CN113637753A - 基于尿液外泌体的膀胱癌标志物lncRNA TERC及其应用 - Google Patents
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Abstract
本发明涉及基于尿液外泌体的膀胱癌标志物lncRNA TERC及其应用,本发明中的靶标分子lncRNA TERC在膀胱癌诊断中兼具了高灵敏度和特异性,具有优异的诊断性能,填补了临床上膀胱癌液体活检诊断标志物的空白,并且样本类型为尿液,无创易获取,患者依从性高。总的来说,本发明为临床提供了一种特异性强、灵敏度高的膀胱癌液体活检诊断标志物。
Description
技术领域
本发明涉及生物医药技术领域,具体地说,涉及基于尿液外泌体的膀胱癌标志物lncRNA TERC及其应用。
背景技术
膀胱癌(bladder cancer,BLCA)是泌尿***常见恶性肿瘤之一,随着人口老龄化加剧、工业化进程加快,膀胱癌的发病率呈逐年增长的趋势。据世界卫生组织国际癌症研究署统计,2020年膀胱癌的发病率占全部恶性肿瘤第十位,其发病率和死亡率均居男性泌尿***恶性肿瘤第二位,仅次于***癌。世界范围内,膀胱癌的发病率呈明显的地区和性别差异。全球年龄标准化发病率男、女性分别为9.0/10万人、2.2/10万人,在欧盟国家中比利时的年龄标准化发病率最高,男、女性分别为31/10万人、6.2/10万人,最低者为芬兰,男、女性分别为18.1/万人、4.3/10万人。膀胱癌分为非肌肉浸润性和肌肉浸润性,80%左右的膀胱癌是非肌肉浸润性,预后较好,但是复发率高达70%,至少1/3的非肌肉浸润性膀胱癌会进展为肌肉浸润性,即使此时进行根治性膀胱全切除手术并辅以放化疗,远处转移的风险也极高且预后不良,五年生存率仅为5%。因此,探索一种膀胱癌快速诊断的方法为临床诊疗提供实验室依据,从而提高患者存活率,仍是当前研究的热点与难点。
目前临床上膀胱癌的诊断金标准仍然是膀胱镜病理活检,但是该方法有创、昂贵,很难让患者做到定期复查随访,患者依从性较差。其他辅助诊断方法主要有NMP-22、BTA、FISH、尿脱落细胞学、影像学检查等。但是,上述检测方法仍存在一定的局限性,如NMP-22胶体金法灵敏度仅45.9%,并且大多数膀胱癌患者的首发症状是无痛性肉眼血尿,而NMP-22容易受血尿影响引起结果假阳性;传统的尿脱落细胞学临床检出率低;影像学检查无法分辨肿瘤分期、分级。因此,寻找一种兼具特异性和灵敏度的、无创的膀胱癌诊断标志物,具有极其重要的意义。
近年来,从体液中寻找一种循环肿瘤标志物已经成为一种新兴的潮流,外泌体作为胞间通讯的信使更是被研究人员寄予厚望。外泌体是直径约30-200nm的杯形小囊泡,具有特定的表面分子特征,它可将蛋白质、脂质和核酸(miRNA、lncRNA、circRNA)等生物活性分子转移到受体细胞,改变受体细胞的生物学行为,在肿瘤发生发展过程中发挥改变肿瘤微环境,介导肿瘤细胞增殖、转移和耐药,促进血管生成等作用。不同于循环肿瘤细胞(CTC)需要新鲜样本才能进行研究,外泌体在保存良好的陈旧样本中仍然形态稳定。从人体体液中提取的裸露的lncRNA极易受RNA酶的影响发生降解,而外泌体中的lncRNA却可以在脂质双分子膜的保护作用下在全身体液稳定循环。以上证据说明了外泌体lncRNA在体液中的稳定性,其作为临床诊断、治疗及预后标志物可能具有广阔的应用前景。
