CN114150061A - 一种用于诊断癌症的分子标志物及试剂盒 - Google Patents
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Abstract
本发明公开了一种用于诊断癌症的分子标志物及试剂盒,尤其是BEND4基因启动子区DNA甲基化作为分子标志物在制备诊断癌症产品中的应用。本发明首次通过MSP的方法在胰腺癌、乳腺癌和食管癌中发现BEND4启动子区频繁发生高甲基化,并发现BEND4参与DNA损伤修复的NHEJ通路,BEND4甲基化是ATM抑制剂结合顺铂等化疗药物杀伤肿瘤细胞的协同致死标志物。本发明还提供了用于检测BEND4基因启动子区甲基化状态的引物、试剂盒。应用本发明引物、试剂盒来检测BEND4基因启动子区高甲基化状态可作为胰腺癌、乳腺癌和食管癌诊断、疗效观察、预后判断、微小残留病检测等的有力手段,而且,操作简便、稳定性好,具有深远的临床意义和推广性。
Description
技术领域
本发明涉及生物技术领域。更具体地,涉及一种用于诊断癌症的分子标志物及试剂盒。
背景技术
胰腺癌(pancreatic cancer,PC)是最难治的癌症之一,被认为在消化系统肿瘤中预后最差。尽管采用不同的手术治疗方案和辅助治疗以及新辅助化疗来治疗PC,但总体5年生存率仍不超过8%(Bray F,Ferlay J.Global cancer statistics2018:GLOBOCANestimates of incidence and mortality worldwide for 36cancers in185countries.CA Cancer J Clin.2018Nov;68(6):394-424.;Siegel RL,Miller KD.CACancer J Clin.2017Jan;67(1):7-30.)。PC患者的高死亡率主要归因于其早期缺乏典型症状和侵袭性局部侵袭。PDAC(pancreatic ductal adenocarcinoma,PDAC)是迄今为止最常见的类型,占原发性胰腺癌的85%以上,因此“胰腺癌”的大多数属性都与这种肿瘤类型有关。PDACs在头部明显比胰腺的其余部分更常见。绝大多数是实体瘤,尽管偶尔它们表现为囊性肿块或显示一些囊性区域。PDAC倾向于非常隐蔽地浸润周围组织,而不会形成致密的肿块病变,在绝大多数情况下,其大小小于7.0厘米。PDAC的特点是显著的结缔组织增生,其中间质成分占肿瘤总体积的70%以上。PDAC的唯一治愈方法是手术切除(Masiak-Segit W,Rawicz-Pruszyński K.Surgical treatment of pancreatic cancer.PolPrzeglChir.2018Apr 30;90(2):45-53.);然而,由于就诊时出现局部晚期或转移性疾病,大约80%的患者无法接受手术。就诊时,只有10%–20%的患者适合切除,30%–40%是不可切除的/局部晚期,超过50%的患者在首次就诊时被发现患有转移性疾病(Shinde RS,Bhandare M.Cutting-edge strategies for borderline resectable pancreaticcancer.Ann Gastroenterol Surg.2019Apr 25;3(4):368-372.)。
食管癌(esophageal cancer,EC)是所有胃肠道恶性肿瘤中侵袭性最强的一种。全球5年总生存率为15%至25%,它是男性癌症相关死亡的第六大原因(Watanabe M,OtakeR.Recent progress in multidisciplinary treatment for patients with esophagealcancer.Surg Today.2020Jan;50(1):12-20.)。中国是食管癌的高危地区。