CN113604570A - circSHKBP1作为非小细胞肺癌诊断标志物的应用 - Google Patents
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Abstract
本发明公开了circSHKBP1在作为非小细胞肺癌诊断标志物的应用。本发明公开了circSHKBP1在制备非小细胞肺癌诊断试剂、诊断试剂盒或芯片中的应用。本发明公开了circSHKBP1在制备非小细胞肺癌预后评估的产品中的应用。本发明还公开了circSHKBP1在非小细胞肺癌患者生存指标评估中的应用。本发明通过实验证明，血清外泌体circSHKBP1可作为非小细胞肺癌诊断的特异性靶点，其在非小细胞肺癌患者中会明显的上调表达，并且高表达的circSHKBP1预示非小细胞肺癌患者预后不良，提供了一种无创的、易获得、可重复的非小细胞肺癌的特异性靶点，在临床诊疗有着重要的价值。
Description
技术领域
本发明属于生物技术领域，特别涉及circSHKBP1作为非小细胞肺癌诊断标志物的应用。
背景技术
肺癌是最常见的恶性肿瘤之一，是世界范围内癌症相关死亡的主要原因。非小细胞肺癌(Non-small cell lung cancer,NSCLC)是肺癌的主要类型，约占所有肺癌病例的85％，生存率低于15％。肺腺癌(LUAD)和肺鳞癌(LUSC)是NSCLC最普遍的亚型。手术切除、化疗和放疗是治疗NSCLC的主要方式，但疗效有限。虽然近年来诊断和治疗有所改善，但NSCLC的预后仍然很差。究竟原因之一，目前仍然缺乏NSCLC特异性的诊疗标志物，大部分患者明确诊断已经处于晚期，或者治疗后容易复发，缺乏预后评估的特异性指标，所以寻找NSCLC潜在的诊疗特异性靶点，对于提高NSCLC诊疗水平有重要的意义。
外泌体是直径40-160nm的细胞外囊泡的特殊亚型。宿主细胞产生的外泌体可被相邻或远处的细胞摄取并影响其生物学功能。外泌体包含不同的生物分子，如非编码RNA(ncRNAs)、DNA、脂质、蛋白质，并与许多疾病，包括癌症有关。含有功能性ncRNAs的外泌体可促进NSCLC的进展。来自癌症相关成纤维细胞的外泌体的ncRNAs使NSCLC细胞具有顺铂耐药。外泌体携带的ncRNAs加速NSCLC的进展和有氧糖酵解。外泌体传递的ncRNAs通过抑制抑癌基因的表达促进NSCLC细胞增殖、迁移和侵袭。因此，外泌体在非小细胞肺癌进展中的作用值得进一步探讨。
其中外泌体携带一种ncNRAs类型，即环状RNA(circRNA)参与了NSCLC的发生发展。circRNA是一类由线性RNA分子产生的具有闭合环状结构的ncRNAs。许多环状circRNA被鉴定并发现参与细胞周期、增殖、侵袭、迁移、凋亡、血管生成、上皮-间充质转化和免疫逃逸等多个生物学过程。所以circRNA是潜在的NSCLC靶点。虽然关于circRNA在NSCLC的机制研究与分子靶点的筛选有了很多的研究，但是仍然缺乏NSCLC特异性的circRNA诊疗靶点。
发明内容
本发明旨在至少解决现有技术中存在的技术问题之一。为此，本发明提出一种将血清外泌体circSHKBP1作为非小细胞肺癌特异性诊疗标志物在非小细胞肺癌诊断及预后评估的应用。
本发明的上述技术目的是通过以下技术方案得以实现的：
本发明提供了circSHKBP1作为非小细胞肺癌诊断标志物的应用。
本发明提供了circSHKBP1在制备非小细胞肺癌诊断试剂、诊断试剂盒或芯片中的应用。
本发明提供了circSHKBP1在制备非小细胞肺癌预后评估的产品中的应用。
本发明提供了circSHKBP1在非小细胞肺癌患者生存指标评估中的应用。
本发明提供的上述应用，是通过检测受试者血清的外泌体中circSHKBP1的表达量来判断。
优选的，所述血清来自于受试者的外周血。
本发明所提供的上述应用，均为仅通过检测受试者外周血清的外泌体中circSHKBP1基因表达水平就能够获得检测结果，申请人意外地发现，该circSHKBP1基因表达水平在非小细胞肺癌患者与正常人之间存在明显的差异表达，因而可以通过对受试者外周血清的外泌体中circSHKBP1在核酸和/或蛋白水平的检测来对受试者是否患有非小细胞肺癌进行诊断，同时还能对非小细胞肺癌患者的预后情况进行有效评估。
其中，诊断是指对受试者是否患有非小细胞肺癌做出判断，预后评估是指对非小细胞肺癌患者的可能出现的病程和结局进行评估，其中circSHKBP1基因表达水平越高，非小细胞肺癌患者的预后情况越差。
本发明还提供了一种试剂盒，包括能够定量检测circSHKBP1表达量的试剂。
优选的，所述试剂盒用于非小细胞肺癌的诊断和/或预后评估。
优选的，所述试剂在核酸和/或蛋白水平上对circSHKBP1进行定量检测。
本领域技术人员应当理解，凡是以检测circSHKBP1蛋白的表达量为目的，用于判断待测样品是否来自非小细胞肺癌患者的诊断试剂盒、评价体系、诊断方法都属于本发明的保护范围。