中国专利文献CN112680522A公开了一种检测膀胱癌的试剂盒,所述试剂盒包括检测尿液脱落细胞中CA9、CCL18、ERBB2、IGF2、LINC00565、MMP12、PPP1R14D和SGK2等8个基因表达水平的试剂。中国专利文献CN112834644A公开了一种膀胱癌相关的组合标志物和检测试剂盒,所述试剂盒提供以3-甲基胞苷,1-甲基腺苷,假尿嘧啶核苷,1-甲基鸟苷,3-甲基尿苷,1-甲基次黄苷,N4-乙酰胞苷和N,N-二甲基鸟苷八种修饰核苷作为组合标志物用于膀胱癌诊断和复发监测。本发明人前期发表的论文“陈晨,商安全,权文强,孙祖俊,李冬.外泌体lncRNA在肿瘤中的研究进展[J].国际检验医学杂志,2021,42(03):360-365.”和“常文婧,李冬,孙祖俊.外泌体长链非编码RNA:肿瘤分子诊断新型标志物[J].国际检验医学杂志,2020,41(15):1793-1799.”中公开了外泌体lncRNA除了作为疾病早期诊断、治疗监测和预后评估等生物标志物外,还可作为肿瘤的治疗靶点,为肿瘤的精准诊疗提供一个新的方向。但是关于本发明基于尿液外泌体的膀胱癌标志物lncRNA TERC及其应用目前还未见报道。
发明内容
本发明的目的是针对现有技术中的不足,提供基于尿液外泌体的膀胱癌标志物lncRNA TERC及其应用。
为实现上述目的,本发明采取的技术方案是:
检测lncRNATERC含量的试剂在制备膀胱癌诊断的试剂或试剂盒中的应用。
优选地,检测样品为组织、尿液外泌体或血浆外泌体。
优选地,所述检测lncRNATERC含量的试剂为特异性识别或扩增lncRNA TERC的试剂。
优选地,所述检测lncRNA TERC含量的试剂为lncRNA TERC的特异性探针或引物。
优选地,所述引物序列为SEQ ID NO:1、SEQ ID NO:2或其完全互补序列。
优选地,所述试剂盒还包括:特异性检测内参基因的检测试剂。
优选地,所述内参基因为18S rRNA。
更优选地,所述18S rRNA的引物序列为SEQ ID NO:3、SEQ ID NO:4或其完全互补序列。
优选地,所述试剂盒还包括:RNA提取试剂、反转录试剂反应试剂、PCR扩增试剂和/或使用说明书。
本发明将膀胱癌患者和健康人尿液进行高通量测序,筛选出差异表达的lncRNATERC,并在尿液外泌体中进行大样本验证,发现lncRNA TERC在膀胱癌患者和健康人尿液外泌体中差异表达并具有一定的诊断价值。本发明优点在于:
1、本发明中的靶标分子lncRNA TERC兼具了高灵敏度(73.44%)和特异性(77.66%),ROC曲线下面积0.811,具有优异的诊断性能,填补了临床上膀胱癌液体活检诊断标志物的空白。
2、本发明的样本类型为尿液,无创易获取,患者依从性高。尿液样本仅需8ml,且取随机尿即可,不受采集时间的影响。
附图说明
图1.差速超速离心法提取尿液外泌体的实验流程。
图2.长链RNA差异表达热图。
图3.长链RNA差异表达火山图。
图4.膀胱癌患者组织、血浆exo、尿液exo中lnc TERC的相对表达水平。
图5.引物熔解曲线。
图6.尿液外泌体lnc TERC的ROC曲线。
具体实施方式
下面结合附图对本发明提供的具体实施方式作详细说明。
实施例1外泌体靶标分子lncRNA——TERC的筛选和提取
1.本发明首先将从上海市同济医院获取的4个临床病理确诊为膀胱癌的患者尿液、3个健康人尿液样本进行外泌体抽提。差速超速离心法提取尿液外泌体的实验流程图,见图1。
提取好的外泌体按照ISEV2018标准,采用透射电镜(TEM)、纳米粒径跟踪分析技术(NTA)和western blot技术进行严格鉴定,鉴定合格的外泌体样本才能进行下游分析。
2.提取外泌体RNA的具体步骤如下:
(1)在外泌体-PBS混合物中加入500μl无水trizol,震荡混匀后室温静置5min使其充***解;
(2)加入100μl氯仿,震荡混匀后室温放置10min,4℃,12000g离心15min;
(3)吸取上层水相至一个新EP管中,切勿吸到下层溶剂;
(4)加入200μl异丙醇混匀,室温静置10min;
(5)4℃,12000g离心10min,弃上清;
(6)加入500μl的75%乙醇,轻柔颠倒以悬浮沉淀;
