CN112501294A - 一种结直肠癌生物标志物及其应用 - Google Patents
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Abstract
本发明属于生物检测技术领域,具体涉及一种结直肠癌生物标志物及其应用,为在结直肠癌(CRC)患者中寻找异常表达的tRNA衍生小片段RNA(tDRs)作为CRC早期诊断的生物标志物,本发明通过检测直肠癌患者和健康体检者的血浆5’‑tRF‑GlyGCC,发现血浆5’‑tRF‑GlyGCC含量可作为潜在的结直肠癌诊断和预后判断的指标,通过检测血浆中的5’‑tRF‑GlyGCC含量,或5’‑tRF‑GlyGCC联合血清CEA和CA199中的一种或多种作为诊断结直肠癌的靶标,不仅具有简单易行且性能优越的特点,而且有助于医师结合临床症状、病史或其他检查信息进行结直肠癌的诊断、早期筛查以及预后判断。
Description
技术领域
本发明属于生物检测技术领域,具体涉及一种结直肠癌生物标志物及其应用。
背景技术
结直肠癌(Colorectal cancer,CRC)是消化系统最常见的恶性肿瘤之一。2018年全球结直肠癌新增病例超过100万例,死亡病例约69.3万例,分别位于恶性肿瘤发病率第3位、病死率第2位。在我国,结直肠癌的发病率和病死率均高居第5位,据我国国家癌症中心的最新数据估计,2015年中国结直肠癌新发病例数达37.6万例,死亡病例数约19.1万,高于世界平均发病率和死亡率。近些年来,我国结直肠癌的发病率和死亡率呈现上升趋势,发病年龄有所提前,严重威胁着我国居民的健康。早期结直肠癌患者进行根治性切除术后的5年内,生存率可达90%,但由于结直肠癌早期症状不明显和结肠镜常规检查普及不足等原因,50%以上癌患者就诊时已到晚期,因此,结直肠癌的早期诊断显得尤为重要。目前,结肠镜检查、大便潜血中肿瘤相关标志物的检测是结直肠癌早期诊断的主要工具,但结肠镜检查存在侵入性、疼痛、费用高、不安全等缺点,而临床常用的粪便潜血相关标志物如癌胚抗原(CEA)和糖类抗原199(CA199)又存在灵敏度低、特异性差等不足。因此,亟需一种简单、灵敏度高、特异性好的结直肠癌分子标志物。
tRNA衍生小片段RNA(tRNA-derived small RNAs,tDRs)来源于前体或成熟体tRNA,长度通常为15-50nt。根据tDRs发生的位置不同,可将其分为两类:tRNAhalves(tiRNAs)和tRNA-derived small RNA fragments(tRFs)。其中,tiRNAs是由angiogenin(ANG)在多种应激条件下在成熟tRNA的反密码子环处特异性切割产生的5’-和3’-tRNA半分子,长度约为29-50nt。tRFs是在特定的细胞或组织中或者在细胞受到胁迫等特定条件下,由特定的核酸酶在tRNA的环上剪切,产生的特定大小的小片段RNA。tRFs在基因的表达和调控上发挥着重要的作用。tDRs的功能具有多样性,如:参与基因的调控表达、前体RNA的加工、核糖体的生物发生、LTR-反转录转座子等。同时,tDRs的异常表达也与人类多种疾病的发生相关,如:癌症、病毒感染、代谢障碍和神经退行性疾病。研究表明,tDRs可促进细胞增殖参与乳腺癌及前列腺癌的发生;tRNA去甲基化酶ALKBH3可诱导生成tDRs并促进肿瘤发展。人源细胞、组织和细胞外液中均含有大量的tDRs,同时tDRs可稳定存在于人的血液和尿液中,且越来越多的研究表明,tDRs可作为潜在的肿瘤诊断生物标志物,因此,在CRC患者中寻找异常表达的tDRs作为CRC早期诊断的生物标志物具有重大意义。
发明内容
