在没有发生转移之前，它是一种可补救的疾病。临床上，已经表明RCC通常对化疗和放射治疗具有抗性，并且肿瘤切除仍然是唯一明确的治疗方法。在治愈性肾切除术后，近55％的RCC患者存活。然而，由于缺乏早期症状，通过成像或穿刺活检，患早期症状和良性和恶性肿块之间的不能正确区别，检测早期阶段的癌细胞很具有挑战性，因此，需要开发针对早期RCC检测的创新的非侵入性方法。
最近，许多分子标记如碳酸酐酶IX(carbonic anhydrase IX，CAIX)，血管内皮生长因子(vascular endothelial growth factor，VEGF)，缺氧诱导因子(hypoxia-inducible factor，HIF)，KI67，P53，P21，PTEN(phosphatase and tensin homolog，磷酸酶和硫素同源物)，E-Cadherin，骨桥蛋白和CD44，CXCR4和其他细胞周期和增殖标记物。这些潜在的RCC生物标志物都没有明显提高当前预后系统的预测准确性，多数没有经过大规模的验证，并且在常规临床实践中没有建议使用上述的标志物。到目前为止，没有鉴定出高效的针对RCC的早期诊断生物标志物，导致疾病的晚期检测和治疗效果差。因此，鉴定可改善RCC患者早期诊断和预后的RCC生物标志物是癌症治疗的重要焦点。
miRNA是长度为18-25个核苷酸的短非编码RNA，可以通过特定目标mRNA的分解或通过抑制其翻译来阻止蛋白质表达，参与调控个体发育、细胞凋亡、增殖及分化等生命活动，在肿瘤的发生和发展过程中发挥着类似于致癌基因或抑癌基因的功能。miRNA的表达谱具有明显的组织特异性，在不同肿瘤中具有特定的表达模式。这些特点都使得miRNA有可能成为肿瘤诊断新的生物学标记和治疗靶标。
虽然许多研究已经评估了RCC患者组织和血清中的miRNA表达，但是能用于肾癌筛查的尿液miRNA生物标志物以及生物标志物组合标志物还未见定论，急需开发适合于肾癌诊断用尿液miRNA标志物。
发明内容
本发明的目的在于提供一种肾癌诊断用尿液miRNA标志物、诊断试剂及试剂盒，相对国际上报道的其它miRNA标志物，具备有更高的人群特异性；所公开的miRNA诊断标志物均为首次提出，相较于其他miRNA分子标志物更可靠。为肾癌诊断提供了一种新的可选途径。
本发明解决其技术问题所采用的技术方案是：
一种肾癌诊断用尿液miRNA标志物，所述尿液miRNA标志物选自hsa-miR-671-3p、hsa-miR-518e-5p、hsa-miR-153-3p、hsa-miR-21-3p、hsa-miR-331-5p、hsa-miR-885-5p中的一种或多种的组合。
作为优选，所述尿液miRNA标志物为hsa-miR-671-3p、hsa-miR-518e-5p、hsa-miR-153-3p、hsa-miR-21-3p、hsa-miR-331-5p和hsa-miR-885-5p的组合。
一种肾癌诊断试剂，所述肾癌诊断试剂包括权利要求1所述尿液miRNA标志物。
一种肾癌诊断用试剂盒，所述肾癌诊断用试剂盒包括权利要求1所述尿液miRNA标志物作为标准品，以及检测权利要求1所述尿液miRNA标志物的相应引物。
本发明的有益效果是：本发明明确6个适用于中国人肾癌的特异性诊断标志物，以及特异性诊断标志物组合。上述miRNA诊断标志物均为首次提出，相较于其他miRNA分子标志物更可靠。
与现有技术相比，本发明的积极和有益效果在于：
(1)本发明首次发现了hsa-miR-671-3p、hsa-miR-518e-5p、hsa-miR-153-3p、hsa-miR-21-3p、hsa-miR-331-5p、hsa-miR-885-5p可作为新的肾癌标志物组合，上述标志物组合在肾癌的早期检测和诊断中有重要的价值；
(2)本发明的miRNA标志物检测，操作简便，可行性强，灵敏度和特异性高，具有临床诊断和指导意义。
(3)本发明可通过尿液检测miRNA表达量，从而达到无创的目的。
附图说明
图1为8个((P<0.05)有差异表达的肾癌miRNA诊断标志物的表达水平热图。miRNA的表达水平(拷贝数/毫升)是以log2标度呈现的并且相对于零平均值标准化。点的颜色表示浓度。基于欧几里德距离针对两个维度(miRNA和样品)进行分层聚类。对于水平维度，使用颜色表示病例-对照的受试者。
