CN117402964A - 食管癌标志物、引物、试剂盒和应用 - Google Patents
食管癌标志物、引物、试剂盒和应用 Download PDFInfo
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Abstract
本发明属于分子生物学领域,具体涉及一种食管癌标志物、引物、试剂盒和应用。本发明公开的食管癌标志物为血清miRNA标志物。该标志物包括:hsa‑miR‑1915‑5p、hsa‑miR‑3177‑5p、hsa‑miR‑5008‑3p、hsa‑miR‑9500、hsa‑miR‑12117中的一种或多种,每种microRNA对应的编码核苷酸序列分别如SEQ ID NO.1至SEQ ID NO.5所示。血清miRNA具有稳定性好,微创易获取、灵敏性和特异性高的特点,本发明提供的血液microRNA作为食管癌筛查或诊断标志物用途在检测中具有非侵入性无创和高效的效果,在制备食管癌筛查、诊断试剂盒中具有广泛的应用前景。
Description
技术领域
本发明属于分子生物学技术领域,具体涉及一种食管癌标志物、引物、试剂盒和应用。
背景技术
食管癌是当前全球最常见的癌症之一,也是癌症致死的最主要病因之一,仅在2020年全球就新增食管癌患者604000例和食管癌死亡病例544000万例。全球将近50%的新增食管癌患者在中国。食管癌对于病人、病人家庭和社会都是一个沉重的负担。因此,食管癌严重威胁人类,特别是中国人民的健康。
食管癌早期通常没有症状,其早期筛查和准确诊断是改善患者预后的一个重要因素。但现有的食管癌筛查和诊断手段都存在众多限制与不足。如传统的食管癌相关肿瘤标志物,由于其敏感性和特异性较低,因此不适合食管癌筛查和辅助诊断,只能用于术后监测复发;食管镜筛查和诊断食管癌准确率高,但其准备过程复杂、检查过程令人痛苦,而且其价格较高,因此不适合食管癌筛查和辅助诊断。因此临床上急需简便无创、高效能的食管癌筛查和诊断方法。
理想的生物学标志物应满足以下特点:1.可通过非侵入性的方法获得并且可用简便的方法将其分离;2.对不同疾病的高度特异性;3.高度诊断敏感性;4.疾病相关机制的生物学合理性;4.表达具有稳定性,以允许临床常规应用。当前越来越多的证据表明血液microRNA具有这些特征,有望成为筛查和诊断疾病的生物学标志物。
微小RNA(miRNA)是一类长度约为19-25个核苷酸的由内源基因编码的非编码RNA单链分子,广泛存在于真核生物细胞中。miRNA基因约占整个基因组的1%,其通过降低靶mRNA的稳定性或抑制翻译效率来调节基因的表达,从而参与包括细胞增殖、分化、凋亡、发育及逆境应答等多种生物学过程。血液中的miRNA经常与蛋白质等形成复合物,因此具有良好的抗RNase降解作用,冷冻、解冻或在室温下长期保存均不影响血液中miRNA的表达水平。此外,不同性别、不同年龄的人群,其血液中miRNA的表达水平十分相似。
随着越来越多的miRNA作为人类疾病的生物标志物及治疗靶点被发现,从中寻找一种能精确诊断食管癌的标志物,对优化临床食管癌诊断策略具有非常重大的意义。
发明内容
有鉴于此,本发明的目的在于提供一种食管癌标志物,以应用于食管癌的筛查或诊断,以及提供对应的引物和试剂盒,其技术方案如下:
第一方面,本发明提供了一种食管癌标志物,包括:hsa-miR-1915-5p、hsa-miR-3177-5p、hsa-miR-5008-3p、hsa-miR-9500、hsa-miR-12117中的至少一种;
其中,所述hsa-miR-1915-5p的碱基序列如SEQ ID NO.1所示;
所述hsa-miR-3177-5p的碱基序列如SEQ ID NO.2所示;
所述hsa-miR-5008-3p的碱基序列如SEQ ID NO.3所示;
所述hsa-miR-9500的碱基序列如SEQ ID NO.4所示;
所述hsa-miR-12117的碱基序列如SEQ ID NO.5所示。
以上列举的食管癌标志物均为血清miRNA,经实验检测,以上列举的食管癌标志物在食管癌患者血清组织中的表达量均与正常人相对应组织的表达量具有统计学差异,hsa-miR-1915-5p、hsa-miR-3177-5p、hsa-miR-5008-3p、hsa-miR-9500、hsa-miR-12117等食管癌标志物可单独用于检测和诊断食管癌,也可以两种标志物以上联合检测和诊断食管癌,同时,该些标志物能及时反映食管癌患者的疾病状态,尤其适用于食管癌的早期诊断、预后判断以及术后复发检测等,避免既往繁杂检测,节约时间人力成本,便于临床医生采取个性化的治疗方案。而且,检测血清样本中microRNA标志物的方法非常成熟,检测过程简便、易于重复,由普通技术员即可完成,可大大降低医院或检测机构的检测投入,同时,检测样本易于获得,临床可操作性强且创伤性小,而且血清microRNA稳定性好,检测便利。