为了克服上述现有技术的不足,本发明提供了一种结直肠癌生物标志物,通过检测血浆中的5’-tRF-GlyGCC含量,或5’-tRF-GlyGCC联合血清CEA和CA199中的一种或多种作为诊断结直肠癌的靶标,不仅具有简单易行且性能优越的特点,而且有助于医师结合临床症状、病史或其他检查信息进行结直肠癌的诊断、早期筛查以及预后判断。
为了实现上述目的,本发明所采用的技术方案是:
本发明第一方面提供一种结直肠癌生物标志物,所述结直肠癌生物标志物为血浆5’-tRF-GlyGCC,所述5’-tRF-GlyGCC的核苷酸序列如SEQ ID NO:1所示。
本发明第二方面提供上述的结直肠癌生物标志物在制备结直肠癌诊断(包括早期诊断)或/和预后判断产品中的应用。
所述应用是指检测结直肠癌患者外周血中5’-tRF-GlyGCC含量,与健康人群相应的指标含量进行比较,其中所述的相应的指标含量,指健康人群检测的血浆样品中5’-tRF-GlyGCC含量。
优选地,所述结直肠癌诊断或/和预后判断产品包括但不限于试剂盒和试剂。
血浆5’-tRF-GlyGCC是一种tDRs片段,本发明通过检测直肠癌患者和健康体检者的血浆5’-tRF-GlyGCC,发现健康血浆与结直肠癌血浆的5’-tRF-GlyGCC含量存在显著性差异;血浆5’-tRF-GlyGCC含量与结直肠癌的进展程度具有相关性;血浆5’-tRF-GlyGCC含量与结直肠癌的转移性具有一定的相关性;血浆5’-tRF-GlyGCC含量与血清ECA含量显著正相关;血浆5’-tRF-GlyGCC含量与血清CA199含量显著正相关。同时,在健康人群与结直肠癌患者中,血浆5’-tRF-GlyGCC的AUC为0.882,其灵敏度为85.71%,特异性为72.22%;而临床常用的血清肿瘤标志物CA199、CEA的AUC分别为0.557、0.762,表明血浆5’-tRF-GlyGCC的诊断性能显著优于CA199和CEA,提示血浆5’-tRF-GlyGCC含量可作为潜在的结直肠癌诊断和预后判断的指标。
本发明第三方面提供了一种结直肠癌诊断或/和预后判断产品,所述产品以血浆5’-tRF-GlyGCC为检测指标。
或者所述结直肠癌诊断或/和预后判断产品以血浆5’-tRF-GlyGCC和血清CEA为检测指标。
或者所述结直肠癌诊断或/和预后判断产品以血浆5’-tRF-GlyGCC和血清CA199为检测指标。
或者所述结直肠癌诊断或/和预后判断产品以血浆5’-tRF-GlyGCC、血清CEA以及CA199为检测指标。
本发明经过研究发现,在健康人群与结直肠癌患者中,临床常用的血清肿瘤标志物CA199、CEA的AUC分别为0.557、0.762,当血浆5’-tRF-GlyGCC分别联合CA199、CEA时,其AUC分别可达0.910、0.921,诊断性能得到进一步提高;当血浆5’-tRF-GlyGCC同时联合CA199和CEA时,其AUC可达0.926,显示出优良的诊断性能。说明本发明检测的血浆中5’-tRF-GlyGCC含量联合血清CEA和CA199含量中的一种或多种,可进一步提高检测的灵敏度和特异性。提示5’-tRF-GlyGCC指标联合血清CEA和/或CA199指标,有助于监测结直肠癌的进展程度、转移情况,并且有助于病程监控、手术治疗效果评估及预后判断。
或者所述结直肠癌诊断或/和预后判断产品包括检测血浆5’-tRF-GlyGCC的引物,所述引物的核苷酸序列如SEQ ID NO:2所示。
本发明第四方面提供一种结直肠癌诊断或/和预后判断试剂盒,包括检测血浆5’-tRF-GlyGCC的引物,5’-tRF-GlyGCC标准品,血浆RNA提取试剂或试剂盒,实时荧光定量试剂盒。
优选地,所述检测血浆5’-tRF-GlyGCC的引物的核苷酸序列如SEQ ID NO:2所示。
本发明第五方面提供采用上述结直肠癌诊断或/和预后判断试剂盒检测及分析的方法:
(1)采集和处理受试者的血浆样品;
(2)采用5’-tRF-GlyGCC标准品建立定量检测的标准曲线;
