CN110628907B - 一组胆囊癌血浆外泌体microRNAs标记物及其应用 - Google Patents
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Abstract
本发明提供一组胆囊癌血浆外泌体microRNAs标记物及其检测方法和应用。本组血浆外泌体microRNAs标记物包括在胆囊癌血浆外泌体中升高表达的miR‑552‑3p、miR‑581、miR‑4433a‑3p以及在胆囊癌血浆外泌体中降低表达的miR‑496以及miR‑203b‑3p。利用本发明所述的检测方法可以增强对胆囊癌诊断的特异性和敏感性。本发明所提供的标记物组合、检测方法和试剂盒能够用于辅助胆囊癌的诊断与鉴别诊断、胆囊癌体检筛查、药物治疗疗效评价、复发转移预警、并发症发生、患者预后判断等方面,具有无创、稳定、早期、检测方法简便、检测时间较短等优点,具有较好的临床应用价值。
Description
技术领域
本发明涉及医学分子生物学领域,尤其涉及一组胆囊癌血浆外泌体microRNAs标记物及应用。
背景技术
胆囊癌在胆囊恶性肿瘤中占首位,在胃肠道肿瘤中位居第5位,近年来发病率上升很快,被认为是侵袭性和致死性最高的恶性肿瘤之一。我国胆囊癌相对高发,其日益成为严重威胁广大人民群众生命的恶性疾病。根据全球肿瘤统计(Global Cancer Statistics,2012),在发达地区,胆囊癌的年龄标准化(ASR,age-standardized rate per 100,000)发病率为2.0,而年龄标准化致死率达到1.4;在不发达地区,年龄标准化发病率为2.4,而年龄标准化致死率高达2.0,这也就意味着,只要发病,患者最终的结局大多都是死亡。胆囊癌常与胆囊良性疾患同时存在,最常见是与胆囊结石及胆囊炎共存,结石的慢性刺激是重要的致病因素。胆囊癌早期无特异的临床症状和体征,大多数患者就诊时已属中晚期,手术切除率低,术后的5年生存率不到5%。综上这些因素,寻找胆囊癌特异性的诊断标志物具有重要的临床意义。
目前,胆囊癌的诊断方法主要包括B超检查、CT扫描、彩色多普勒血流显像等等,但是这些方法的诊断精确度对于胆囊癌诊断尤其是早期诊断来说并不理想。直接取活检或抽取胆汁查找癌细胞也应用于胆囊癌的诊断,但是活检由于其自身的缺陷如不能反映患者整体情况、有加速转移的风险、结果报告滞后以及患者创伤较大等并不适合广泛应用。同时,传统的胆囊癌诊断血清学标志物CA199等的敏感性、特异性很低,不能满足临床需求。近年来,体液活检技术受到临床上越来越多的重视和推广,被“麻省理工大学科技评论”评为“2015十大突破”之一。体液活检是指通过患者的血液或者尿液等对癌症等疾病做出诊断。体液活检的检测内容主要包括血液中游离的循环肿瘤细胞(Circulating tumor cells,CTCs)、循环肿瘤DNA(Circulating tumor DNA,ctDNA)、循环RNA(Circulating RNA)以及近年来的研究热点之一的外泌体(Exosomes)。
外泌体是一种可由机体中的多种细胞分泌的直径为40-100nm的均一、双层膜结构小囊泡,其广泛地分布于细胞间隙、细胞外液,乃至被分泌至唾液、血浆和乳汁等体液中。外泌体是Johnstone RM等在研究网织红细胞向成熟红细胞转变过程中发现的,其内容物十分丰富。目前的研究发现,不同的细胞和组织来源的外泌体中确定包含有约9769种蛋白质、3408种mRNA和2838种微小RNA(microRNA)以及1116种脂质等等。与正常细胞比较,大多数类型的肿瘤细胞包括黑色素瘤、结直肠癌、乳腺癌、肺癌、肝癌和前列腺癌细胞等都可以分泌外泌体,并且其外泌体量更大,内容物差异显著。因此,外泌体被认为可以作为有效的肿瘤诊断标志物。肿瘤外泌体与肿瘤的发生、发展及预后密切相关,其影响肿瘤的增殖、细胞外基质重塑、侵袭、转移、血管生成、免疫逃逸以及耐药等多个过程。