图2为肾癌miRNA诊断标志物组合在各队列的AUC图；其中，图2-A为8个肾癌miRNA诊断标志物组合在各队列的AUC图，图2-B为6个肾癌miRNA诊断标志物组合在各队列的AUC图。
图3为6个肾癌miRNA诊断标志物组合在对照和癌症预测值的线箱图。
具体实施方式
下面通过具体实施例，对本发明的技术方案作进一步的具体说明。
本发明中，若非特指，所采用的原料和设备等均可从市场购得或是本领域常用的。下述实施例中的方法，如无特别说明，均为本领域的常规方法。
针对肾癌检测方法包括以下步骤：a)测定人类来源的被检测样本和参考样本中hsa-miR-671-3p，hsa-miR-518e-5p，hsa-miR-153-3p，hsa-miR-21-3p，hsa-miR-331-5p，hsa-miR-885-5p；b)比较被检测样本中与参考样本中hsa-miR-671-3p，hsa-miR-518e-5p，hsa-miR-153-3p，hsa-miR-21-3p，hsa-miR-331-5p，hsa-miR-885-5p的表达水平；其中，被检测样本中上述miRNA的任一种的表达水平与参考样本中相应miRNA的表达水平相比出现显著变化，表明该个体可能患有肾癌。
具体来讲，被检测肾癌中任意一种miRNA的表达水平比参考样本中miRNA的表达水平发生显著变化时，样本提供者可能患有肾癌。在较佳的实施方案中，被检测尿液样本中任意8种miRNA的表达水平比参考样本中miRNA的表达水平发生显著变化时，样本提供者可能患有肾癌。在更佳的实施方案中，被检测尿液样本中6种miRNA的表达水平比参考样本中miRNA的表达水平发生显著变化时，样本提供者可能患有肾癌。
本发明中，参考样本来自健康人源尿液样本。
另一方面，本发明还提供了一种检测肾癌microRNA的方法。具体地说，该方法包括逆转录和实时荧光定量聚合酶链式反应(RT-qPCR)。更具体地，在实时荧光定量聚合酶链式反应中，使用的引物组合为hsa-miR-671-3p，hsa-miR-518e-5p，hsa-miR-153-3p，hsa-miR-21-3p，hsa-miR-331-5p，hsa-miR-885-5p检测的逆转录引物和PCR引物组合。
可以根据相关miRNA的序列，对本发明的引物序列做出适当调整和修改，这些经过修改的引物序列仍可以用于检测所述miRNA标志物。本发明也包括这些等同的技术方案。
本发明使用RT-qPCR方法，相较于基于数百个分子的芯片，具备更可靠的临床可行性和实用性，而且成本低，更易于推广。另外，对于上述miRNA的检测方法，不限于本发明提供的RT-qPCR法，其他方法例如测序法，微阵列，RNA印迹，生物发光和探针法等也可用于检测所述的miRNA标志物。
申请人在研究中发现任意1种可以用于诊断肾癌的miRNA标志物组合，任意2种-5种可以用于诊断肾癌的miRNA标志物组合，利用上述标志物，肾癌可以被可靠地鉴定。
本发明公开了hsa-miR-671-3p，hsa-miR-518e-5p，hsa-miR-153-3p，hsa-miR-21-3p，hsa-miR-331-5p，hsa-miR-885-5p组合在内的肾癌标志物组合，hsa-miR-671-3p，hsa-miR-518e-5p，hsa-miR-153-3p，hsa-miR-21-3p，hsa-miR-331-5p，hsa-miR-885-5p有多重核酸分子组成，每种核酸分子编码至少一个miRNA序列。具体序列信息如下表1所示。
本发明公开的所有miRNA序列均已经储存在miRBase数据库中(http:// www.mirbase.org/)。
表1
本发明公开了1种肾癌诊断标志物组合，包含编码hsa-miR-671-3p，hsa-miR-518e-5p，hsa-miR-153-3p，hsa-miR-21-3p，hsa-miR-331-5p，hsa-miR-885-5p的多个核酸分子。编码hsa-miR-671-3p，hsa-miR-518e-5p，hsa-miR-153-3p，hsa-miR-21-3p，hsa-miR-331-5p，hsa-miR-885-5p的任何一个或任何两个或任何三个或任何四个或任何五个或任何六个核酸分子在诊断为肾癌患者的尿液中的表达比对对照中的表达相比发生显著变化。肾癌诊断最优的实施方案中，所述的多个核酸分子包括hsa-miR-671-3p，hsa-miR-518e-5p，hsa-miR-153-3p，hsa-miR-21-3p，hsa-miR-331-5p，hsa-miR-885-5p的6个核酸分子组合。