(3)采用所述试剂盒检测所述血浆样品中的5’-tRF-GlyGCC含量;
(4)将检测的结直肠癌患者血浆中的5’-tRF-GlyGCC含量,单独或联合以下任一种或多种指标:血清CEA含量、血清CA199含量,与健康人群中相应生物标志物的含量或表达含量进行比较,通过统计学分析对血浆5’-tRF-GlyGCC或5’-tRF-GlyGCC联合CEA和/或CA199作为结直肠癌检测指标的性能进行评价,评价的参数包括临界值含量、受试者工作特征曲线下面积(area under the ROC curve,AUC)、灵敏度、特异性、p值及95%置信区间。为实现结直肠癌的检测或早期检测以及预后判断提供特异性的中间结果,并提供一种快捷简便的无创伤性检测手段。
本发明检测的血浆中5’-tRF-GlyGCC含量联合血清CEA和CA199含量中的一种或多种,可进一步提高检测的灵敏度和特异性。
本发明利用检测5’-tRF-GlyGCC和检测5’-tRF-GlyGCC与CEA和CA199中的任一种或多种的组合制备出适用于临床诊疗用途的试剂或试剂盒,为实现结直肠癌的检测(包括早期检测)或预后判断提供特异性的中间结果以及快捷简便的无创伤性检测手段。
本发明所用的术语“健康体检者”是指无基础疾病的健康人群。
本发明利用试剂或试剂盒检测血浆5’-tRF-GlyGCC含量,通过分析上述指标中一种或多种联合的结果,可用评估检测对象结直肠癌的患病风险,对于结直肠癌的筛查及后续的诊疗方案具有重要的指导意义。
事实上,本发明通过试剂或试剂盒检测血浆5’-tRF-GlyGCC含量还可用于监测结直肠癌的进展程度、转移情况,有助于病程监控、手术治疗效果评估及预后判断。
与现有技术相比,本发明的有益效果是:
本发明提供一种结直肠癌生物标志物,所述结直肠癌生物标志物为血浆5’-tRF-GlyGCC,所述5’-tRF-GlyGCC的核苷酸序列如SEQ ID NO:1所示,通过检测血浆中的5’-tRF-GlyGCC含量来诊断结直肠癌具有以下优点:
(1)血浆5’-tRF-GlyGCC含量在结直肠癌的检测中具有较高的检测灵敏度和特异性,可作为检测结直肠癌的优良指标;
(2)血浆5’-tRF-GlyGCC含量在结直肠癌患者及健康人群之间存在差异,血浆5’-tRF-GlyGCC含量可作为检测结直肠癌的一个优良指标,且与CEA、CA199中的一种或多种联合检测可进一步提高其检测的灵敏度及特异性,其诊断性能显著优于目前临床上使用的血清肿瘤标志物CEA和CA199,从而大大提高了检测的准确性;
(3)血浆5’-tRF-GlyGCC含量与结直肠癌恶性程度如分期、转移具有相关性,有望作为结直肠癌病程监控、预后判断的评价指标;
(4)本发明检测的血浆指标相对其它组织较易获得,与结肠镜检查相比,属于无创性,极大地方便了医疗人员的使用,更减轻了患者的痛苦;
(5)血浆5’-tRF-GlyGCC含量的检测可以通过RNA提取技术、荧光定量PCR技术等常规的分子生物学手段进行,不但易于操作,重复性好,而且还增加了针对性和实用性,提高了结直肠癌检测的灵敏度和特异性。
综上所述可见,检测血浆5’-tRF-GlyGCC含量具有简单易行且性能优越的特点,同时通过5’-tRF-GlyGCC指标或联合常见的血清肿瘤标志物作为诊断结直肠癌的靶标,有助于医师结合临床症状、病史或其他检查信息进行结直肠癌的诊断、早期筛查以及预后判断。
附图说明
图1为血浆5’-tRF-GlyGCC检测的工作原理流程图;
图2为5’-tRF-GlyGCC定量检测的标准曲线;
图3为血浆5’-tRF-GlyGCC的检测结果;