microRNA是长约19-25nt的非编码小分子RNA,通过与靶基因mRNA的3'非翻译区(UTR)特异性结合负向调控靶基因的表达从而发挥其生物学功能。microRNA与肿瘤关系十分密切,相关研究一直是过去20年来的热点之一。近年来的研究表明,microRNA也可以被包装进外泌体中,进而被运输到相应的受体细胞中调节其生物功能,被认为是细胞间通信新的信号分子,这种由外泌体介导的细胞间信息传递在肿瘤的进展过程中扮演着十分重要的作用。在一项肝癌研究中,研究者发现miR-429能够被包裹在外泌体中,从高表达的肝癌细胞中分泌出去并进入周围环境中低表达的细胞内,促进该细胞的恶性转化。并且,最重要的是,分泌出来的microRNA可以被吸收进入循环中,从而成为肿瘤诊断可能的新型标志物。外泌体microRNA有成为肿瘤诊断标志物的理论与应用价值,但是其能否用于胆囊癌的诊断目前尚不十分明确。
发明内容
本发明为解决现有技术中的上述问题创造性地提出一组胆囊癌血浆外泌体microRNAs标记物及其检测方法、应用。
为了解决上述的技术问题,本发明所采取的技术措施包括:
本发明的第一个方面是提供一组胆囊癌血浆外泌体microRNAs标记物,该组标记物包括miR-552-3p、miR-581、miR-4433a-3p、miR-496、miR-203b-3p中的至少一种,所述人源microRNAs的成熟体序列分别如下:
miR-552-3p 5’-AACAGGUGACUGGUUAGACAA-3’(SEQ ID No.1);
miR-581 5’-UCUUGUGUUCUCUAGAUCAGU-3’(SEQ ID No.2);
miR-4433a-3p 5’-ACAGGAGUGGGGGUGGGACAU-3’(SEQ ID No.3);
miR-496 5’-UGAGUAUUACAUGGCCAAUCUC-3’(SEQ ID No.4);
miR-203b-3p 5’-UUGAACUGUUAAGAACCACUGGA-3’(SEQ ID No.5)。
进一步地,该组胆囊癌血浆外泌体microRNAs可用做胆囊癌诊断标记物。
进一步地,部分microRNAs呈现升高趋势,部分microRNAs呈现下降趋势。
进一步地,呈现上升趋势的microRNAs为miR-552-3p、miR-581、miR-4433a-3p。
进一步地,呈现下降趋势的microRNAs为miR-496、miR-203b-3p。
本发明的第二个方面是提供一种用于非诊断和治疗目的的检测如上述胆囊癌血浆外泌体microRNAs标记物的方法,所述方法包括:
(1)血液样本收集及处理;
(2)血浆外泌体分离及RNA抽提;
(3)外泌体中RNA定量及逆转录;
(4)外泌体中microRNA定量及分析,包括:Real-time PCR反应和数据分析。
进一步地,Real-time PCR方法以U6为内参。
进一步地,PCR的配制体系如下:
进一步地,所述标记物为miR-552-3p、miR-581、miR-4433a-3p、miR-496、miR-203b-3p的联合。
本发明的第三个方面是提供一种任一上述的胆囊癌血浆外泌体microRNAs标记物在制备用于诊断胆囊癌的试剂盒中的应用。
本发明采用上述技术方案,与现有技术相比,具有如下技术效果:本发明提出了一组胆囊癌血浆外泌体microRNAs标记物及其检测方法、应用,利用本发明所述的5种microRNAs联合集可以增强对胆囊癌诊断的特异性和敏感性。本发明所提供的标记物组合、检测方法能够用于辅助胆囊癌的诊断与鉴别诊断、胆囊癌体检筛查、药物治疗疗效评价、复发转移预警、并发症发生、患者预后判断等方面,具有无创、稳定、早期、检测方法简便、检测时间较短等优点,具有较好的临床应用价值。
附图说明
图1为本发明一实施例中的外泌体电镜鉴定图。
图2为本发明一实施例中的胆囊癌患者血浆外泌体中microRNAs差异表达图。
图3为本发明一实施例中的胆囊癌与慢性胆囊炎对比血浆外泌体中升高表达的microRNA图。
图4本发明一实施例中的胆囊癌与慢性胆囊炎对比血浆外泌体中降低表达的microRNA图。
图5本发明一实施例中的ROC分析5种microRNAs联合集对于胆囊癌诊断效果图。
