CN117604109A - 用于膀胱癌诊断和预后判断的生物标志物及其应用 - Google Patents
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Abstract
本发明涉及生物医药技术领域,特别是涉及一种用于膀胱癌诊断和预后判断的生物标志物及其应用。更具体地,本发明涉及一种用于膀胱癌的诊断和预后判断的生物标志物,所述生物标志物是KANK2和/或TBX1。本发明人已发现,KANK2在膀胱癌患者中的表达水平明显低于健康人群中的表达水平,并且TBX1在膀胱癌患者中的表达水平明显高于健康人群中的表达水平。更特别地,本发明人发现,KANK2的低水平表达和/或TBX1的高水平表达在用于诊断膀胱癌时具有良好的灵敏度和特异性,并且与膀胱癌患者的预后相关,因此可以作为用于膀胱癌诊断和预后判断的生物标志物。
Description
技术领域
本发明涉及生物医药技术领域,特别是涉及一种用于膀胱癌诊断和预后判断的生物标志物及其应用。
背景技术
膀胱癌是泌尿系统最常见的恶性肿瘤之一,严重威胁着生命健康。从组织病理学上讲,膀胱癌包括尿路上皮膀胱癌、鳞状细胞癌、腺细胞癌、脐尿管癌等。其中膀胱尿路上皮癌最为常见,约占膀胱癌的90%以上。根据肿瘤是否浸润膀胱肌层,膀胱癌分为非肌肉浸润性疾病(NMIBC)和肌肉浸润性疾病(MIBC)。几乎80%的膀胱癌表现为非肌肉浸润性疾病,其中约60%的疾病局限于膀胱粘膜,约30%的疾病侵入膀胱粘膜下层,约10%的疾病表现为原位癌。膀胱癌的5年生存率与确诊时的疾病分期有关,原位癌的5年生存率高达95.8%,而转移性膀胱癌的5年生存率低至4.6%,这说明准确、及时的诊断对于膀胱癌患者的预后是至关重要的。
临床上已发现多种可以用于膀胱癌诊断、病理分型和临床分期、判断预后和疗效的肿瘤标志物,但目前常用的膀胱癌标志物的诊断效能均不理想,尚未发现一种特异的肿瘤标志物对膀胱癌诊断有较高的敏感度以及特异性。
循环肿瘤细胞是从原发肿瘤或者转移肿瘤上脱落下来、释放进入血液循环中的一群肿瘤细胞亚群。近些年的研究发现,一方面,循环肿瘤细胞在肿瘤发生的很早期就可能出现在患者的外周血中,这为癌症的早期诊断提供了帮助。另一方面,这些循环肿瘤细胞还可以用来预测癌症患者的预后,循环肿瘤细胞的发现往往预示着肿瘤的复发或转移,这也提示患者的预后不良。如何使用循环肿瘤细胞来对癌症尤其是特定癌症例如膀胱癌进行诊断或者预后,这也是我们未来在循环肿瘤细胞系列探索中的一个重要方向。使用循环肿瘤细胞进行诊断或预后的一大好处是其可以有效替代肿瘤活检,对于那些无法进行病理组织活检的患者,这是一个很好的替代指标,它可以帮助临床医生实时地对癌症的生物学特征进行动态监测和判断。然而,由于循环肿瘤细胞数量稀少,利用其作为诊断癌症尤其是特定癌症例如膀胱癌的手段存在挑战,并非所有的癌症相关标志物都可以在循环肿瘤细胞中被检测到。
因此,寻找新的膀胱癌诊断相关标志物尤其是适用于借助循环肿瘤细胞进行诊断的生物标志物具有重要的临床价值。
发明内容
为了解决上述问题,本发明人已发现,KANK2在膀胱癌患者中的表达水平明显低于健康人群中的表达水平,并且TBX1在膀胱癌患者中的表达水平明显高于健康人群中的表达水平。更特别地,本发明人发现,KANK2的低水平表达和/或TBX1的高水平表达在用于诊断膀胱癌时具有良好的灵敏度和特异性,并且与膀胱癌患者的预后相关,因此可以作为用于膀胱癌诊断和预后判断的生物标志物。