2015年全国食管癌发病率居所有恶性肿瘤第五位,死亡率居第四位(Li J,Ma S.History and currentsituation of neoadjuvant treatment for locally advanced esophagealcancer.Thorac Cancer.2021Sep;12(17):2293-2299.)。手术、化疗和放疗仍是食管癌的主要治疗方法,但术后症状如食欲减退、早期饱腹、吞咽困难、误吸和反流等可影响患者的生活质量(Elliott JA,Docherty NG.Weight Loss,Satiety,and the Postprandial GutHormone Response After Esophagectomy:A Prospective Study.Ann Surg.2017Jul;266(1):82-90.)。食管癌的两种主要组织学亚型是鳞状细胞癌和腺癌,在中国食管鳞癌占食管癌的90%以上,而欧洲国家及南北美洲以腺癌为主。不同病理类型食管鳞癌的危险因素也不同,包括性别、种族、吸烟、饮酒、饮食、营养状况和遗传因素。通常食管癌被诊断时已处于晚期,因此更好地了解食管癌发病机制的分子机制对于食管癌患者的临床诊断和治疗至关重要。
乳腺癌(Breast cancer,BC)是最常见的癌症,也是全球女性癌症死亡的主要原因之一。乳腺癌是一种复杂的异质性疾病,根据组织学特征分为激素受体阳性(雌激素受体ER和孕激素受体PR)、人表皮生长因子受体2过表达(HER2+)和三阴性乳腺癌(TNBC)(NaginiS.Breast Cancer:Current Molecular Therapeutic Targets and NewPlayers.Anticancer Agents Med Chem.2017;17(2):152-163.)。乳腺癌发病的确切机制尚不清楚,表达HR的BC是最普遍的BC类型,在发达国家占60-70%的BC病例,仅为绝经前妇女。因此,激素治疗是最常用的治疗方法。三阴性乳腺癌是乳腺癌中恶性程度最高的亚型,占所有乳腺癌病例的10%–20%(Tian T,Zhao Y.Circular RNA:A potentialdiagnostic,prognostic,and therapeutic biomarker for human triple-negativebreast cancer.MolTher Nucleic Acids.2021Jul 2;26:63-80.)。遗传因素和环境因素可能会影响乳腺癌的发生和进展,乳腺癌的危险因素包括年龄增加、种族、月经初潮史、乳房特征、生殖模式、激素使用、酒精使用、烟草使用、饮食、身体活动和身体习惯(Winters S,Martin C.Breast Cancer Epidemiology,Prevention,andScreening.ProgMolBiolTransl Sci.2017;151:1-32.)。
随着后基因组时代的到来,表观遗传学(Epigenetics)已成为生命学科的研究前沿。在肿瘤领域中,表观遗传学研究主要集中DNA甲基化及组蛋白乙酰化上。DNA甲基化修饰是基因组表观遗传调控中主要的方式,人类肿瘤的发生、发展与DNA甲基化的异常有关,基因启动子区CpG岛高甲基化可导致抑癌基因表达沉默进而导致肿瘤发生。DNA甲基化是指在甲基化转移酶的作用下,以s-腺苷甲硫氨酸为甲基供体,将甲基转移到CG二核苷酸胞嘧啶的5号碳原子上,形成5甲基胞嘧啶。DNA甲基化异常是细胞癌变过程中早期、频发事件,因此,特异基因的甲基化可作为癌症早期诊断的分子标志物、治疗靶点和判断预后手段。
本申请意在筛选出具有强敏感性、高特异性的胰腺癌、食管癌或乳腺癌诊断分子标志物,以及寻找有效的检测方法,建立有效监控手段,从而早诊早治,实现重要的临床应用价值。
发明内容
本发明的第一个目的在于提供一种用于胰腺癌、食管癌或乳腺癌诊断的分子标志物,该分子标志物具有敏感性强、特异性高的特点,及分子标志物在制备诊断胰腺癌、食管癌或乳腺癌产品中的应用。