图6本发明一实施例中的传统胆囊癌诊断血清学标志物表达水平检测图。
图7本发明一实施例中的ROC分析CEA和CA199对于胆囊癌诊断效果对照图。
具体实施方式
下面通过具体实施例和附图对本发明进行详细和具体的介绍,以使更好的理解本发明,但是下述实施例并不限制本发明范围。
实施例一
本实施例为一组胆囊癌血浆外泌体microRNAs标记物及其应用。基本操作方法如下:
一、血液样本收集及处理
1.抗凝管(拱东,含EDTA,货号GD050EK2)收集胆囊癌患者或慢性胆囊炎患者静脉血10毫升(mL)。
2.4度条件下,3000rpm离心10min,收集上清,之后再离心一次,重新收集上清,收集的上清即为血浆样本。10mL静脉血收集血浆体积约5mL,置于负80℃冰箱保存。
二、血浆外泌体分离及RNA抽提
利用差速离心法收集胆囊癌病人及慢性胆囊炎病人血浆中外泌体。其具体流程如下:4度条件下,300g,5min→收集上清→1200g,20min→收集上清→10000g,30min→收集上清→110000g,2h→收集沉淀物→重悬沉淀物→利用电镜观察外泌体形状及大小→利用TRIZOL法抽提外泌体中的总RNA。
三、外泌体中RNA定量及逆转录
参照iScript cDNA Synthesis Kit(BIO-RAD,货号:1708891)逆转录试剂盒,操作按照说明进行,经过反复实践改良,具体操作过程如下:
1.使用“Nanodrop”机器测定血浆外泌体中的RNA浓度:
打开取样臂,无尘纸清洁测量基座→吸取1微升(μL)DEPC水作为空白样品调零,滴加在测量基座表面→在软件中点击“空白”进行调零→调零完成后,使用无尘纸拭去基座和取样臂上的液滴,吸取1μL待测RNA样品,滴加在测量基座表面→在软件中点击“检测”测定样品RNA浓度,记为c(ng/μL)。
2.逆转录反应体系配置:
每个反应使用100-200ng总RNA进行逆转。例如,使用200ng总RNA进行逆转,RNA模板体积x=200/c:
5×iScript Reaction Mix,4μL;
iScript Reverse Transcriptase,1μL;
Nuclease-free water,15-xμL;RNA template,xμL;
Total volume,20μL。
3.反应程序如下:
使用Eppendorf Mastercycler nexus GSX1机器对逆转录反应管进行下列加热孵育程序:Priming,25℃,5分钟→Reverse transcription,46℃,20分钟→RTinactivation,95℃,1分钟→Optional step,4℃,保持。
4.反应结束后,逆转录产物(cDNA)为20μL,可置于-80℃保存或用ddH2O稀释30倍后用于后续Real-time PCR实验。
四、外泌体中microRNA定量及分析
使用Real-time PCR方法对血浆外泌体中的microRNA进行定量分析。以定量miR-552-3p为例,其余血浆外泌体中microRNA定量方法与此相同:
1.引物:血浆外泌体中microRNA定量以U6为内参,目的microRNA及U6引物使用QIAGEN引物套餐,包括序列特异性的上游引物(Forward primer)以及通用的下游引物(Reverse primer)的混合物。
2.PCR反应体系配置(微升,μL)
按照表1所示配制PCR体系,配制全程将eppendorf管置于冰上,PCR孔板每个反应孔设置2个复孔。
表1
*PCR mix:FastStart Universal SYBR Green Master(Roche,货号:4913850001)
3.PCR反应
水平离心机1000rpm离心10秒,将侧壁液体离心至孔底,使用Light Cycler Real-Time PCR(Roche)机器进行荧光定量PCR。程序如下(表2):
表2
4.数据分析
使用Light Cycler软件进行CT值读取,分别计算慢性胆囊炎组和胆囊癌组2-△CT=2-CT待检-(-CT内参)。均一化:2-△△CT=2-△CT/慢性胆囊炎组2-△CT,以慢性胆囊炎组2-△△CT平均值作为基准线,绘制散点图。在本项研究中,我们将由此方法计算得出的倍数关系进行log10转化,使用转化后的数据绘制散点图。