(7)4℃,8000g离心10min,弃上清,室温晾干5min,底部白色沉淀即为RNA,加入10-20μlDEPC水溶解RNA;
(8)60℃煮10min,并进行RNA质检。
3.由上海康成生物科技有限公司进行高通量测序,具体流程如下:通过NanoDrop2000进行浓度和纯度测定,取至少1μg总RNA进行oligo dT富集(rRNA去除)处理后,按照标准流程构建文库,使用Illumina Novaseq 6000测序仪进行测序。分析并计算基因水平和转录本水平表达差异情况,筛选出实验组与对照组间差异表达的基因,比较方案为fold change>1.5,P value(F test)≤0.05,且组内FPKM均值≥0.5。
长链RNA差异表达热图见图2,长链RNA差异表达火山图见图3,最终筛选得到差异表达的lncRNA TERC,log2FC=3.892,P value=0.045。
4.我们在膀胱癌和癌旁组织样本,血浆外泌体及尿液外泌体样本中分别检测lncTERC的相对表达水平。组织和血浆样本来自:上海市同济医院泌尿外科;尿液样本来自:上海市同济医院和复旦大学附属肿瘤医院。发现lnc TERC在肿瘤患者的组织、血浆外泌体和尿液外泌体中均表现为上调,并且血浆外泌体中lnc TERC个体差异大,lnc TERC在尿液外泌体中比在血浆外泌体中表达更加稳定(图4)。同时尿液外泌体是一种无创检测指标,为临床医生诊疗与患者随访带来更大的便利性。对于外泌体样本我们采用18S作为qPCR的内参,TERC与18S的引物序列如下:
TERC-F:GTGGTGGCCATTTTTTGTCTAAC(SEQ ID NO:1)
TERC-R:TGCTCTAGAATGAACGGTGGAA(SEQ ID NO:2)
18S-F:CGTTCTTAGTTGGTGGAGCG(SEQ ID NO:3)
18S-R:CGCTGAGCCAGTCAGTGTAG(SEQ ID NO:4)
引物熔解曲线如图5所示。
因此,尿液外泌体lncTERC可以作为一种极佳的生物标志物用于诊断膀胱癌。ROC曲线如图6所示,lncTERC在膀胱癌患者肿瘤组织中(相比正常/癌旁组织)高表达,在膀胱癌患者血浆外泌体和尿液外泌体中同样高表达。尤其是尿液外泌体中显著上调。诊断阈值5.011,诊断灵敏度73.44%,特异度77.66%,曲线下面积(AUC)0.811,诊断性能优于目前临床使用诊断标志物NMP-22、BTA,并且尿液是一种无创、易获取样本,真正意义上实现了无创“液体活检”。
实施例2基于尿液外泌体lncRNA TERC诊断膀胱癌的方法
1.尿液外泌体提取(PEG沉淀法)
(1)用15ml无菌离心管收集8ml待提取的尿液样品,置4度保存;
(2)将样品4℃,3000g离心15min,弃沉淀收取上清;
(3)小心将上清移入另一干净15ml离心管中;
(4)将样品4℃,10000g离心20min,弃沉淀收取上清;
(5)小心将上清移入另一干净15ml离心管中;
(6)在8ml尿液上清中加入2ml PEG6000(比例为4:1),盖紧离心管盖,上下颠倒混匀1min左右,使液体充分混匀,将液体均匀分装至5个2mlEP管中;
(7)置4℃冰箱静置过夜(至少10小时);
(8)将样品4℃,10000g离心60min,小心除净上清,勿触及侧壁的半透明凝胶状沉淀,此即为外泌体。
2.外泌体RNA提取
(1)在EP管中分别加入100μl无水Trizol,共5个EP管;震荡混匀后室温静置5min使其充***解外泌体;瞬时离心,将5个EP管中Trizol全部移至1个1.5mlEP管中,此时总体积500μl;
(2)加入100μl氯仿,震荡混匀后室温放置10min;4℃,12000g离心15min;
(3)吸取上层水相至一个新EP管中,切勿吸到下层溶剂;
