CN110656172A - 一种预测小细胞肺癌对ep化疗方案敏感性的分子标志物及试剂盒 - Google Patents
一种预测小细胞肺癌对ep化疗方案敏感性的分子标志物及试剂盒 Download PDFInfo
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Abstract
本发明属于医学预测、诊断技术领域，具体涉及一种预测小细胞肺癌对EP化疗方案敏感性的分子标志物及试剂盒，EP化疗方案包括化疗药物足叶乙甙和顺铂，足叶乙甙的分子标志物包括基因CSMD3、PCLO、RYR1、EPB41L3；顺铂的分子标志物包括基因RYR2、APOB、DNAH14、RELN、LAMA3、MLL3。试剂盒包括CSMD3、PCLO、RYR1、EPB41L3以及RYR2、APOB、DNAH14、RELN、LAMA3、MLL3的正向引物和反向引物，逆转录反应体系试剂，定量PCR反应体系试剂以及酶基因GAPDH内参引物，本发明可应用于开发EP化疗方案敏感性检测试剂盒，预测患者对EP化疗方案的敏感性。
Description
技术领域
本发明属于医学预测、诊断技术领域，具体涉及一种预测小细胞肺癌对EP化疗方案敏感性的分子标志物及试剂盒。
背景技术
肺癌已成为严重人类生命和健康的常见病，统计资料显示肺癌在我国城市恶性肿瘤的发病中居于首位，且其发病率仍呈明显上升趋势，预估中国肺癌发病人数至2025年将位居世界第一。小细胞肺癌(SCLC)的发病率约占全部肺癌患者的15-20％，是肺癌中侵袭性最强的亚型，EP(Etoposide+Cisplatin)(足叶乙甙和顺铂)化疗方案目前仍是SCLC的标准治疗模式，尽管大部分患者在治疗初期对化疗药物敏感，但大约6月时间就会因耐药而复发，5年生存期不足10％，临床预后很差。早期且准确地预测小细胞肺癌患者对EP方案的敏感性，指导临床为小细胞肺癌患者制定个体化治疗方案。
研究发现肿瘤化疗耐药机制主要涉及DNA修复基因、药物代谢相关基因及凋亡相关基因参与，EP方案包含化疗药物足叶乙甙和顺铂，已有报道在多种肿瘤中发现一些基因在顺铂或足叶乙甙耐药细胞表达异常，可作为预测耐药的分子标志物，如核苷酸切除修复交叉互补组1(excision repair cross-complementation group1，ERCC1)在蛋白水平或mRNA水平高表达与非小细胞肺癌、头颈部鳞癌、食道癌、胃癌、膀胱癌、子宫颈癌、卵巢癌等肿瘤患者顺铂耐药相关。乳腺癌1号基因(Breast cancer 1，BRCA1)高表达能够诱导顺铂耐药及增强肿瘤对抗微管蛋白药物的敏感性，RAP80是BRCA1复合物进行同源染色体重组修复的必需结构单元，其mRNA表达水平可提高BRCA1低表达的非小细胞肺癌患者对化疗的疗效预测。XAF1被认为是凋亡相关通路中发挥着重要作用的凋亡相关蛋白，与足叶乙甙、5-氟尿嘧啶(5-flurouracil，5-FU)等化疗药物耐药有关。
由于小细胞肺癌具有特异的基因组学改变，其对足叶乙甙和顺铂耐药的机制也与其它肿瘤有一定差异。文献报道药物代谢相关基因谷胱甘肽合成酶(GSS)变异位点rs725521和DNA双链断裂修复基因RAD52的变异位点rs10774474，都是影响小细胞肺癌患者铂类药物化疗疗效的独立因素，是预测小细胞肺癌患者铂类药物化疗疗效的重要标志物。本发明可早期预测小细胞肺癌患者对EP化疗方案是否敏感，为临床开展个体化治疗提供重要依据，目前尚未见相关文献报道。
发明内容
本发明的目的在于提供一种预测小细胞肺癌对EP化疗方案敏感性的分子标志物及试剂盒。
本发明的技术方案如下：
一种预测小细胞肺癌对EP化疗方案敏感性的分子标志物，EP化疗方案包括化疗药物足叶乙甙和顺铂，足叶乙甙的分子标志物包括基因CSMD3、PCLO、RYR1、EPB41L3；顺铂的分子标志物包括基因RYR2、APOB、DNAH14、RELN、LAMA3、MLL3；