图3中,A为健康对照组(n=90)及结直肠癌组(n=105)血浆中的5’-tRF-GlyGCC含量;B为健康组、I期、II期、III期、IV期血浆中的5’-tRF-GlyGCC含量;C为I与II期、III与IV期血浆中的5’-tRF-GlyGCC含量;D为无转移及转移结直肠癌患者血浆中的5’-tRF-GlyGCC含量;E为CEA<5ng/mL及CEA≥5ng/mL结直肠癌患者血浆中的5’-tRF-GlyGCC含量;F为CA199<37U/mL及CA199≥37U/mL结直肠癌患者血浆中的5’-tRF-GlyGCC含量;G为结直肠患者血清CEA含量及血浆5’-tRF-GlyGCC含量的相关性分析;H为结直肠患者血清CA199含量及血浆5’-tRF-GlyGCC含量的相关性分析。
图4为血浆5’-tRF-GlyGCC在结直肠癌检测中的性能评价。
图4中,A为血浆5’-tRF-GlyGCC在结直肠癌患者与健康人群中的ROC曲线分析;B为血清CEA在结直肠癌患者与健康人群中的ROC曲线分析;C为血清CA199在结直肠癌患者与健康人群中的ROC曲线分析;D为血浆5’-tRF-GlyGCC联合血清CEA在结直肠癌患者与健康人群中的ROC曲线分析;E为血浆5’-tRF-GlyGCC联合血清CA199在结直肠癌患者与健康人群中的ROC曲线分析;F为血浆5’-tRF-GlyGCC联合血清CEA、血清CA199在结直肠癌患者与健康人群中的ROC曲线分析。
具体实施方式
下面对本发明的具体实施方式作进一步说明。在此需要说明的是,对于这些实施方式的说明用于帮助理解本发明,但并不构成对本发明的限定。此外,下面所描述的本发明各个实施方式中所涉及的技术特征只要彼此之间未构成冲突就可以相互组合。
下述实施例中的实验方法,如无特殊说明,均为常规方法,下述实施例中所用的试验材料,如无特殊说明,均为可通过常规的商业途径购买得到的。
实施例1血浆tDRs 5’-tRF-GlyGCC与结直肠癌的关系
按照图1所示的操作流程对结直肠癌患者和与健康人群的血浆5’-tRF-GlyGCC进行检测,具体包括以下步骤:
(1)血浆样本的收集与保存
CRC血浆样本收集自中山大学附属第六医院,包括105例结直肠癌患者、90例健康体检者。
分别用EDTA抗凝管采集1mL新鲜血液,于1小时内置于离心机以3500rpm/min的转速室温离心5分钟,离心后的上清即为血浆,吸取血浆置新的1.5mL的RNAase-free的EP管中,作好标记并进行下一步实验或冻存于-80℃冰箱。
(2)血浆样品中RNA提取
1)将血浆样本从-80℃冰箱中取出,并将样品置于冰上解冻,然后以5000rpm/min的转速离心5分钟,用移液器吸取125μL上清液于新的1.5mL的RNAase-free的EP管中,用Trizol LS(Invitrogen,Carlsbad,CA,USA)提取血浆中的总RNA,并将其溶解于10μL DEPC水中,具体操作参考试剂说明书。
2)用Nanodrop2000检测RNA浓度及纯度:检测前先用DEPC水对仪器进行调零,用RNAase-free无菌枪头取上述RNA样品1μL,滴加于Nanodrop2000(Thermo)样品检测孔的中间位置,采用点击检测的方式对RNA浓度及纯度进行检测,被检测样本显示为单峰,260/280的范围为1.80~2.20的样品记录为合格,并用于后续检测。
(3)5’-tRF-GlyGCC定量检测的标准曲线制作
1)取200ng 5’-tRF-GlyGCC标准品[生工生物工程(上海)公司,序列为:5’-GCAUGGGUGGUUCAGUGGUAGAAUUCUCGCC-3’(SEQ ID NO:1)],应用Mir-XTM miRNA FirstStrand synthesis kit(Takara)按照其操作说明书进行逆转录。
2)将上述逆转录后的样品分成40,10,1,0.1,0.01,0.001,0.0001和0.00001ng 8个质量梯度,然后每个梯度设置3个复孔,分别进行实时荧光定量PCR(Quantitative real-time PCR)检测,最后以质量梯度为横坐标,CT值为纵坐标绘制标准曲线。制作得到如入2所示的5’-tRF-GlyGCC定量检测的标准曲线。