通过构建受试者工作特征曲线(Receiver operating characteristics,ROC)来评价组合标记物的敏感性、特异性和曲线下面积(AUC)。诊断的最优截断值(Cut off)评价:敏感性和特异性的总和最大化,最小化整体误差(Square root of the sum[1–sensitivity]2+[1–specificity]2),截断值与ROC曲线左上角的距离最短。结合使用5种血浆外泌体microRNAs表达值,根据二元逻辑回归得到的方程,建立了胆囊癌的预测概率新变量logit(P)=1.628×miR-552-3p+1.543×miR-581+2.485×miR-4433a-3p-3.228×miR-496-3.127×miR-203b-3p-2.254。
应用实施例一验证该组胆囊癌血浆外泌体microRNAs标记物对胆囊癌的诊断效果
步骤1.分离了各8例胆囊癌患者及慢性胆囊炎患者血浆中的外泌体,之后利用电镜对分离的外泌体形态进行了观察鉴定,从图1可以看出,分离的膜结构小体的体积符合外泌体的特征。
步骤2.利用高通量二代测序技术检测了8例胆囊癌患者及8例慢性胆囊炎患者外泌体中microRNAs的表达。和慢性胆囊炎患者相比较,胆囊癌患者血浆外泌体中有352种microRNAs下调以及235种上调(图2)。
步骤3.将筛选出来的升高和降低(胆囊癌和慢性胆囊炎相比较)候选miRNAs的前10位分别扩大样本量进行单独验证,利用实时定量PCR的方法,分别检测了其在30例胆囊癌和30例慢性胆囊炎患者血浆外泌体中的表达情况。在升高表达的10种miRNAs中,miR-552-3p、miR-581、miR-4433a-3p以及miR-372-3p仍然呈现升高表达趋势(图3)。在降低表达的miRNAs中,miR-496、miR-551b-3p以及miR-203b-3p仍然呈现降低表达趋势(图4)。这些结果提示,这7种miRNAs可以作为胆囊癌诊断新的标记物。
步骤4.联合应用上述7种通过大样本验证的microRNAs表达值数据(miR-552-3p,miR-581,miR-372-3p,miR-4433a-3p,miR-551b-3p,miR-496,miR-203b-3p),建立logisticregression方程,其中有5个microRNAs通过了logistic regression方程,并建立了胆囊癌的预测概率新变量logit(P)=1.628×miR-552-3p+1.543×miR-581+2.485×miR-4433a-3p-3.228×miR-496-3.127×miR-203b-3p-2.254。利用预测概率新变量logit(P)做ROC曲线,这5种microRNAs集合的AUC=0.920,OR=36.000,当选定阈值(Cut off)=0.5779时,其诊断敏感性=80%,特异性=90%(图5)。说明其具有良好的诊断效用。
步骤5.进一步的检测了胆囊癌和慢性胆囊炎相比较的传统血清标志物的差异,检测了包栝CEA,AFP,CA199,CA242,CA74,CA25以及CA15等7种常用的传统标志物。通过对30例胆囊癌和30例慢性胆囊炎进行检测,发现CEA和CA199呈现差异性表达,而其他5种标志物差异并不显著(图6)。
步骤6.通过ROC曲线分析,传统胆囊癌诊断的血清学标志物CEA和CA199也能够显著区分胆囊癌和慢性胆囊炎,AUC分别为0.755和0.569(图7),但是,二者的诊断效能均低于包含5种外泌体microRNAs的标志物集合(AUC=0.920)。