一种预测小细胞肺癌对EP化疗方案敏感性的试剂盒，试剂盒包括CSMD3、PCLO、RYR1、EPB41L3以及RYR2、APOB、DNAH14、RELN、LAMA3、MLL3的正向引物和反向引物；
正向引物和反向引物的序列如SEQ ID 1～40所示：
SEQ ID No.1和No.3为CSMD3的正向引物，SEQ ID No.5和No.7为PCLO的正向引物，SEQ ID No.9和No.11为RYR1的正向引物，SEQ ID No.13和No.15为EPB41L的正向引物，SEQID No.17和No.19为RYR2的正向引物，SEQ ID No.21和No.23为APOB的正向引物，SEQ IDNo.25和No.27为DNAH14的正向引物，SEQ ID No.29和No.31为RELN的正向引物，SEQ IDNo.33和No.35为LAMA3的正向引物，SEQ ID No.37和No.39为MLL3的正向引物；
SEQ ID No.2和No.4为CSMD3反向引物，SEQ ID No.6和No.8为PCLO反向引物，SEQID No.10和No.12为RYR1的反向引物，SEQ ID No.14和No.16为EPB41L的反向引物浓度，SEQID No.18和No.20为RYR2的反向引物，SEQ ID No.22和No.24为APOB的反向引物，SEQ IDNo.26和No.28为DNAH14的反向引物，SEQ ID No.30和No.32为RELN的反向引物，SEQ IDNo.34和No.36为LAMA3的反向引物，SEQ ID No.38和No.40为MLL3的反向引物，浓度为10pmol/μL；
试剂盒还包括逆转录反应体系试剂、定量PCR反应体系试剂以及酶基因GAPDH内参引物，内参引物序列如SEQ ID 41～42所示。
一种预测小细胞肺癌对EP化疗方案敏感性的分子标志物的应用，应用于开发EP化疗方案敏感性检测试剂盒，预测患者对EP化疗方案的敏感性。
本发明的有益效果如下：
(1)本发明的的RYR2、APOB、DNAH14、RELN、LAMA3、MLL3六个基因组合为顺铂分子标志物以及CSMD3、PCLO、RYR1、EPB41L3四个基因的组合为足叶乙甙分子标志物，经过临床样本验证，能较好预测小细胞肺癌患者对EP化疗方案的敏感性；
(2)本发明的分子标志物首次应用于开发EP化疗方案敏感性检测试剂盒，可用于指导临床开展个体化治疗；提供的相应的试剂盒可以运用实时荧光定量PCR方法，检测快速方便、检测灵敏度。
说明书附图
图1为54株小细胞肺癌细胞对足叶乙甙的半数致死量(IC50)图；
图2为52株小细胞肺癌细胞对顺铂的半数致死量(IC50)图；
图3为CSMD3、PCLO、RYR1和EPB41L3单指标及四个指标组合的ROC曲线图；
图4为RYR2、APOB、DNAH14、RELN、LAMA3、MLL3单指标及六个指标组合的ROC曲线图；
具体实施方式
以下通过具体的实施案例以及附图说明对本发明作进一步详细的描述，应理解这些实施例仅用于说明本发明而不用于限制本发明的保护范围，在阅读了本发明之后，本领域技术人员对本发明的各种等价形式的修改均落于本申请所附权利要求所附权利要求所限定。
实施例1
一种预测小细胞肺癌对EP化疗方案敏感性的试剂盒：
A)特异性引物：
1μL F1引物，SEQ ID No.1和No.3为CSMD3的正向引物，SEQ ID No.5和No.7为PCLO的正向引物，SEQ ID No.9和No.11为RYR1的正向引物，SEQ ID No.13和No.15为EPB41L的正向引物，SEQ ID No.17和No.19为RYR2的正向引物，SEQ ID No.21和No.23为APOB的正向引物，SEQ ID No.25和No.27为DNAH14的正向引物，SEQ ID No.29和No.31为RELN的正向引物，SEQ ID No.33和No.35为LAMA3的正向引物，SEQ ID No.37和No.39为MLL3的正向引物，浓度为10pmol/μL；