第二方面,本发明提供了一种检测上述食管癌标志物的引物,所述引物包括反转录引物和/或上游引物;
其中,所述hsa-miR-1915-5p的上游引物的碱基序列如SEQ ID NO.6所示;
所述hsa-miR-3177-5p的上游引物的碱基序列如SEQ ID NO.7所示;
所述hsa-miR-5008-3p的上游引物的碱基序列如SEQ ID NO.8所示;
所述hsa-miR-9500的上游引物的碱基序列如SEQ ID NO.9所示;
所述hsa-miR-12117的上游引物的碱基序列如SEQ ID NO.10所示;
所述hsa-miR-1915-5p的逆转录引物的碱基序列如SEQ ID NO.11所示;
所述hsa-miR-3177-5p的逆转录引物的碱基序列如SEQ ID NO.12所示;
所述hsa-miR-5008-3p的逆转录引物的碱基序列如SEQ ID NO.13所示;
所述hsa-miR-9500的逆转录引物的碱基序列如SEQ ID NO.14所示;
所述hsa-miR-12117的逆转录引物的碱基序列如SEQ ID NO.15所示。
第三方面,本发明提供了一种试剂盒,包括:上述引物。
作为一种实施方式,所述试剂盒还包括:通用反向引物,各食管癌标志物的通用反向引物的碱基序列如SEQ ID NO.18所示。
作为一种实施方式,所述试剂盒还包括:探针,所述探针的碱基序列如SEQ IDNO.19所示。
作为一种实施方式,所述试剂盒还包括:RNA提取试剂;
所述RNA提取试剂包括TRIzolTMLS、Beyozol、PureZOL、AurumTM中的至少一种。
本发明提供的试剂盒,包括:用于检测上述食管癌标志物的引物,通过利用逆转录反应技术和PCR反应技术,能够实现检测血清中的食管癌标志物,从而达到筛查或诊断食管癌的目的,方法成熟,检测过程简便、易于重复,有利于实现食管癌的早期诊断、预后判断以及术后复发检测等。
采用上述试剂盒检测食管癌时,检测方法包括:
1)采集血清样本;
2)提取样本中总的RNA;
3)以提取的RNA为模板合成cDNA;
4)通过引物和探针进行RT-qPCR反应,获得待测血清标本中目标引物和外参引物的荧光信号到达设定阈值时所经历的循环数,即CT值;
5)根据2-ΔΔCq计算方法,通过外参引物计算出该血清样本中目标引物的ΔCT值。
第四方面,本发明还提供了前述食管癌标志物或上述引物或上述试剂盒在制备抗食管癌药物中的应用。
所述的食管癌包括食管腺癌和食管鳞状细胞癌,优选食管鳞状细胞癌。
将本发明以上提供的食管癌标志物、试剂盒应用于制备抗食管癌药物,有助于节约时间,节省人力成本,缩短抗食管癌药物的研发和生产周期。
附图说明
图1为hsa-miR-1915-5p在食管癌患者及健康对照组血清中的表达图;
图2为hsa-miR-1915-5p的ROC曲线;
图3为hsa-miR-3177-5p在食管癌患者及健康对照组血清中的表达图;
图4为hsa-miR-3177-5p的ROC曲线;
图5为hsa-miR-5008-3p在食管癌患者及健康对照组血清中的表达图;
图6为hsa-miR-5008-3p的ROC曲线;
图7为hsa-miR-9500在食管癌患者及健康对照组血清中的表达图;
图8为hsa-miR-9500的ROC曲线;
图9为hsa-miR-12117在食管癌患者及健康对照组血清中的表达图;
图10为hsa-miR-12117的ROC曲线。
具体实施方式
为了使本发明要解决的技术问题、技术方案及有益效果更加清楚明白,以下结合实施例,对本发明进行进一步详细说明。应当理解,此处所描述的具体实施例仅仅用以解释本发明,并不用于限定本发明。
实施例1
(1)血清样品的采集与制备
在取得所有志愿者的知情同意下,收集已确诊的食管鳞状细胞癌患者血清120例,体检的健康人血清120例,各取外周血2ml,并在2小时内于4℃下离心,转速为3600g,离心10分钟,取上层血清,储存于-80℃冰箱保存备用。
(2)血清样品中RNA的提取及cDNA的合成
取200uL步骤(1)提取的血清,加入0.1pM线虫cel-miR-54作为外参,再按1:5的比例加入1ml的TRIzol充分混匀,依照TRIzol提供的方案提取总RNA。按照逆转录试剂盒的方法,在普通PCR仪上以提取的总RNA(Purified RNA)为模版,加入试剂盒提供的逆转录酶(Sensiscript Reverse Transcriptase)、dNTP(dNTP Mix)以及反应缓冲液(10×Sensiscript Reverse Transcription Buffer),microRNA标志物和cel-miR-54的逆转录引物混合物(Reverse transcription Mix),将RNA逆转录成cDNA。-20℃保存待用。