如本文所用,KANK2是KN 基序和锚蛋白重复结构域 2(KN motif and ankyrinrepeat domains 2,KANK2)的简称,其NCBI Gene ID为25959。
如本文所用,TBX1是T-box转录因子1(T-box transcription factor 1,TBX1)的简称,其NCBI Gene ID为6899。
具体地,本发明提供了一种用于膀胱癌诊断的生物标志物,其中所述生物标志物是KANK2和/或TBX1。
在其他方面,本发明还提供了一种用于膀胱癌预后判断的生物标志物,其中所述生物标志物是KANK2和/或TBX1。
在其他方面,本发明还提供了一种用于膀胱癌诊断的试剂盒,所述试剂盒包含用于检测KANK2和/或TBX1表达的试剂。
在其他方面,本发明还提供了一种用于膀胱癌预后判断的试剂盒,所述试剂盒包含用于检测KANK2和/或TBX1表达的试剂。
在其他方面,本发明还提供了检测KANK2和/或TBX1表达的试剂在制备用于膀胱癌的诊断的工具中的用途。
在其他方面,本发明还提供了检测KANK2和/或TBX1表达的试剂在制备用于膀胱癌的预后判断的工具中的用途。
进一步地,所述膀胱癌的诊断包括以下步骤:
(1)收集待测受试者的样本,和收集对照样本;
(2)检测并比较待测受试者样本和对照样本中KANK2和/或TBX1的表达水平;
如果待测受试者的样本中KANK2的表达水平与对照样本中KANK2的表达水平相比降低,和/或待测受试者的样本中TBX1的表达水平与对照样本中TBX1的表达水平相比升高,则诊断所述待测受试者患有膀胱癌或处于患有膀胱癌的风险中。
进一步地,所述对照样本来源于健康人群或待测受试者的健康组织。
进一步地,所述膀胱癌的预后判断包括以下步骤:
(1)收集预后膀胱癌患者的样本为待测组,并以预前膀胱癌患者的样本为对照组;
(2)检测并比较待测组和对照组的样本中KANK2和/或TBX1的表达水平;
如果待测组样本中KANK2的表达水平与对照组样本中KANK2的表达水平相比升高,和/或待测组样本中TBX1的表达水平与对照组样本中TBX1的表达水平相比降低,则判断待测组的预后良好。
如本文所用,所述受试者包括哺乳动物,优选为灵长类哺乳动物,更优选为人。
如本文所用,所述待测受试者的样本包括受试者的临床生物样本,包括但不限于血清、血浆、全血、分泌物、棉拭子、脓液、体液、组织、器官、石蜡切片、肿瘤组织、活检样本、循环肿瘤细胞、循环肿瘤DNA或尿液脱落细胞中的一种或几种。在优选的实施方案中,所述待测受试者的样本包括待测受试者的膀胱组织,例如膀胱活检样本,所述对照样本来源于健康受试者的膀胱组织,例如膀胱活检样本,或待测受试者的健康组织,例如癌旁组织。在优选的实施方案中,所述待测受试者的样本为循环肿瘤细胞。
如本文所用,所述预后和预前膀胱癌患者的样本包括受试者的临床生物样本,包括但不限于血清、血浆、全血、分泌物、棉拭子、脓液、体液、组织、器官、石蜡切片、肿瘤组织、活检样本、循环肿瘤细胞、循环肿瘤DNA或尿液脱落细胞中的一种或几种。在优选的实施方案中,所述预后和预前膀胱癌患者的样本包括待测受试者的膀胱组织,例如膀胱活检样本。在优选的实施方案中,所述预后和预前膀胱癌患者的样本为循环肿瘤细胞。
如本文所用,检测待测受试者的样本中KANK2和/或TBX1的表达的试剂没有特别限制,并且是本领域技术人员熟知且容易获得的用于检测受试者样本中KANK2和/或TBX1在mRNA或蛋白质水平上的表达的试剂。例如,用于检测受试者样本中KANK2和/或TBX1的表达的试剂可以包括用于实时荧光定量PCR、酶联免疫吸附法(ELISA)、蛋白/肽段芯片检测、化学发光、免疫印迹、微珠免疫检测、微流控免疫的相应试剂。