本发明的第二个目的在于提供一种检测上述分子标志物的物质在制备诊断胰腺癌、食管癌或乳腺癌产品中的应用。
本发明的第三个目的在于提供一种基于上述分子标志物的诊断胰腺癌、食管癌或乳腺癌的试剂盒。
为达到上述目的,本发明采用下述技术方案:
本发明提供了BEND4基因启动子区DNA片段作为分子标志物在制备诊断癌症产品中的应用,其中,所述癌症主要为胰腺癌、食管癌或乳腺癌。
根据本发明的具体实施方式,作为分子标志物的所述BEND4基因启动子区DNA片段的核苷酸序列如SEQ ID No.1所示(硫化处理前原始DNA序列,BEND4基因TSS附近CG密集区域),且包含至少一个甲基化位点。
BEN结构域是一个新发现保守与特异DNA序列相结合的结构域,其在发育过程中主要发挥转录抑制作用。BEND4是该家族中的一员。发明人在不同肿瘤的细胞系中筛选了BEND基因家族不同成员的表达情况,发现BEND4在多种肿瘤细胞中表达缺失,然后分析了其表达调控机制,通过MSP的方法在胰腺癌、乳腺癌和食管癌中发现BEND4启动子区频繁发生高甲基化。因此推断BEND4基因的启动子区高甲基化状态可能是一个新的胰腺癌、乳腺癌和食管癌分子标志物,BEND4基因可能是一个新的胰腺癌、乳腺癌和食管癌相关抑癌候选基因。以此为坚实的理论依据,本发明人应用MS-PCR的方法对大量临床胰腺癌、食管癌和乳腺癌及正常胰腺、乳腺、食管标本进行检测,明确了其BEND4基因启动子区甲基化状态。且首次发现,BEND4参与DNA损伤修复的NHEJ通路,BEND4甲基化是ATM抑制剂结合顺铂等化疗药物杀伤肿瘤细胞的协同致死标志物。
本发明还提供了检测BEND4基因启动子区DNA片段甲基化状态的物质在制备诊断癌症产品中的应用,其中,所述癌症主要为胰腺癌、食管癌或乳腺癌。
可选的,所述BEND4基因启动子区DNA片段的核苷酸序列如SEQ ID No.1所示,且包含至少一个甲基化位点。
本发明还提供了一种诊断癌症的试剂盒,包括用于检测BEND4基因启动子区甲基化状态的物质,所述癌症主要为胰腺癌、食管癌或乳腺癌。
进一步的,所述用于检测BEND4基因启动子区甲基化状态的物质针对BEND4基因启动子区核苷酸序列如SEQ ID No.1所示的DNA序列。
进一步的,所述物质包括甲基化引物对和非甲基化引物对。
优选的,所述甲基化引物对的上游引物为5'TCGTATGAGTTTAGAGGTCGCGATGTTC 3',如SEQ ID No.2所示,下游引物为5'TCTTCTACCAATCGAAAATTACTCTCCG 3',如SEQ ID No.3所示;所述非甲基化引物对的上游引物为5'GTTTTGTATGAGTTTAGAGGTTGTGATGTTT 3',如SEQID No.4所示,下游引物为5'ATTCTTCTACCAATCAAAAATTACTCTCCA 3',如SEQ ID No.5所示。
利用上述甲基化引物对,以硫化修饰后的DNA为模板进行MS-PCR扩增,如果BEND4基因启动子区存在甲基化,则会得到138bp大小的片段;如果BEND4基因正常即未甲基化,则不产生扩增产物。基于此,本申请将该引物对称为甲基化引物对。所述甲基化引物对,其上游引物的Tm64.61,GC含量46.43%,下游引物的Tm 61.09,GC含量39.29%。
利用上述非甲基化引物对,以硫化修饰后的DNA为模板进行MS-PCR扩增,如果BEND4基因正常即未甲基化,则会得到143bp大小的片段。基于此,本申请将该引物对称为非甲基化引物对。所述非甲基化引物对,其上游引物的Tm 61.25,GC含量32.26%,下游引物的Tm59.66,GC含量30%。
本发明中,用甲基化引物对进行扩增,有扩增产物,而用非甲基化引物进行扩增则无扩增产物为完全甲基化;用甲基化及非甲基化引物进行扩增,均有扩增产物,为部分甲基化;用非甲基化引物进行扩增,有扩增产物,用甲基化引物进行扩增,无扩增产物,为无甲基化。部分甲基化及完全甲基化均判定为甲基化。
进一步的,所述试剂盒还包括甲基化阳性对照PCR模板和非甲基化阳性对照PCR模板。