1μL R1引物，SEQ ID No.2和No.4为CSMD3反向引物，SEQ ID No.6和No.8为PCLO反向引物，SEQ ID No.10和No.12为RYR1的反向引物，SEQ ID No.14和No.16为EPB41L的反向引物浓度，SEQ ID No.18和No.20为RYR2的反向引物，SEQ ID No.22和No.24为APOB的反向引物，SEQ ID No.26和No.28为DNAH14的反向引物，SEQ ID No.30和No.32为RELN的反向引物，SEQ ID No.34和No.36为LAMA3的反向引物，SEQ ID No.38和No.40为MLL3的反向引物，浓度为10pmol/μL。
B)逆转录反应体系试剂：
20μL 5×
Buffer(for Real Time)，5μL 
RT Enzyme MixI，5μL Oligo dT Primer(50μM)，5μL Random 6mers(100μM)，Template RNA4×105copies，100μL RNase Free dH2O；
C)定量PCR反应体系试剂：20μL Taq DNA聚合酶，20U/μL；100μL 10×PCR缓冲液；20μL 10mM dNTP混合液；80μL 10×LC-green PLUS饱和荧光染料；640μL无核酸酶纯水；
D)20μL酶基因GAPDH内参引物各0.5μL，浓度为10pmol/μL，SEQ ID NO.41为正向引物，SEQ ID NO.42为反向引物。
实施例2
1)利用Genomics of Drug Sensitivity in Cancer(GDSC)数据库中54株小细胞肺癌细胞系的二代测序结果，如图1，与这些细胞对足叶乙甙的半数致死量(IC50)进行比对分析；
2)利用GDSC数据库中52株小细胞肺癌细胞系的二代测序结果，如图2，与这些细胞对顺铂的半数致死量(IC50)进行比对分析；
挑选出突变频率大于或等于10％的基因，相对高频的突变基因共有399个。
以下为分析结果：
1.根据GDSC数据库对SCLC的足叶乙甙耐药性定义为，大于等于16μM为耐药细胞系，小于则为敏感细胞系，54株细胞系的IC50分布见图1，在本发明中35株为敏感细胞株，19株为耐药细胞株；
2.对SCLC的顺铂耐药性定义为，大于等于10μM为耐药细胞系，小于则为敏感细胞系，52株细胞系的IC50分布见图2，在本发明中11株为敏感细胞株，41株为耐药细胞株。
3.利用ROC曲线，分别计算每个突变基因单独对细胞耐药性的预测，得到每个基因的曲线下面积，敏感度，特异性等诊断性检验指标，随机挑选出最适合诊断的高频突变组合预测足叶乙甙敏感性或顺铂敏感性：
以大于等于16μM的耐药细胞系为标准，小于16μM的敏感细胞系为对照，绘制ROC曲线来评估CSMD3、PCLO、RYR1和EPB41L3四个基因对EP化疗方案中足叶乙甙敏感性的判断能力。就单个基因而言，CSMD3以69.7％的AUC将化疗药物敏感组和非敏感组分开；PCLO以59.1％的AUC将药物敏感组和非敏感组分开；RYR1以63.1％的AUC将药物敏感组和非敏感组分开；EPB41L3以61.0％的AUC将药物敏感组和非敏感组分开；
对4个标志物的联合分析发现，这4种基因组合以80.4％的AUC将药物敏感组和非敏感组分开，敏感度：85.0％，特异性：70.6％，准确性：77.8％，预测足叶乙甙敏感性效能最高；
各个变量预测药物敏感性的ROC曲线下面积(见表1、图3)。
表1 CSMD3、PCLO、RYR1及EPB41L3预测足叶乙甙敏感性的ROC曲线下面积
| Genes | AUC | SE | 95％CI |
| CSMD3 | 0.697 | 0.077 | 0.546-0.848 |
| PCLO | 0.591 | 0.083 | 0.429-0.756 |
| RYR1 | 0.631 | 0.082 | 0.469-0.792 |
| EPB41L3 | 0.610 | 0.083 | 0.447-0.774 |