本发明的有益效果
本发明人已发现,KANK2在膀胱癌患者中的表达水平明显低于健康人群中的表达水平,并且TBX1在膀胱癌患者中的表达水平明显高于健康人群中的表达水平。更特别地,本发明人发现,KANK2的低水平表达和/或TBX1的高水平表达,尤其是这两者在联合使用时,在用于诊断膀胱癌时具有良好的灵敏度和特异性,并且与膀胱癌患者的预后相关,因此可以作为用于膀胱癌诊断和预后判断的生物标志物。进一步,本发明还发现可以通过收获受试者的循环肿瘤细胞并检测其中KANK2和TBX1的表达水平来对膀胱癌进行诊断和预后判断。
附图说明
图1显示了KANK2和TBX1在膀胱癌组织样本和癌旁正常组织样本中的表达水平。
图2显示了KANK2和TBX1在膀胱癌患者循环肿瘤细胞中的表达水平。
图3显示了KANK2和TBX1在人膀胱癌细胞T24和人正常膀胱上皮细胞SV-HUC-1中的表达水平。
图4显示了在过表达KANK2和/或干扰TBX1表达后,人膀胱癌细胞T24的迁移能力和侵袭能力的变化。
图5显示了KANK2和TBX1单独和联合地在膀胱癌患者与健康人群中的ROC曲线分析。
图6显示了KANK2和TBX1单独和联合地在膀胱癌患者与健康人群中的Kaplan-Meier生存曲线分析。
具体实施方式
以下结合具体实施例来进一步说明本发明,但实施例并不对本发明做任何形式的限定。除非特别说明,本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。
实施例1:人膀胱癌与配对正常组织的表达谱芯片分析
肿瘤基因组图谱(TCGA)计划由美国National Cancer Institute(NCI)和National Human Genome Research Institute(NHGRI)于2006年联合启动的项目,利用大规模测序为主的基因组分析技术,针对36种癌症进行大规模实验,TCGA基因组分析中心(GCC)比对肿瘤和正常组织,寻找与各癌症或者亚型相关的基因突变、扩增或者缺失。为理解癌症的分子机制,提高人们对癌症发病分子基础的科学认识提供帮助。
TCGA标准方法下载110对膀胱癌组织和正常组织的全基因表达谱数据及临床信息,统计分析采用R语言(3.1.1版本)软件,需安装及加载的程序包(heatmap,venndiagram,hist等),然后用DESeq和edgeR程序包进行分析,找出差异表达的基因。判断标准:(1)癌/癌旁的表达量<-2,(2)P<0.05,(3)在膀胱癌中未被报道。最终筛选到在膀胱癌中显著低表达的基因KANK2和在膀胱癌中显著高表达的基因TBX1。
实施例2:KANK2在膀胱癌中低表达并且TBX1在膀胱癌中高表达
收集临床58例膀胱癌组织样本和34例癌旁正常组织样本,分别使用TRIzol法提取上述膀胱癌组织样本和癌旁正常组织样本的RNA,利用RT-qPCR方法分别检测KANK2和TBX1的mRNA水平。结果如图1所述,其证实KANK2在膀胱癌中低表达并且TBX1在膀胱癌中高表达。
实施例3:膀胱癌患者的循环肿瘤细胞中KANK2和TBX1表达水平的检测
1)抽取膀胱癌患者静脉血10mL于ACD抗凝管中,常规离心分离血浆备用;
2)对血浆中CTC细胞进行富集及分离,具体步骤为:通过向血浆中加入样本密度分离液(Cytelligen)提取血浆中单细胞层,之后加入免疫细胞去除磁珠对所提取单细胞层中的CD45+免疫细胞进行去除,通过差向富集对单细胞层中CTC进行浓缩及富集;