其中,所述甲基化阳性对照PCR模板为硫化修饰后的BEND4基因启动子区100%甲基化的肿瘤细胞DNA,例如胰腺癌、乳腺癌、食管癌细胞;所述非甲基化阳性对照PCR模板为硫化修饰后的BEND4基因启动子区100%非甲基化的正常淋巴细胞或正常组织细胞DNA,例如正常胰腺、乳腺、食管组织细胞。
进一步的,所述试剂盒还包括MS-PCR反应所需试剂,例如10×MSP buffer缓冲液,20mMdNTP,Hot start taq酶,去离子水等。
进一步的,利用本发明的试剂盒进行癌症诊断时,尤其是胰腺癌、乳腺癌或食管癌,可以使用现有技术中已知的MS-PCR方法。
优选的,反应条件如下:
95℃下预变性5min,接着进入循环,在95℃下变性30s,在60℃下退火30s,在72℃下延伸40s,共35个循环,之后,继续在72℃下延伸5min。
本发明的有益效果如下:
利用本发明所述分子标志物,检测物质(引物)及试剂盒检测胰腺癌、食管癌或乳腺癌的组织标本,在肿瘤细胞中BEND4基因启动子区呈高甲基化状态,甲基化率在胰腺癌、乳腺癌及食管癌中分别约为53.92%、66.6%%、65.67%,而在正常组织细胞中,BEND4基因启动子区呈非甲基化状态,表明该基因启动子区的甲基化状态对于胰腺癌、乳腺癌和食管癌细胞具有特异性;同时应用本发明所述引物及试剂盒、反应体系及反应条件可使检测胰腺癌、乳腺癌和食管癌细胞的灵敏度达到0.1%,即1000个细胞中有1个癌细胞就能够被检测出,这说明BEND4基因启动子区甲基化状态可作为胰腺癌、乳腺癌和食管癌等的一个新的分子标志。我们探讨了胰腺癌细胞对ATM抑制剂(AZD0156)敏感性,结果发现,顺铂药物诱导下,BEND4甲基化的胰腺癌细胞(SW1990)的IC50值为0.19±0.07μM,BEND4非甲基化的胰腺癌细胞(CFpac1)的IC50值为8.62±1.31μM,BEND4甲基化的胰腺癌细胞对AZD0156更加敏感(P<0.05)。本试剂盒首次发现BEND4参与DNA损伤修复的NHEJ通路,BEND4甲基化是ATM抑制剂结合顺铂等化疗药物杀伤肿瘤细胞的协同致死标志物。因此,应用本发明分子标志物、引物或试剂盒来检测BEND4基因启动子区高甲基化状态可作为胰腺癌、乳腺癌和食管癌诊断、疗效观察、预后判断、微小残留病检测等的有力手段,而且,操作简便、稳定性好,具有深远的临床意义和推广性。
附图说明
图1示出以硫化修饰后的正常胰腺、乳腺、食管细胞DNA,硫化修饰后的胰腺癌、乳腺癌和食管癌细胞DNA、去离子水(阴性对照)为模板,分别用甲基化引物对及非甲基化引物对进行MS-PCR,扩增产物琼脂糖凝胶电泳结果图;其中,
DNA Marker(100bp Ladder)
PC1~PC5为胰腺癌;BC1~BC5为乳腺癌;EC1~EC7为食管癌;PN1~PN2为正常胰腺;BN1~BN2为正常乳腺;EN1~EN2为正常食管;
另有IVD标记的为甲基化对照,NL标记的为非甲基化对照,H2O阴性对照,U代表非甲基化结果;M代表甲基化结果。
图2示出将胰腺癌细胞系SW1990的DNA(BEND4基因启动子区100%甲基化)与正常淋巴细胞的DNA(BEND4基因启动子区100%非甲基化)按比例混合,进行硫化修饰,此后用甲基化引物进行扩增,扩增产物的琼脂糖凝胶电泳结果图;其中,
第1组:100%SW1990细胞DNA+0%正常细胞DNA;
第2组:50%SW1990细胞DNA+50%正常细胞DNA;
第3组:5%SW1990细胞DNA+95%正常细胞DNA;
第4组:1%SW1990细胞DNA+99%正常细胞DNA;
第5组:0.1%SW1990细胞DNA+99.9%正常细胞DNA;
第6组:0%SW1990细胞DNA+100%正常细胞DNA。
具体实施方式
为了更清楚地说明本发明,下面结合优选实施例和附图对本发明做进一步的说明。本领域技术人员应当理解,下面所具体描述的内容是说明性的而非限制性的,不应以此限制本发明的保护范围。
实施例1