步骤7.分别探讨了2种传统的胆囊癌血清学诊断指标以及新型血浆外泌体microRNAs指标对于诊断胆囊癌的效能,并探讨了其联合应用的可能性,各标志物具体的诊断表现以及联合使用的诊断表现如下(表3)。从表中可以看出,在诊断胆囊癌方面,5种血浆外泌体microRNAs集合标记物(miR-552-3p,miR-581,miR-4433a-3p,miR-496,miR-203b-3p)的诊断效能最佳(AUC=0.920),优于传统胆囊癌血清学诊断指标CEA(AUC=0.755)和CA199(AUC=0.569),并且优于单个血浆外泌体microRNA标记物。这5种血浆外泌体microRNAs集合标记物联合CEA及CA199,对于AUC指标并无显著提升(AUC=0.91),但是能够提高诊断的敏感性(由80%提高至96.67%),提示新型标记物可与传统诊断标记物联合使用。
表3传统胆囊癌血清学诊断指标以及新型血浆外泌体microRNAs集合指标对于诊断胆囊癌的效能
通过上述的实施例可以知道,本发明的一组特异性血浆外泌体microRNAs组合可以有效地诊断胆囊癌;利用本发明提供的检测该组胆囊癌血浆外泌体microRNAs的方法,大大提高了诊断的敏感度和特异性。并且,该检测方法为无创的体液活检方法,通过检测方法的优化,具有稳定、早期、简便、检测时间较短等优点,具有较好的临床应用价值。
以上对本发明的具体实施例进行了详细描述,但其只是作为范例,本发明并不限制于以上描述的具体实施例。对于本领域技术人员而言,任何对本发明进行的等同修改和替代也都在本发明的范畴之中。因此,在不脱离本发明的精神和范围下所作的均等变换和修改,都应涵盖在本发明的范围内。
序列表
<110> 张宝华
李亮
<120> 一组胆囊癌血浆外泌体microRNAs标记物及其应用
<140> IPI192972
<141> 2019-09-09
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
<212> RNA
<213> miR-552-3p的成熟体序列(人源)
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<210> 2
<211> 21
<212> RNA
<213> miR-581的成熟体序列(人源)
<400> 2
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<210> 3
<211> 21
<212> RNA
<213> miR-4433a-3p的成熟体序列(人源)
<400> 3
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<210> 4
<211> 22
<212> RNA
<213> miR-496的成熟体序列(人源)
<400> 4
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<210> 5
<211> 23
<212> RNA
<213> miR-203b-3p的成熟体序列(人源)
<400> 5
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Claims (1)
1.检测胆囊癌血浆外泌体microRNAs标记物表达水平的引物在制备用于区分胆囊癌和慢性胆囊炎的试剂盒中的应用,其特征在于,所述microRNAs标记物由miR-552-3p、miR-581、miR-4433a-3p、miR-496以及miR-203b-3p组成,所述miR-552-3p、所述miR-581、所述miR-4433a-3p、所述miR-496以及所述miR-203b-3p的序列分别如SEQ ID No.1~SEQ IDNo.5所示。
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---|
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胆囊癌外周血循环肿瘤细胞和外泌体的检测及其临床应用;温志坚;《中国优秀博硕士学位论文全文数据库(硕士) 医药卫生科技辑》;20180115;摘要、表11 * |
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