实施例1：验证队列验证用于肾癌诊断的8个miRNA组合
一：肾癌诊断标志物组合研发队列尿液样本要求，采集和抽提
肾癌诊断标志物研究中使用了2组样本，分别为病例组合对照组，病例组包含69例肾癌样本，对照组为88例健康人尿液样本，发现和验证了用于检测早期肾癌的生物标记物和生物标记物组合。研发队列中的肾癌病例和健康样本均来浙江大学医学院附属第二医院。
二：逆转录-实时荧光PCR操作过程及结果
按照Zymo血清血浆cfDNA快速抽提试剂盒操作步骤进行抽提，抽提完成后，利用MIRXE反转录试剂盒操作步骤进行反转录，反转录完成后qPCR扩增进行质检。
质检完成后进行样本qPCR扩增，检测312miRNA的表达量，癌症和健康对照中全部312个miRNA的表达没有存在明显的分级，进一步研究发现了用于肾癌检测的8个miRNA生物标记物，校正之后发现8个miRNAs的p值小于0.05，其中在肾癌受试者中上调了3个，下调了5个。在研发队列中提取这8个miRNA绘制Heatmap热图，观察到癌症和对照受试者之间存在miRNA表达量的显著差异，如图1所示。
三：验证队列验证上述8个miRNA
病例-对照队列采用交叉验证((Leave-one-out Cross-validation)的方法检测这8种尿液miRNA生物标志物。验证队列样本为157个样本，诊断效能AUC＝0.681，如图2-A所示。
实施例6：验证队列验证用于肾癌诊断的最优6个miRNA组合
研发队列尿液样本要求，采集和抽提，逆转录-实时荧光PCR操作过程及结果和实施例1中描述相同，基于前面筛选出的8个miRNA，分别使用Lasso、Stepwise以及穷举法等算法进行miRNA组合的优化，最终确定了最优的6个miRNA组合，又使用交叉验证(Leave-one-out Cross-validation)的方法验证hsa-miR-671-3p，hsa-miR-518e-5p，hsa-miR-153-3p，hsa-miR-21-3p，hsa-miR-331-5p，hsa-miR-885-5p在内的6种尿液生物标志物组合。验证队列样本为157个样本，6个miRNA组合在验证队列中的诊断效能AUC＝0.703，如2-B所示。模型判读的结果如图3所示，可以看到癌症患者的模型输出概率显著高于对照受试者，说明模型可以较好地对癌症患者和对照受试者进行预测分类。
基于上述对比确认6个miRNA组合为诊断肾癌的最优组合。
本发明建立了一个完整的工作流程，用于发现和验证尿液miRNA生物标志物组合，及成功确定了用于检测肾癌的生物标志物及生物标志物组合。
以上所述的实施例只是本发明的一种较佳的方案，并非对本发明作任何形式上的限制，在不超出权利要求所记载的技术方案的前提下还有其它的变体及改型。
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<120> 肾癌诊断用尿液miRNA标志物、诊断试剂及试剂盒
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Claims (4)
1.一种肾癌诊断用尿液miRNA标志物，其特征在于，所述尿液miRNA标志物选自hsa-miR-671-3p、hsa-miR-518e-5p、hsa-miR-153-3p、hsa-miR-21-3p、hsa-miR-331-5p、hsa-miR-885-5p中的一种或多种的组合。
2.根据权利要求1所述的肾癌诊断用尿液miRNA标志物，其特征在于，所述尿液miRNA标志物为hsa-miR-671-3p、hsa-miR-518e-5p、hsa-miR-153-3p、hsa-miR-21-3p、hsa-miR-331-5p和hsa-miR-885-5p的组合。
3.一种肾癌诊断试剂，其特征在于，所述肾癌诊断试剂包括权利要求1所述尿液miRNA标志物。
4.一种肾癌诊断用试剂盒，其特征在于，所述肾癌诊断用试剂盒包括权利要求1所述尿液miRNA标志物作为标准品，以及检测权利要求1所述尿液miRNA标志物的相应引物。
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