表1cDNA合成反应体系的组成
表2使用的逆转录引物
(3)RT-qPCR检测
血清样本逆转录的cDNA,按照Golden HS TaqMan荧光实时定量PCR试剂盒的方法,进行荧光实时定量PCR,在LightCycler480(Roche)荧光定量PCR仪读出各个样本的Ct值。qRT-RCR运用仪器为LightCycler480(Roche),反应条件为:①预变性,95℃,5分钟;②变性,95℃,10秒;退火,60℃,30秒,共40个循环。
根据2-ΔΔCq计算方法,以cel-miR-54作外参计算出该血清样本的ΔCT值,根据公式ΔCT(检测)=CT(microRNA,检测)-CT(外参,检测);ΔCT(校准)=CT(食管癌,校准)-CT(对照组,校准);ΔΔCT=ΔCT(检测)-ΔCT(校准),根据得到的CT值,按2^-ΔΔCT值进行目的基因的相对定量检测。
采用2^-ΔΔCT法整理数据,数据分析和图形处理采用GraphPad Prism 8.3.0软件分析,运用Two-tailed Student’s test统计学方法对所有数据进行检测,数据以平均值±标准差表示,P<0.05表示有统计学意义。
表3实时荧光定量PCR反应体系
表4本实施例的食管癌筛查或诊断标志物、上游引物和探针序列
实施例2食管癌患者与正常人血清中hsa-miR-1915-5p表达对比
收集已确诊的食管鳞状细胞癌患者血清120例,体检的健康人血清120例。在取得所有志愿者的知情同意下,分别取各例外周血参照实施例1的方法进行定量分析,结果图1-2所示。
如图1-2所示,食管癌患者(Cancer)血清中hsa-miR-1915-5p表达水平较正常人(Control)的显著下调(P<0.0001)(图1);通过ROC曲线得到所对应的曲线下面积为0.8669(95%CI:0.8206-0.9133,P<0.0001).(图2),灵敏度为83.33%,特异度为79.17%。
本实施例的实验结果表明,食管癌患者血清中hsa-miR-1915-5p相比于正常人的表达下调,差异有统计学意义。将循环hsa-miR-1915-5p对食管癌患者的诊断效能进行ROC曲线分析,其结果显示AUC=0.8669,灵敏度为83.33%,特异度为79.17%。
实施例3食管癌患者与正常人血清中hsa-miR-3177-5p表达对比
收集已确诊的食管鳞状细胞癌患者血清120例,体检的健康人血清120例。在取得所有志愿者的知情同意下,分别取各例外周血参照实施例1的方法进行定量分析,结果如图3-4所示。
食管癌患者(Cancer)血清中hsa-miR-3177-5p表达水平较正常人(Control)的显著上调(P<0.0001)(图3);通过ROC曲线得到所对应的曲线下面积为0.7138(95%CI:0.6494-0.7781,P<0.0001).(图4),灵敏度为70.83%,特异度为56.67%。
本实施例的实验结果表明,食管癌患者血清中hsa-miR-3177-5p相比于正常人表达上调,差异有统计学意义。将循环hsa-miR-3177-5p对食管癌患者的诊断效能进行ROC曲线分析,其结果显示AUC=0.7138,灵敏度为70.83%,特异度为56.67%。
实施例4食管癌患者与正常人血清中hsa-miR-5008-3p表达对比
收集已确诊的食管鳞状细胞癌患者血清120例,体检的健康人血清120例。在取得所有志愿者的知情同意下,分别取各例外周血参照实施例1的方法进行定量分析,结果如图5-6所示。
食管癌患者(Cancer)血清中hsa-miR-5008-3p表达水平较正常人(Control)的显著下调(P<0.0001)(图5);通过ROC曲线得到所对应的曲线下面积为0.8571(95%CI:0.8092-0.9049,P<0.0001)(图6),灵敏度为83.33%,特异度为75.83%。
本实施例的实验结果表明,食管癌患者血清中hsa-miR-5008-3p相比于正常人组表达下调,差异有统计学意义。将循环hsa-miR-5008-3p对食管癌患者的诊断效能进行ROC曲线分析,其结果显示AUC=0.8571,灵敏度为83.33%,特异度为75.83%。
实施例5食管癌患者与正常人血清中hsa-miR-9500表达对比