1、模板的制备(基因组DNA的提取及硫化修饰过程)
DNA的制备:获取胰腺癌、乳腺癌、食管癌标本及正常上述组织标本。在本实施例中结胰腺癌5例(PC1-PC5)、乳腺癌5例(BC1-BC5)、食管癌7例(EC1-EC7)、正常胰腺2例(PN1-PN2)、正常乳腺2例(BN1-BN2)、正常食管2例(EN1-EN2),应用酚-氯仿抽提的方法分别提取基因组DNA,紫外分光光度仪测定吸光度(A)值确定其含量及纯度。
亚硫酸盐修饰:参考Herman(J.G.Herman,J.R.Graff,S.Myohanen,B.D.NelkinandS.B.Baylin,Methylation-specific PCR:a novel PCR assay formethylationstatus of CpG islands,Proc.Natl.Acad.Sci.USA93(1996),9821–9826.)等报道的方法加以改进。取上述制备的基因组DNA,经稀释后,应用紫外分光光度计测定DNA浓度,并精确取2μgDNA,加去离子水至终体积50μL,加入2M的NaOH 5.5μL,37℃金属浴15min。之后加入新鲜配制的10mM的氢醌30μL及3M亚硫酸氢钠520μL混合均匀,50℃孵育16h。此后应用DNA纯化试剂盒(promega公司产品)纯化并回收DNA,应用50μL去离子水洗脱DNA,向离心管内提前加入5.5μL 3M的NaOH,13000rpm,离心1min,室温静置4min。加入1μL糖原和17μL 7.5M乙酸铵,用3倍体积的无水乙醇沉淀DNA,最终硫化修饰后的DNA重新溶解在20μL去离子水中,得到DNA模板,立即使用或-20℃保存。
2、MS-PCR扩增
以甲基化引物对进行扩增的PCR反应体系,以总体积25μL计,包括:
模板(硫化修饰后的DNA):2μL;
甲基化引物(50pmol/μL):
上游引物(5'TCGTATGAGTTTAGAGGTCGCGATGTTC3'):0.5μL,
下游引物(5'TCTTCTACCAATCGAAAATTACTCTCCG 3'):0.5μL;
10×MSP buffer(缓冲液)2.5μL;
20mMdNTP:1.25μL;
Hot start taq酶:0.5μL;
去离子水:17.75μL。
以非甲基化引物对进行扩增的PCR反应体系,以总体积25μL计,包括:
与甲基化引物进行扩增的PCR反应体系所不同的是:引物不同,在该体系中为非甲基化引物(50pmol/μL):
上游引物(5'GTTTTGTATGAGTTTAGAGGTTGTGATGTTT 3'):0.5μL,
下游引物(5'ATTCTTCTACCAATCAAAAATTACTCTCCA 3'):0.5μL;
其余均与甲基化引物对进行扩增的PCR反应体系中的相同。
利用上面建立的MS-PCR反应体系对制备的硫化DNA模板:胰腺癌5例(PC1-PC5)、乳腺癌5例(BC1-BC5)、食管癌5例(EC1-EC5)、正常胰腺2例(PN1-PN2)、正常乳腺2例(BN1-BN2)、正常食管2例(EN1-EN2),甲基化阳性对照(IVD)和非甲基化对照(NL-正常外周血DNA),分别进行扩增,并以去离子水作为阴性对照,扩增程序为:95℃下预变性5min,接着进入循环,在95℃下变性30s,在60℃下退火30s,在72℃下延伸40s,共35个循环,之后,继续在72℃下延伸5min。
3.PCR反应产物的检测
PCR产物进行2%琼脂糖凝胶电泳,紫外透射分析仪检测并照相。
4.结果
结果见图1,用甲基化引物进行扩增,胰腺癌PC1、PC2、PC4,乳腺癌BC2、BC3、BC5,食管癌EC1、EC2、EC4、EC5有扩增产物;而正常胰腺(PN1-2)、正常乳腺(BN1-2)、正常食管(NE1-2),胰腺癌PC3、PC5,乳腺癌BC1、BC4,食管癌EC3、EC6、EC7无扩增产物。而用非甲基化引物进行扩增,全部标本组织均有扩增产物。因此,胰腺癌PC1、PC2、PC4,乳腺癌BC2、BC3、BC5,食管癌EC1、EC2、EC4、EC5为甲基化;正常胰腺(PN1-2)、正常乳腺(BN1-2)、正常食管(NE1-2),胰腺癌PC3、PC5,乳腺癌BC1、BC4,食管癌EC3、EC6、EC7为非甲基化。对照体系反应正常,结果可信。