| CSMD3+PCLO+RYR1+EPB41L3 | 0.804 | 0.064 | 0.679-0.930 |
以大于等于10μM的耐药细胞系为标准，小于10μM的敏感细胞系为对照，绘制ROC曲线来评估RYR2、APOB、DNAH14、RELN、LAMA3、MLL3六个基因对EP化疗方案中顺铂敏感性的判断能力。就单个基因而言，RYR2以63.4％的AUC将化疗药物敏感组和非敏感组分开；APOB以59.1％的AUC将药物敏感组和非敏感组分开；DNAH14以63.4％的AUC将药物敏感组和非敏感组分开；RELN以63.4％的AUC将药物敏感组和非敏感组分开；LAMA3以61.0％的AUC将药物敏感组和非敏感组分开；MLL3以61.0％的AUC将药物敏感组和非敏感组分开；
对6个标志物的联合分析发现，这6种基因组合以90.9％的AUC将药物敏感组和非敏感组分开，敏感度：90.2％，特异性：81.8％，准确性：77.8％，预测顺铂敏感性效能最高；
各个变量预测药物敏感性的ROC曲线下面积(见表2、图4)。
表2 RYR2、APOB、DNAH14、RELN、LAMA3及MLL3预测顺铂敏感性的ROC曲线下面积
| Genes | AUC | SE | 95％CI |
| RYR2 | 0.653 | 0.088 | 0.480-0.826 |
| APOB | 0.634 | 0.083 | 0.471-0.797 |
| DNAH14 | 0.634 | 0.083 | 0.471-0.797 |
| RELN | 0.634 | 0.083 | 0.471-0.797 |
| LAMA3 | 0.610 | 0.086 | 0.441-0.779 |
| MLL3 | 0.610 | 0.086 | 0.441-0.779 |
| RYR2+APOB+DNAH14+RELN+LAMA3+MLL3 | 0.909 | 0.044 | 0.823-0.995 |
因此，本发明证明了采用CSMD3、PCLO、RYR1和EPB41L3这四个基因组合，能够较好地预测EP化疗方案中足叶乙甙敏感性；RYR2、APOB、DNAH14、RELN、LAMA3、MLL3六个基因组合，能够较好地预测EP化疗方案中顺铂敏感性。
本发明的设计构思不局限于此，凡未脱离本发明技术方案的内容，依据本发明的技术实质对以上实施例所作的任何形式的简单修改、等同变化与改型，仍属于本发明技术方案的保护范围。
序列表
<110> 南方医科大学珠江医院
<120> 一种预测小细胞肺癌对EP化疗敏感性的分子标志物及试剂盒
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Claims (4)
1.一种预测小细胞肺癌对EP化疗方案敏感性的分子标志物，其特征在于，EP化疗方案包括化疗药物足叶乙甙和顺铂，足叶乙甙的分子标志物包括基因CSMD3、PCLO、RYR1、EPB41L3；顺铂的分子标志物包括基因RYR2、APOB、DNAH14、RELN、LAMA3、MLL3。
2.一种由权利要求1所述的预测小细胞肺癌对EP化疗方案敏感性的试剂盒，其特征在于，试剂盒包括特异性引物：CSMD3、PCLO、RYR1、EPB41L3以及RYR2、APOB、DNAH14、RELN、LAMA3、MLL3的正向引物和反向引物，正向引物和反向引物的序列如SEQ ID 1~40所示。
3.由权利要求2所述的预测小细胞肺癌对EP化疗方案敏感性的试剂盒，其特征在于，试剂盒还包括逆转录反应体系试剂、定量PCR反应体系试剂以及酶基因GAPDH内参引物，内参引物序列如SEQ ID 41~42所示。
4.一种由权利要求1所述的预测小细胞肺癌对EP化疗方案敏感性的分子标志物的应用，其特征在于，应用于开发EP化疗方案敏感性检测试剂盒，预测患者对EP化疗方案的敏感性。
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