收集已确诊的食管鳞状细胞癌患者血清120例,体检的健康人血清120例。在取得所有志愿者的知情同意下,分别取各例外周血参照实施例1的方法进行定量分析,结果如图7-8所示。
食管癌患者(Cancer)血清中hsa-miR-9500表达水平较正常人(Control)的显著下调(P<0.0001)(图7);通过ROC曲线得到所对应的曲线下面积为0.8535(95%CI:0.8036-0.9033,P<0.0001)(图8),灵敏度为82.5%,特异度为73.33%。
本实施例的实验结果表明,食管癌患者血清中hsa-miR-9500相比于正常人表达下调,差异有统计学意义。将循环hsa-miR-9500对食管癌患者的诊断效能进行ROC曲线分析,其结果显示AUC=0.8535(图8),灵敏度为82.5%,特异度为73.33%。
实施例6食管癌患者与正常人血清中hsa-miR-12117表达对比
收集已确诊的食管鳞状细胞癌患者血清120例,体检的健康人血清120例。在取得所有志愿者的知情同意下,分别取各例外周血参照实施例1的方法进行定量分析,结果如图9-10所示。
如图9-10所示,食管癌患者(Cancer)血清中hsa-miR-12117表达水平较正常人(Control)的显著下调(P<0.0001)(图9);通过ROC曲线得到所对应的曲线下面积为0.7536(95%CI:0.6935-0.8138,P<0.0001)(图10),灵敏度为75.83%,特异度为60.83%。
本实施例的实验结果表明,食管癌患者血清中hsa-miR-12117相比于正常人表达下调,差异有统计学意义。将循环hsa-miR-12117对食管癌患者的诊断效能进行ROC曲线分析,其结果显示AUC=0.7536(图10),灵敏度为75.83%,特异度为60.83%。
以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等,均应包含在本发明的保护范围之内。
Claims (8)
1.一种食管癌标志物,其特征在于,包括:hsa-miR-1915-5p、hsa-miR-3177-5p、hsa-miR-5008-3p、hsa-miR-9500、hsa-miR-12117中的至少一种;
其中,所述hsa-miR-1915-5p的碱基序列如SEQ ID NO.1所示;
所述hsa-miR-3177-5p的碱基序列如SEQ ID NO.2所示;
所述hsa-miR-5008-3p的碱基序列如SEQ ID NO.3所示;
所述hsa-miR-9500的碱基序列如SEQ ID NO.4所示;
所述hsa-miR-12117的碱基序列如SEQ ID NO.5所示。
2.一种检测权利要求1所述食管癌标志物的引物,其特征在于,所述引物包括反转录引物和/或上游引物;
其中,所述hsa-miR-1915-5p的上游引物的碱基序列如SEQ ID NO.6所示;
所述hsa-miR-3177-5p的上游引物的碱基序列如SEQ ID NO.7所示;
所述hsa-miR-5008-3p的上游引物的碱基序列如SEQ ID NO.8所示;
所述hsa-miR-9500的上游引物的碱基序列如SEQ ID NO.9所示;
所述hsa-miR-12117的上游引物的碱基序列如SEQ ID NO.10所示;
所述hsa-miR-1915-5p的逆转录引物的碱基序列如SEQ ID NO.11所示;
所述hsa-miR-3177-5p的逆转录引物的碱基序列如SEQ ID NO.12所示;
所述hsa-miR-5008-3p的逆转录引物的碱基序列如SEQ ID NO.13所示;
所述hsa-miR-9500的逆转录引物的碱基序列如SEQ ID NO.14所示;
所述hsa-miR-12117的逆转录引物的碱基序列如SEQ ID NO.15所示。
3.一种试剂盒,其特征在于,包括:权利要求2所述的引物。
4.根据权利要求3所述的试剂盒,其特征在于,所述试剂盒还包括:通用反向引物,各食管癌标志物的通用反向引物的碱基序列如SEQ ID NO.18所示。
5.根据权利要求3所述的试剂盒,其特征在于,所述试剂盒还包括:探针,所述探针的碱基序列如SEQ ID NO.19所示。
6.根据权利要求3所述的试剂盒,其特征在于,所述试剂盒还包括:RNA提取试剂;
所述RNA提取试剂包括TRIzolTMLS、Beyozol、PureZOL、AurumTM中的至少一种。
7.权利要求1所述的食管癌标志物或权利要求2所述的引物或权利要求3至6任一项所述的试剂盒在制备抗食管癌药物中的应用。
8.如权利要求7所述的应用,所述食管癌为食管鳞状细胞癌。
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