CN106244696A - 一种与肺癌易感性相关的生物标志物及应用 - Google Patents

一种与肺癌易感性相关的生物标志物及应用 Download PDF

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CN106244696A
CN106244696A CN201610685601.XA CN201610685601A CN106244696A CN 106244696 A CN106244696 A CN 106244696A CN 201610685601 A CN201610685601 A CN 201610685601A CN 106244696 A CN106244696 A CN 106244696A
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曹亚
李江江
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Central South University
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Abstract

本发明属于生物技术的基因领域，具体涉及一种与肺癌易感性相关的单核苷酸多态性位点及其应用。本发明提供与肺癌易感性相关的遗传区域15q26.1上的部分基因序列，其核苷酸序列如SEQ.ID.NO.1所示；还提供遗传区域15q26.1上的单核苷多态性位点rs11540478,即SEQ.ID.NO.1序列261位C/T；rs11540478碱基为T的个体为肺癌易感人群，rs11540478碱基为C的个体为非易感人群。还提供检测单核苷酸多态性位点的特异性核酸引物：SEQ.ID.NO.2、SEQ.ID.NO.3，其特异性地扩增出单核苷酸多态性位点的扩增产物。

Description

一种与肺癌易感性相关的生物标志物及应用
技术领域
本发明属于生物技术的基因领域，具体涉及一种与肺癌易感性相关的生物标志物及应用。
背景技术
随着经济社会的发展与疾病谱的改变，我国肺癌等肿瘤的发病率明显上升。根据中国肿瘤登记年报(2012)，我国全国肿瘤登记地区恶性肿瘤发病第一位的是肺癌。其发病率为53.57/10万，人口标化后为25.34/10万，五年生存率只有10％-15％。肺癌分为小细胞肺癌和非小细胞肺癌。在我国，约80％的肺癌被诊断为非小细胞肺癌。吸烟是肺癌发生的重要风险因素，近来的研究表明遗传因素也是肺癌发生的重要原因。人类遗传因素的主要表现形式为单核苷酸多态性(Single Nucleotide Polymorphisms，SNP)。SNP主要是指在基因组水平上由单个核苷酸的变异所引起的DNA序列多态性。它是人类可遗传的变异中最常见的一种。目前的研究已经发现了多个肺癌易感性SNP位点。
异柠檬酸脱氢酶2(isocitrate dehydrogenase 2,IDH2)是细胞代谢途径中参与三羧酸循环的酶，位于染色体15q26.1，亚细胞定位于线粒体基质。其生理功能是正向催化异柠檬酸氧化产生α-酮戊二酸，同时还原NADP+产生NADPH。IDH2的逆向反应催化α-酮戊二酸产生异柠檬酸，并消耗NADPH产生NADP+。
近期的研究已报道IDH2突变产生致癌代谢物2-羟基戊二酸，能抑制胶质瘤分化和促进肿瘤形成。IDH2也可以在低氧条件通过谷氨酰胺代谢经过a-酮戊二酸产生柠檬酸，从而促进细胞增殖和增加细胞活力。IDH2的编码区域频率大于1％的SNP位点为rs11540478，目前对IDH2基因的肿瘤易感性研究尚未有文献报道。
发明内容
本发明提供了一种与肺癌易感性相关的生物标志物，所述生物标志物为遗传区域15q26.1上的基因序列，该基因序列如SEQ ID NO.1所示。该生物标志物可用于制备检测肺癌易感性试剂、试剂盒或药物。另外，本发明还提供了扩增上述生物标志物的引物对，所述引物对序列如SEQ ID NO.2和SEQ ID NO.3所示。所述引物对可以用于制备检测肺癌易感性的试剂盒。
下面对本发明作进一步说明：
本发明研究表明，遗传区域15q26.1与肺癌的易感性相关。与肺癌易感性相关的遗传区域15q26.1上的IDH2部分基因序列的核苷酸序列如SEQ.1D.NO.1所示，通过http://www.ncbi.nlm.nih.gov数据库获得，其第261位为rs11540478C/T。IDH2rs11540478C>T多态与肺癌发生相关，即使在调整年龄、性别、吸烟多种混杂因素后，这种相关性仍然存在。因此这个SNP的多态可用于评估个体肺癌的易感性。
本发明所述的引物对能特异性地扩增出单核苷酸多态性位点的扩增产物。本发明关于与肺癌易感性相关的遗传区域15q26.1基因序列，其获得rs11540478序列的具体步骤如下：通过http://www.ncbi.nlm.nih.gov数据库，在遗传区域15q26.1上选择单核苷酸多态性位点rs11540478；依据相应的碱基序列合成引物：5′-GCTGGGGGAGGCTGAGTAGAG-3′(SEQ.1D.NO.2),5′-GCCCTGCAC TCACCTTCTGG-3′(SEQ.1D.NO.3)进行PCR扩增，进一步通过Sanger测序或者限制性片段长度多态性酶切(PCR-RFLP)的方法获取SNP rs11540478分型数据。其中rs11540478碱基为T的个体为肺癌易感人群。rs11540478碱基为C的个体为肺癌非易感人群。
总之，本发明提供了一种与肺癌易感性相关的生物标志物，该生物标志物为遗传区域15q26.1的部分基因序列，通过rs11540478单核苷酸多态性位点的遗传研究，提出一个可作为判定前肺癌高危人群的易感SNP位点，以利于肺癌高危人群的筛查，预防和治疗。
具体实施方式
实施例1.肺癌及健康人群IDH2rs11540478位点的基因分型。
本部分研究对601例健康人及262例肺癌患者样本外周血IDH2rs11540478位点分别采用Sanger测序法和PCR-RFLP方法进行基因分型，确定IDH2rs11540478是肺癌的易感性位点。
实验方法：
1.gDNA提取
由检测者签署知情同意书，常规方法抽取被检测者EDTA抗凝血5ml。用哺乳动物DNA快速提取方法(分子克隆实验手册)提取血液标本的基因组DNA。
2.基因分型检测
检测肺癌IDH2rs11540478的试剂为Taq DNA Mix(promega公司)和去离子水；特异性弓|物对的序列如SEQ ID NO.2和SEQ ID NO.3所示，反应体系共20ul，分别为Taq DNAMix 10ul，正义引物和反义引物(浓度10uM)各1ul共2ul，DNA模板(50ng/ul)2ul，去离子水6ul。
反应条件为95℃2min；95℃0.5min，96℃0.5min，72℃lmin，45个循环；72℃5min,4℃5min。
基因分型方法为健康人群直接送华大基因进行Sanger测序法测序,肺癌人群采用PCR-RFLP方法进行基因分型。
PCR-RFLP方法反应体系共20ul，分别为PCR产物5ul，AvaII酶(Fermentas)2ul，AvaII酶缓冲液2ul，去离子水11ul。经过37度孵育1小时后，琼脂糖凝胶电泳根据条带位置进行基因分型。
实验结果：
通过我们对601例健康人及262例肺癌患者样本外周血进行基因分型后我们发现：携带IDH2rs11540478CT和TT基因型的个体比携带rs11540478CC基因型的个体罹患肺癌的风险要高1.44倍，如表1所示。
表1
a数据通过非条件Logistic回归分析校准年龄，性别和吸烟状况。