实施例2临床标本检测
取胰腺癌临床标本204例,乳腺癌标本12例,食管癌标本67例,进行MS-PCR扩增,模板制备、PCR扩增体系及条件、扩增产物的检测均与实施例1中的相同,检测结果请见下表:
实施例3敏感度实验
将胰腺癌细胞系SW1990的DNA(BEND4基因启动子区100%甲基化)与正常淋巴细胞的DNA(BEND4基因启动子区100%非甲基化)按比例混合,进行硫化修饰(方法同实施例1),再进行MS-PCR。PCR产物进行2%琼脂糖凝胶电泳,紫外透射分析仪测定并照相。
分组:第1组:100%SW1990细胞DNA+0%正常细胞DNA
第2组:50%SW1990细胞DNA+50%正常细胞DNA
第3组:5%SW1990细胞DNA+95%正常细胞DNA
第4组:1%SW1990细胞DNA+99%正常细胞DNA
第5组:0.1%SW1990细胞DNA+99.9%正常细胞DNA
第6组:0%SW1990细胞DNA+100%正常细胞DNA
结果请见图2:在1000个正常细胞中有1个胰腺癌细胞就可以被检测出,即用本发明引物及反应体系、条件检测胰腺癌细胞BEND4基因启动子区的甲基化状态其灵敏度可达到0.1%,灵敏度较高。
实施例4ATM抑制剂结合顺铂等化疗药物杀伤肿瘤细胞的协同致死标志物的检测
将胰腺癌细胞SW1990(BEND4基因启动子区100%甲基化)与CFpac1(BEND4基因启动子区100%非甲基化)进行药物敏感性实验,通过设置不同的AZD0156浓度梯度SW1990(0,0.25,0.5,1,2,4,8,16,32,64μM)和CFpac1(0,0.078,0.156,0.313,0.625,1.25,2,5,10,20μM)处理48小时后使用MTT实验检测490nm波长下的吸光度值,计算细胞对ATM抑制剂(AZD0156)的敏感性,即IC50值(细胞的半数致死量)。检测结果见下表:
胰腺细胞 | IC50(μM) |
SW1990 | 0.19±0.07 |
CFpac1 | 8.62±1.31 |
实施例5诊断胰腺癌、乳腺癌或食管癌的试剂盒组成
试剂盒包括以下成分,其中,进行1次MS-PCR的用量为:
1、甲基化引物:核苷酸序列如SEQ ID No.2所示的上游引物,核苷酸序列如SEQ IDNo.3所示的下游引物,各为0.5μL。
2、非甲基化引物:核苷酸序列如SEQ ID No.4所示的上游引物,核苷酸序列如SEQID No.5所示的下游引物,各为0.5μL。
3、反应液2份,每份反应液包括:
(1)10x MSP buffer(缓冲液)2.5μL;
(2)20mMdNTP:1.25μL;
(3)Hot start taq酶:0.5μL;
(4)去离子水:17.75μL。
一个试剂盒可以包括上述各成分进行多次MS-PCR的用量,如25次、50次、100次等,各成分的具体量视情况需要而定。
为防止出现MS-PCR扩增产物的假阳性,试剂盒还可包括:
a、甲基化阳性对照PCR模板:经甲基转移酶处理过,硫化修饰后的BEND4基因启动子区为100%甲基化的肿瘤细胞DNA,进行1次MS-PCR其用量为:2μL。
b、非甲基化阳性对照PCR模板:硫化修饰后的BEND4基因启动子区为100%非甲基化的正常淋巴细胞DNA,进行1次MS-PCR其用量为:2μL。
c、双阴性的体系对照:ddH2O,用以评估体系是否存在PCR产物的污染,如ddH2O检测的结果为双阴性(M和U均为阴性),则体系结果可信。
显然,本发明的上述实施例仅是为清楚地说明本发明所作的举例,而并非是对本发明的实施方式的限定,对于所属领域的普通技术人员来说,在上述说明的基础上还可以做出其它不同形式的变化或变动,这里无法对所有的实施方式予以穷举,凡是属于本发明的技术方案所引伸出的显而易见的变化或变动仍处于本发明的保护范围之列。
