CN101423869B - 一种检测用生物标记物及应用 - Google Patents
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Abstract
本发明提供一种检测用生物标记物,以检测样本中microRNA-375的表达量作为生物标记,以miR-375表达量小于等于0.2为临界值,miR-375表达量小于等于0.2的认为是肿瘤组织,大于0.2的认为是非肿瘤组织。本发明提供的生物标记物检测胃癌的特异性72.7%,敏感性82.8%,具有敏感性和特异性高,操作方便等特点,有较高的参考价值,可在检测胃癌组织中的应用。本发明有助于揭示肿瘤发生发展的分子生物学机制,为肿瘤的个体化靶向治疗奠定基础。
Description
技术领域
本发明属生物技术领域,涉及一种胃癌生物标记物及其在胃癌诊断中的应用。
背景技术
胃癌是严重危害我国人民生命健康的重大疾病,其年龄标化发病率和死亡率均居我国常见恶性肿瘤年龄标化发病率和死亡率的第二位。由于绝大多数胃癌患者缺乏特异性临床症状,目前早期胃癌的诊断率较低,其病例仅占胃癌手术病例的10-20%,就诊的患者大多处于胃癌的中晚期,其五年生存率仍徘徊于20-30%。
胃癌的发生是机体遗传因素和外界环境因素相互作用、多基因参与、多步骤发生的过程。近年研究结果显示,各分子事件之间存在以关键调控基因为重要节点的多中心调控网络,这些重要节点通过各种信号传导通路参与正常胃粘膜到胃癌这一复杂的演变过程。目前胃癌的临床诊断主要依据组织学形态观察结果,而未应用胃癌特征性分子事件变化对上述病变进行分型和分级,因此上述病变的分型和分级具有较大的人为主观性,且无法为患者提供个性化治疗。而现有的肿瘤标记物如CEA、CA19-9、CA50、CA125在胃癌中的阳性率约60%,但敏感性和特异性均不高,并与其他肿瘤存在交叉。因此,我们迫切需要了解胃癌发生发展的分子机制,寻找新的生物标记物和药物靶标来早期诊断胃癌和有效治疗胃癌。
MicroRNA(miRNA)是一类长度约19~25个核苷酸的非编码单链小分子RNA,广泛存在于植物、线虫、果蝇和哺乳动物等生物中,主要通过不完全互补的方式与编码蛋白mRNA的3’-UTR区结合引起靶mRNA的降解、活性降低或翻译受抑,从而在转录后水平对基因表达进行调控。研究表明miRNA几乎在所有的生物学过程中发挥重要的调控作用,包括个体发育、细胞增殖、分化、代谢、凋亡和应激等。约50%的已知人类miRNA基因定位于和肿瘤相关的染色体区域,而不断累积的研究成果也已充分证实miRNA的异常表达与肿瘤的发生、发展密切相关。
最新的数据显示肿瘤组织普遍具有特征性的miRNA表达谱,即肿瘤细胞某几种miRNA表达水平常与同一组织中的正常细胞存在显著的差异,而特征性的miRNA异常表达可望作为有效工具用于肿瘤的诊断、病理分级、临床分期、预后和对治疗的反应性等,显示了良好的临床应用前景。因此,开发一种可辅助胃癌诊断的miRNA作为生物标记物,具有广泛的科研价值和临床应用前景。
发明内容
本发明的目的在于提供一种检测用生物标记物,以检测样本中microRNA-375(miR-375)的表达量作为生物标记,以miR-375表达量小于等于0.2为临界值。
该生物标记物在胃癌组织中的表达量约为正常组织的四分之一。利用组织中miR-375的表达量作为生物标记,检测胃癌的特异性达72.7%,敏感性达82.8%。
本发明的另一个目的在于提供所述生物标记物在检测胃癌组织中的应用。
取氮冻存的组织1-3块,使用mirVanaTM miRNA Isolation试剂盒(美国Ambion公司)提取总RNA,再使用miR-375特异的Taqman MicroRNA Assays(美国Applied Biosystems公司)试剂盒进行实时荧光定量PCR检测;采用相对定量的方法,得到目标组织相对于参照组织miR-375的含量,参照组织为病理确诊后的正常胃组织;以0.2为临界值,miR-375表达量小于等于0.2的组织检测认为是肿瘤组织,大于0.2的组织检测认为是非肿瘤组织。
本发明的有益效果在于:1.提供的该生物标记物在胃癌组织中的表达量仅为正常组织的四分之一,该生物标记物的特异性72.7%,敏感性82.8%,具有敏感性和特异性高,操作方便等特点,有较高的诊断参考价值。2.有助于揭示肿瘤发生发展的分子生物学机制,为肿瘤的个体化靶向治疗奠定基础。
附图说明
图1:手术胃癌组织中miR-375的相对定量。
图2:胃镜组织中miR-375的相对定量。
图3:胃镜组织中miR-375的相对定量(非肿瘤组vs.肿瘤组)。
具体实施方式
本发明结合实施例和附图作进一步的说明。
实施例1
1、组织样品的准备
收集液氮冻存的胃癌手术切除样本共34例,每例为肿瘤组织及癌旁非肿瘤组织各一块,并收集液氮冻存的胃镜样本60例(29例胃癌组织和31例慢性浅表性胃炎组织)。