(4)加入200μl异丙醇后颠倒混匀,瞬时离心后再加入1μl糖原,颠倒混匀,室温静置10min;
(5)4℃,12000g离心10min,弃上清;
(6)加入500μl的75%乙醇,轻柔颠倒以悬浮沉淀;
(7)4℃,8000g离心10min,弃上清,室温晾干5min,底部白色沉淀即为RNA,加入10μl DEPC水溶解RNA;
(8)60℃煮10min,并测定RNA浓度纯度。
3.qRT-PCR(Takara RR036A)
(1)配制逆转录体系:
试剂 | 使用量 |
5×PrimeScript RT Master Mix(Perfect Real Time) | 2μl |
Total RNA | 10μl |
瞬时离心混匀后进行反转录反应:
37℃,15min(反转录反应);
85℃,5s(反转录酶的失活反应);
4℃。
(2)选择SYBR Green Premix Pro Taq HS qPCR KitⅡ(货号AG11702)进行RealTime PCR反应:
配制PCR体系:
瞬时离心混匀后进行PCR反应:
按照诊断阈值5.011,如尿液外泌体中lncRNA TERC相对于18S rRNA的相对表达量>5.011,则患有膀胱癌;反之,尿液外泌体中lncRNA TERC相对于18S rRNA的相对表达量≤5.011,则不患有膀胱癌。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员,在不脱离本发明方法的前提下,还可以做出若干改进和补充,这些改进和补充也应视为本发明的保护范围。
SEQUENCE LISTING
<110> 上海市同济医院
<120> 基于尿液外泌体的膀胱癌标志物lncRNA TERC及其应用
<130> /
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 23
<212> DNA
<213> 人工序列
<400> 1
gtggtggcca ttttttgtct aac 23
<210> 2
<211> 22
<212> DNA
<213> 人工序列
<400> 2
tgctctagaa tgaacggtgg aa 22
<210> 3
<211> 20
<212> DNA
<213> 人工序列
<400> 3
cgttcttagt tggtggagcg 20
<210> 4
<211> 20
<212> DNA
<213> 人工序列
<400> 4
cgctgagcca gtcagtgtag 20
Claims (9)
1.检测lncRNA TERC含量的试剂在制备膀胱癌诊断的试剂或试剂盒中的应用。
2.根据权利要求1所述的应用,其特征在于,检测样品为组织、尿液外泌体或血浆外泌体。
3.根据权利要求1所述的应用,其特征在于,所述检测lncRNA TERC含量的试剂为特异性识别或扩增lncRNA TERC的试剂。
4.根据权利要求1所述的应用,其特征在于,所述检测lncRNA TERC含量的试剂为lncRNA TERC的特异性探针或引物。
5.根据权利要求4所述的引物,其特征在于,所述引物的序列为SEQ ID NO:1、SEQ IDNO:2或其完全互补序列。
6.根据权利要求1所述的应用,其特征在于,所述试剂盒还包括:特异性检测内参基因的检测试剂。
7.根据权利要求6所述的应用,其特征在于,所述内参基因为18S rRNA。
8.根据权利要求7所述的应用,其特征在于,所述18S rRNA的引物序列为SEQ ID NO:3、SEQ ID NO:4或其完全互补序列。
9.根据权利要求1所述的应用,其特征在于,所述试剂盒还包括:RNA提取试剂、反转录试剂反应试剂、PCR扩增试剂和/或使用说明书。
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