SEQUENCE LISTING
<110> 中国人民解放军总医院第一医学中心
<120> 一种用于诊断癌症的分子标志物及试剂盒
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<213> BEND4基因启动子区DNA片段
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cgtcagtgca acacagccgc acactcccga gtgtctgact ttgggcaatt cccttcaacc 60
tctcagagcc tcggttttct catctttata atggggggga aatcatggtg tcatgcacgt 120
tctcggggcc cctacgggaa acaaatgaaa acgtgaaaaa gttctacgcc ttgcgagctt 180
ttatgttccg catgagctca gaggccgcga tgctcgggga aagcaggacc ccaaagcccc 240
gtaaacaccg cgcgaccacc cgggccaaga tcttcaagag gttcttttca gaaggatcgg 300
agagcaattc ccgattggta gaagaacttg ctgtaataca cacgtactct gacgaccccg 360
ccccaacgac tagcccctcc tctgtgcaac cccgagagtt tggggtcatg cagggggcgc 420
cacgagctcg tttcggaagc cggaccccgc ccgcagccgc agaagcctcg agtccacatc 480
tgggtctcta aagtctcgcc gtagccagat cccggatccc caccttcttc accagttccc 540
gggcgtcctc caggtcctcg ctttccccct tcccccgctc cccgtcggag gcgcggagcc 600
gccaggcaga ccccgcgggc ggctgcggcg ccaggccccg ccccgccccc attatcatta 660
gcagatatta ccctaaacac ttgctcttta cctcctttct ccctccaggc attggcgagg 720
cagcctgtca atcaggagct cgggcggcag ccccccgcgc gggggctcgg cgatgccagc 780
ctcagcgaca ggcggcggcg gcggcggcca cggcacagac acacaccctc ccacacgcgc 840
gcaccagggc agacccggcg ggcaggcggc ggaggcaccc tcggagcccg gcgcccggcg 900
gggaggggac gtgctccgag ggaccggccc cgaggcgccg gatggaggaa gagatgcagc 960
cggcagagga ggggcccagc gtccccaaaa tctacaagca gcgcagcccc tacagcgtcc 1020
tcaagacgtt ccccagcaag agaccggcgc tggccaagcg ctacgagcga cccaccctgg 1080
tggagctgcc gcacgtgcgg gcgcccccgc cgcccccgcc gcccttcgcg ccgcacgccg 1140
ccgtctccat cagcagcagc gagccgccgc cgcagcagtt ccaggcgcag agctcctacc 1200
cccccgggcc cggccgggcc gccgccgccg cttcgtcgtc gtcgccgtcc tgcacgcccg 1260