3)离心收获富集的CTC细胞,加入1ml的RNA裂解液吸至无酶EP管中;向EP管中加入200ul氯仿,剧烈震荡15秒,室温静止3min,反复3次;12000×g、4℃离心15min;将上层水相加入新的无酶的EP管,在EP管中加入等体积异丙醇,颠倒、混匀,静置10min;12000×g、4℃离心15min;弃掉EP管液体,加入75%的乙醇1ml,震荡EP管;12000×g、4℃离心5min;弃净上清后室温放置干燥;加入适量DEPC水溶解RNA;检测RNA的纯度和浓度并通过RT-qPCR检测CTC细胞中KANK2和TBX1的表达,并将其与收获自正常膀胱组织的细胞中KANK2和TBX1的表达进行比较,结果如图2所述,其证实KANK2在膀胱癌患者CTC细胞中低表达并且TBX1在膀胱癌患者CTC细胞中高表达。
实施例4:KANK2和TBX1影响膀胱癌细胞的侵袭及迁移
将人膀胱癌细胞T24和人正常膀胱上皮细胞SV-HUC-1用含10%胎牛血清的RPMI-1640培养基(含100U·mL-1青霉素和0.1mg·mL-1链霉素),于37℃,5%CO2恒温培养箱中培养。
将培养的细胞消化收集后如实施例3所述提取RNA并通过RT-qPCR检测乳腺细胞及癌细胞中KANK2和TBX1的表达。结果如图3所示,其显示KANK2在人膀胱癌细胞T24中的表达显著低于在人正常膀胱上皮细胞SV-HUC-1中的表达,并且TBX1在人膀胱癌细胞T24中的表达显著高于在人正常膀胱上皮细胞SV-HUC-1中的表达。
构建KANK2的cDNA序列(SEQ ID No.1),连接于稳定表达质粒并进行病毒包装和转染膀胱癌细胞以得到过表达KANK2的膀胱癌细胞,通过siRNA干扰TBX1在膀胱癌细胞中的表达(siRNA序列:siNC:5’-UUCUCCGAACGUGUCACGUUCAUACTT-3’(SEQ ID No.2);siTBX1:5’-UCCAGCCCGUGGCUCACGCAGCUCUTT-3’(SEQ ID No.3)),然后进行Transwell细胞迁移及侵袭实验验证。结果如图4所示,其显示在过表达KANK2或干扰TBX1基因的表达后,人膀胱癌细胞T24的迁移能力和侵袭能力显著下降,其中在过表达KANK2的同时干扰TBX1的表达时下降更为显著。
实施例5:KANK2和TBX1的膀胱癌诊断价值
利用实施例2测定的KANK2和TBX1在膀胱癌组织样本和癌旁正常组织样本中的mRNA水平通过受试者工作曲线(ROC)对KANK2和TBX1的独立诊断及联合诊断实验结果进行分析。结果如图5所示,其显示KANK2(灵敏度73.81%,特异性54.76%)和TBX1(灵敏度52.38%,特异性95.24%)mRNA表达对膀胱癌具有独立诊断效果,但联合诊断效果更佳,ROC曲线下面积AUC(area under the ROC curve)=0.9235,灵敏度可达78.57%,特异性可达97.62%。从此结果可知,单独的KANK2诊断灵敏度较高但特异性不足,单独的TBX1诊断特异性较高但灵敏度不足,而两者联合使用时可以实现较高的灵敏度和特异性。因此,KANK2和TBX1可以用于单独和联合诊断膀胱癌。
实施例6:KANK2和TBX1与膀胱癌临床预后的关系