(4)5’-tRF-GlyGCC的实时荧光定量PCR检测
1)每个血浆样本吸取1μL总RNA,使用Mir-XTM miRNA First Strand synthesiskit(Takara)逆转录试剂盒进行逆转录,根据其说明书操作。
反应体系:
组分 | 用量 |
RNA | 1μL |
2×mRQ Buffer | 2.5μL |
mRQ Enzyme | 0.625μL |
加DEPC水至 | 5μL |
反应条件:将上述体系混匀,瞬时离心把EP管壁上的液滴离心到管底,37℃水浴1小时,接着85℃水浴5分钟终止反应。
2)将上述样本用DEPC水稀释至25uL。
3)PCR扩增:用TB GREEN Premix Ex TaqTM(Takara)试剂盒,根据其说明书操作。
反应体系:
组分 | 用量 |
2×TB GREEN Premix Ex Taq | 5μL |
Forward Primer(10μM) | 0.5μL |
mRQ 3’universal primer(10μM) | 0.5μL |
cDNA | 1μL |
DEPC水 | 3μL |
PCR扩增条件:
Stage 1:95℃,30s,1Cycle;
Stage 2:95℃,10s→60℃,30s,40Cycles;
Stage 3:95℃,5s→60℃,60s,1Cycle;
Stage 4:50℃,30s,1Cycle。
PCR扩增完成后,在设备上计算分析扩增曲线、融解曲线和循环数,溶解曲线要呈现单峰,如果不符合要求,数据不可信。
4)采用CT值代入标准曲线公式计算5’-tRF-GlyGCC的绝对表达量,然后除以血浆用量体积计算每个血浆样本5’-tRF-GlyGCC的绝对含量,CT值指循环阈值。
实时荧光定量PCR上游引物由生工生物工程(上海)公司合成,序列如下:
(5)血清CEA和CA199含量的测定
血清CEA和CA199肿瘤标志物均在患者治疗前检测(前述105例结直肠癌患者),使用化学发光酶免疫分析(CLEIA)的方法测定,所有实验均在中山大学附属第六医院检验科完成。
(6)检测结果分析
对105例结直肠癌患者、90例健康体检者的血浆5’-tRF-GlyGCC的检测结果如图3所示。从图3的结果可以看出,图3A中健康组与结直肠癌组的血浆5’-tRF-GlyGCC含量存在显著性差异(p<0.0001);图3B、图3C中显示了血浆5’-tRF-GlyGCC含量与结直肠癌TNM分期有关,I期、II期、Ⅲ期、IV期结直肠癌患者血浆5’-tRF-GlyGCC含量显著高于健康组,Ⅲ与IV期结直肠癌患者血浆5’-tRF-GlyGCC含量显著高于I与II期患者,表明血浆5’-tRF-GlyGCC含量与结直肠癌的进展程度具有相关性;图3D中显示肿瘤转移结直肠癌患者的血浆5’-tRF-GlyGCC含量较非转移结直肠癌患者的血浆5’-tRF-GlyGCC含量显著提高,提示血浆5’-tRF-GlyGCC含量与结直肠癌的转移性具有一定的相关性;图3E、图3G中显示了结直肠癌患者血浆5’-tRF-GlyGCC含量与血清ECA含量显著正相关;图3F、图3H中显示了结直肠癌患者血浆5’-tRF-GlyGCC含量与血清CA199含量显著正相关,以上结果表明血浆5’-tRF-GlyGCC含量可作为潜在的结直肠癌诊断和预后判断的指标。