ccacatccca gggccacttg aggactccgg cgcagccgcc gcccgcgtcc cccgccgcct 1320
cctcgtcgtc ttcgttcgcc gctgtcgtca ggtatggccc aggcgcggcg gcggccgccg 1380
gcaccggcgg cacgggtagc gacagcgcca gcctggagct cagcgcaggt accaactctc 1440
cgcacaactt tccttcccgc cacggccggg ggcagcggga ggggacccag gaaggagaag 1500
cggggggccg ccgacactta cccgtgctgg gccgtgtcgg gatgtcgggg cgcacctggg 1560
gcctccccgc gcacccggaa acttcgagag gattctccag cgccgccgcg cctgggcttc 1620
cgacggcgcg cgcctaacag gtggctccgc ggcttggatc ccgcgccggg aagcacggct 1680
gtggcagtcg gcgaccagga tcgctcgggc gtggtccgga gtggggccag acgccccccc 1740
gggggatagc ggcggccgct cgggaagaag ctggagcttc tcgggccgcc ccgctgggac 1800
gccctcg 1807
<210> 2
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tcgtatgagt ttagaggtcg cgatgttc 28
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attcttctac caatcaaaaa ttactctcca 30
Claims (10)
1.BEND4基因启动子区DNA片段作为分子标志物在制备诊断癌症产品中的应用,其特征在于,所述癌症为胰腺癌、食管癌或乳腺癌。
2.根据权利要求1所述的应用,其特征在于,所述BEND4基因启动子区DNA片段的核苷酸序列如SEQ ID No.1所示,且包含至少一个甲基化位点。
3.检测BEND4基因启动子区DNA片段甲基化状态的物质在制备诊断癌症产品中的应用,其特征在于,所述癌症为胰腺癌、食管癌或乳腺癌。
4.根据权利要求3所述的应用,其特征在于,所述BEND4基因启动子区DNA片段的核苷酸序列如SEQ ID No.1所示,且包含至少一个甲基化位点。
5.一种诊断癌症的试剂盒,其特征在于:包括用于检测BEND4基因启动子区甲基化状态的物质,所述癌症为胰腺癌、食管癌或乳腺癌。
6.根据权利要求5所述的试剂盒,其特征在于,所述用于检测BEND4基因启动子区甲基化状态的物质针对BEND4基因启动子区核苷酸序列如SEQ ID No.1所示的DNA序列。
7.根据权利要求6所述的试剂盒,其特征在于,所述物质包括甲基化引物对和非甲基化引物对;优选的,所述甲基化引物对的上游引物核苷酸序列如SEQ ID No.2所示,下游引物核苷酸序列如SEQ ID No.3所示;所述非甲基化引物对的上游引物核苷酸序列如SEQ IDNo.4所示,下游引物核苷酸序列如SEQ ID No.5所示。
8.根据权利要求6所述的试剂盒,其特征在于,所述试剂盒还包括甲基化阳性对照PCR模板和非甲基化阳性对照PCR模板。
9.根据权利要求8所述的试剂盒,其特征在于,所述甲基化阳性对照PCR模板为硫化修饰后的BEND4基因启动子区100%甲基化的肿瘤细胞DNA;所述非甲基化阳性对照PCR模板为硫化修饰后的BEND4基因启动子区100%非甲基化的正常淋巴细胞或正常组织细胞DNA。
10.根据权利要求6所述的试剂盒,其特征在于,所述试剂盒还包括MS-PCR反应所需试剂。
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