Claims (4)

1.一种与肺癌易感性相关的生物标志物，其特征在于，所述生物标志物为遗传区域15q26.1上的基因序列，该基因序列如SEQ ID NO.1所示。
2.如权利要求1所述的生物标志物在制备检测肺癌易感性试剂、试剂盒或药物中的应用。
3.用于扩增权利要求1所述生物标志物的引物对，其特征在于，所述引物对序列如SEQID NO.2和SEQ ID NO.3所示。
4.检测肺癌易感性的试剂盒，其特征在于，所述试剂盒中含有序列如SEQ ID NO.2和SEQ ID NO.3所示的引物对。
CN201610685601.XA 2016-08-18 2016-08-18 一种与肺癌易感性相关的生物标志物及应用 Pending CN106244696A (zh)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106591450A (zh) * 2016-12-13 2017-04-26 江苏省疾病预防控制中心 铅暴露工人岗前入职筛选基因标志物及其应用
CN107119113A (zh) * 2017-04-01 2017-09-01 江苏省疾病预防控制中心 噪声暴露工人岗前入职筛选基因标志物检测方法及其应用
CN111808955A (zh) * 2020-06-15 2020-10-23 南通大学 一种与肺癌相关的外周血regQTLs生物标志物及其应用

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CN101974636A (zh) * 2010-11-09 2011-02-16 广州医学院 通过map2k4基因检测肺癌易感性的试剂盒及其用途

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CN101974636A (zh) * 2010-11-09 2011-02-16 广州医学院 通过map2k4基因检测肺癌易感性的试剂盒及其用途

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XU GUO等: "Genetic variants in genes of tricarboxylic acid cycle key enzymes are associated with prognosis of patients with non-small cell lung cancer", 《LUNG CANCER》 *
郭旭: "肿瘤代谢相关基因单核苷酸多态性与非小细胞肺癌与预后相关性研究及其机制", 《中国博士学位论文全文数据库（医药卫生科技辑）》 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106591450A (zh) * 2016-12-13 2017-04-26 江苏省疾病预防控制中心 铅暴露工人岗前入职筛选基因标志物及其应用
CN106591450B (zh) * 2016-12-13 2020-03-31 江苏省疾病预防控制中心 铅暴露工人岗前入职筛选基因标志物及其应用
CN107119113A (zh) * 2017-04-01 2017-09-01 江苏省疾病预防控制中心 噪声暴露工人岗前入职筛选基因标志物检测方法及其应用
CN111808955A (zh) * 2020-06-15 2020-10-23 南通大学 一种与肺癌相关的外周血regQTLs生物标志物及其应用

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