对受试者检测的血清肿瘤标志物CEA、CA199与本发明检测的指标作对比,血浆5’-tRF-GlyGCC在结直肠癌检测中的性能评价如图4及表1所示。从图4和表1的结果可以看出,在健康人群与结直肠癌患者中,血浆5’-tRF-GlyGCC的AUC为0.882,其灵敏度为85.71%,特异性为72.22%;而临床常用的血清肿瘤标志物CA199、CEA的AUC分别为0.557、0.762,表明血浆5’-tRF-GlyGCC的诊断性能显著优于CA199和CEA;当血浆5’-tRF-GlyGCC分别联合CA199、CEA时,其AUC分别可达0.910、0.921,诊断性能得到进一步提高;当血浆5’-tRF-GlyGCC同时联合CA199和CEA时,其AUC可达0.926,显示出优良的诊断性能。
综上所述可见,本发明通过检测血浆5’-tRF-GlyGCC含量、血清CEA和CA199含量,通过分析上述指标中一种或多种联合的结果,有助于医师结合临床症状、病史或其他检查信息进行结直肠癌的诊断,对于结直肠癌的筛查及后续的诊疗方案具有重要的指导意义。同时,本发明通过试剂或试剂盒检测血浆5’-tRF-GlyGCC含量还可用于监测结直肠癌的进展程度、转移情况,有助于病程监控、手术治疗效果评估及预后判断。此外,检测血浆5’-tRF-GlyGCC具有简单易行且性能优越的特点,通过常用的血浆RNA提取及实时定量荧光PCR试剂及试剂盒即可进行检测,而上述这些生物学技术已广泛投入实践使用并有成熟的技术支持,因此,仅仅需要病人的血浆而不需要任何其它组织,通过精简的技术即可检测到血浆RNA5’-tRF-GlyGCC的含量,不仅有助于拓展结直肠癌的检测指标,提高检测的灵敏度和特异性,而且能丰富结直肠癌的检测手段。可见,血浆5’-tRF-GlyGCC有望成为诊断结直肠癌的重要生物标志物,具有极重要的临床应用潜力和价值。
表1为各检测指标的性能参数
以上对本发明的实施方式作了详细说明,但本发明不限于所描述的实施方式。对于本领域的技术人员而言,在不脱离本发明原理和精神的情况下,对这些实施方式进行多种变化、修改、替换和变型,仍落入本发明的保护范围内。
序列表
<110> 中山大学
<120> 一种结直肠癌生物标志物及其应用
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 31
<212> RNA
<213> 5’-tRF-GlyGCC(tRNA)
<400> 1
gcaugggugg uucaguggua gaauucucgc c 31
<210> 2
<211> 32
<212> DNA
<213> F引物(人工序列)
<400> 2
ggcaggcgag aattctacca ctgaaccacc aa 32
Claims (8)
1.一种结直肠癌生物标志物,其特征在于,所述结直肠癌生物标志物为血浆5’-tRF-GlyGCC,所述5’-tRF-GlyGCC的核苷酸序列如SEQ ID NO:1所示。
2.权利要求1所述的结直肠癌生物标志物在制备结直肠癌诊断或/和预后判断产品中的应用。
3.根据权利要求2所述的应用,其特征在于,所述结直肠癌诊断或/和预后判断产品包括但不限于试剂盒和试剂。
4.一种结直肠癌诊断或/和预后判断产品,其特征在于,所述产品以血浆5’-tRF-GlyGCC为检测指标。
5.一种结直肠癌诊断或/和预后判断产品,其特征在于,所述产品以血浆5’-tRF-GlyGCC和血清CEA或CA199中的一种或多种联合作为检测指标。
6.一种结直肠癌诊断或/和预后判断产品,其特征在于,所述产品包括检测血浆5’-tRF-GlyGCC的引物,所述引物的核苷酸序列如SEQ ID NO:2所示。
7.一种结直肠癌诊断或/和预后判断试剂盒,其特征在于,包括检测血浆5’-tRF-GlyGCC的引物,5’-tRF-GlyGCC标准品,血浆RNA提取试剂或试剂盒,实时荧光定量试剂盒。
8.根据权利要求9所述的一种结直肠癌诊断或/和预后判断试剂盒,其特征在于,所述检测血浆5’-tRF-GlyGCC的引物的核苷酸序列如SEQ ID NO:2所示。
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