2、组织总RNA提取
使用mirVanaTM miRNA Isolation Kit(Catalog#1560,1561,Ambion)试剂盒,根据试剂盒所提供的操作步骤,提取上述34例胃癌手术切除样本及60例胃镜样本的总RNA(每块组织取50毫克),分装-80℃冰箱冻存,直至检测。
3、Real-time PCR检测miR-375表达量
使用hsa-miR-375 Taqman MicroRNA Assays(Applied Biosystems,P/N:4373151)检测组织样本中miR-375的表达量,Real-time PCR在Real-Time PCRDetection System(ABI 7500)上进行。逆转录PCR使用10ng总RNA和茎环样引物(looper primer),20μL Real-time PCR体系中包含1.33逆转录PCR产物,10μL 2×Taqman Universal PCR Master Mix,No AmpErase UNG(P/N 4369016,Applied Biosystems),1μL real time primer(20×Taqman MicroRNA Assays Mix,P/N4373027)。反应在96孔板中进行,95℃,10分钟,继以4个循环的95℃,15秒60℃,1分钟。使用mRNA U6(RNU6B,P/N:4373381;Applied Biosystems)作为内参。MiRNA的相对定量使用2-ΔΔCt算法计算。手术肿瘤样本以同患者邻近非瘤样本作为参照,胃镜样本以病理鉴定为正常的胃组织作为参照。所得相对定量(Relative Quantitation,RQ)取log10对数,以便于使用t-检验统计。结果显示,在33例受检手术样本中,胃癌组织中miR-375的表达量在87.9%的样本中较癌旁非瘤组织有明显的降低,平均降低倍数为3.94,差异有显著性(参见图1,p<0.01)。而在胃镜样本中,肿瘤组miR-375的表达量较非肿瘤组亦有明显的降低,肿瘤组和非肿瘤组miR-375的表达量的几何平均数分别为0.07497和0.33977,平均下降倍数为4.53,具统计学差异(参见图2,3,p<0.05)。
4、以miR-375作为胃癌检测的辅助生物标记物
以病理形态学检查结果为金标准,60位患者中共29位患者的被检组织为肿瘤组织,31位患者为非肿瘤组织。Real-time PCR检测组织中miR-375的相对定量(以编号611的组织中miR-375的RQ值为参照)。以0.2为临界值,miR-375的表达量小于等于临界值的组织诊断为肿瘤组织,大于0.2的组织诊断为非肿瘤组织。以miR-375的相对含量作为诊断标准,胃癌组织中>0.2的共5例,<=0.2共24例,非胃癌组织中>0.2的共22例,<=0.2共9例。miR-375作为诊断指标的特异性为72.7%(24/33),敏感性为82.8%(24/29)。具体检测结果参见表1。
表1:miR-375诊断实验结果
Claims (2)
1.一种生物标记物的制备方法,其特征在于通过以下步骤实现:取氮冻存的组织1-3块,使用mirVanaTM miRNA Isolation试剂盒提取总RNA,再使用miR-375特异的Taqman MicroRNA Assays试剂盒进行实时荧光定量PCR检测;采用相对定量的方法,得到目标组织相对于参照组织miR-375的含量,参照组织为病理确诊后的正常胃组织;以0.2为临界值,miR-375表达量小于等于0.2的组织检测认为是肿瘤组织,大于0.2的组织检测认为是非肿瘤组织。
2.根据权利要求1所述的一种生物标记物的制备方法,其特征在于:所述实时荧光定量PCR体系中包含1.33逆转录PCR产物,反应在96孔板中进行,反应条件是:95℃,10分钟,继以4个循环的95℃,15秒60℃,1分钟。
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Arti Gaur,et al.Characterization of MicroRNA Expression Levels and Their Biological Correlates in Human Cancer Cell Lines.《Cancer Res》.2007,第67卷(第6期),2456-2468. * |
包阿东,等.微小RNA 功能与癌症形成的关联分析.《中国畜牧兽医》.2008,第35卷(第2期),49-53. * |
王迎昕,等.m i RNA与消化系统肿瘤的关系.《国际内科学杂志》.2008,第35卷(第10期),609-612. * |
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