利用实施例2测定的KANK2和TBX1在膀胱癌组织样本和癌旁正常组织样本中的mRNA水平,统计分析KANK2和TBX1与膀胱癌患者的整体生存率的关系。结果如图6所示,可以看出,在KANK2高表达(KANK2 High+TBX1 High)和TBX1低表达(KANK2 Low +TBX1 Low)的膀胱癌患者组的五年总体生存率明显高于KANK2低表达且TBX1高表达(KANK2Low+TBX1High)的膀胱癌患者组,并且KANK2高表达且同时TBX1低表达(KANK2 High+TBX1 Low)的膀胱癌患者组的五年总体生存率最高。这说明:KANK2低表达和/或TBX1高表达会导致膀胱癌患者的不良预后。
需要说明的是,本发明的说明书及其附图中给出了本发明的较佳的实施例,但是,本发明可以通过许多不同的形式来实现,并不限于本说明书所描述的实施例,这些实施例不作为对本发明内容的额外限制,提供这些实施例的目的是使对本发明的公开内容的理解更加透彻全面。并且,上述各技术特征继续相互组合,形成未在上面列举的各种实施例,均视为本发明说明书记载的范围;进一步地,对本领域普通技术人员来说,可以根据上述说明加以改进或变换,而所有这些改进和变换都应属于本发明所附权利要求的保护范围。
Claims (10)
1.一种用于膀胱癌诊断的生物标志物,其特征在于,所述生物标志物是KANK2和/或TBX1。
2.一种用于膀胱癌预后判断的生物标志物,其特征在于,所述生物标志物是KANK2和/或TBX1。
3.一种用于膀胱癌诊断的试剂盒,其特征在于,所述试剂盒包含用于检测KANK2和/或TBX1表达的试剂。
4.一种用于膀胱癌预后判断的试剂盒,其特征在于,所述试剂盒包含用于检测KANK2和/或TBX1表达的试剂。
5.检测KANK2和/或TBX1表达的试剂在制备用于膀胱癌的诊断的工具中的用途。
6.检测KANK2和/或TBX1表达的试剂在制备用于膀胱癌的预后判断的工具中的用途。
7.根据权利要求5所述的用途,其特征在于,所述膀胱癌的诊断包括以下步骤:
(1)收集待测受试者的样本,和收集对照样本;
(2)检测并比较待测受试者样本和对照样本中KANK2和/或TBX1的表达水平;
如果待测受试者的样本中KANK2的表达水平与对照样本中KANK2的表达水平相比降低,和/或待测受试者的样本中TBX1的表达水平与对照样本中TBX1的表达水平相比升高,则诊断所述待测受试者患有膀胱癌或处于患有膀胱癌的风险中。
8.根据权利要求7所述的用途,其特征在于,所述对照样本来源于健康人群或待测受试者的健康组织,
所述待测受试者的样本为血清、血浆、全血、分泌物、棉拭子、脓液、体液、组织、器官、石蜡切片、肿瘤组织、活检样本、循环肿瘤细胞、循环肿瘤DNA或尿液脱落细胞中的一种或几种。
9.根据权利要求6所述的用途,其特征在于,所述膀胱癌的预后判断包括以下步骤:
(1)收集预后膀胱癌患者的样本为待测组,并以预前膀胱癌患者的样本为对照组;
(2)检测并比较待测组和对照组的样本中KANK2和/或TBX1的表达水平;
如果待测组样本中KANK2的表达水平与对照组样本中KANK2的表达水平相比升高,和/或待测组样本中TBX1的表达水平与对照组样本中TBX1的表达水平相比降低,则判断待测组的预后良好。
10.根据权利要求9所述的用途,其特征在于,所述预后和预前膀胱癌患者的样本为血清、血浆、全血、分泌物、棉拭子、脓液、体液、组织、器官、石蜡切片、肿瘤组织、活检样本、循环肿瘤细胞、循环肿瘤DNA或尿液脱落细胞中的一种或几种。
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