本发明的有益效果是：本发明发现血清外泌体circSHKBP1可作为非小细胞肺癌诊断的特异性靶点，其在非小细胞肺癌患者中会明显的上调表达，并且高表达的circSHKBP1预示非小细胞肺癌患者预后不良，提供了一种无创的、易获得、可重复的非小细胞肺癌的特异性靶点，在临床诊疗有着重要的价值。
附图说明
本发明的上述和/或附加的方面和优点从结合下面附图对实施例的描述中将变得明显和容易理解，其中：
图1为本发明实施例1中RT-PCR测试图；
图2为本发明实施例1中Sanger测序图；
图3为本发明实施例2中非小细胞肺癌外泌体形态和大小测试图；
图4为本发明实施例2中非小细胞肺癌外泌体粒径分布图；
图5为本发明实施例2中circSHKBP1在非小细胞肺癌患者外周血血清外泌体RNA及正常对照外周血血清外泌体的RNA中的相对表达量对比图。
图6为本发明实施例3中非小细胞肺癌患者外周血清的外泌体中circSHKBP1表达水平与患者的总生存期的关系图。
具体实施方式
下面结合具体实施例对本发明做进一步的说明。
实施例1：
1.样本收集
非小细胞肺癌患者外周血血清来源：全部由深圳市龙华区人民医院提供，均为首诊肺癌，诊断明确，病理清楚。
正常对照外周血血清来源：正常对照外周血血清全部由深圳市龙华区人民医院体检中心提供，均为健康人群常规查体获得。
分别提取非小细胞肺癌患者外周血血清外泌体RNA和正常对照外周血血清外泌体的RNA。
2.PCR检测
对从非小细胞肺癌患者外周血血清外泌体提取的RNA进行PCR检测，其中收敛引物用于检测SHKBP1的线性形式，而包含反向剪接连接的发散引物仅用于扩增circSHKBP1的圆形形式，实验结果如图1所示，由图1可知扩增引物对cDNA有明显的结合，但对基因组DNA(gDNA)没有产物，说明检测的circSHKBP1不是由基因重组产生的。
3.Sanger测序
对从非小细胞肺癌患者外周血血清外泌体提取的RNA进行Sanger测序，测序结果如图2所示，由图2可知扩增的circSHKBP1的头-尾反向剪接连接，表明circSHKBP1来源于SHKBP1基因的第11外显子和第12外显子。
实施例2：
测试circSHKBP1在非小细胞肺癌患者外周血血清外泌体RNA及正常对照外周血血清外泌体的RNA中的表达量，其中透射电镜观察的非小细胞肺癌外泌体形态和大小测试结果见图3，动态光散射检测非小细胞肺癌外泌体粒径分布见图4，circSHKBP1在非小细胞肺癌患者外周血血清外泌体RNA及正常对照外周血血清外泌体的RNA中的相对表达量对比图见图5。
实施例3：
在大数据中，下载大量非小细胞肺癌的患者的生存数据，用生存曲线方法(Kaplan-Meier)分析和比较非小细胞肺癌患者外周血清的外泌体中circSHKBP1表达水平与患者的总生存期(over survival,OS)的关系图，关系图如图6所示，由图6可知，非小细胞肺癌患者外周血清的外泌体中circSHKBP1表达水平与患者的总生存期呈负相关，即非小细胞肺癌患者外周血清的外泌体中circSHKBP1表达水平越高，患者的总生存期越短，非小细胞肺癌患者外周血清的外泌体中circSHKBP1表达水平越高，患者的总生存期越长。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。
Claims (9)
1.circSHKBP1在作为非小细胞肺癌诊断标志物的应用。
2.circSHKBP1在制备非小细胞肺癌诊断试剂、诊断试剂盒或芯片中的应用。
3.circSHKBP1在制备非小细胞肺癌预后评估的产品中的应用。
4.circSHKBP1在非小细胞肺癌患者生存指标评估中的应用。
5.根据权利要求1-4任一项所述的应用，其特征在于：通过检测受试者血清的外泌体中circSHKBP1的表达量来判断。
6.根据权利要求5所述的应用，其特征在于：所述血清来自于受试者的外周血。
7.一种试剂盒，其特征在于，包括能够定量检测circSHKBP1表达量的试剂。
8.根据权利要求7所述的一种试剂盒，其特征在于，所述试剂盒用于非小细胞肺癌的诊断和/或预后评估。
9.根据权利要求7所述的一种试剂盒，其特征在于，所述试剂在核酸和/或蛋白水平上对circSHKBP1进行定量检测。
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| US20160109453A1 (en) * | 2013-05-24 | 2016-04-21 | Ait Austrian Institute Of Technology Gmbh | Lung Cancer Diagnostic Method and Means |
Non-Patent Citations (5)
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| JINGJING PAN等: "Detection of serum long non-coding RNA UCA1 and circular RNAs for the diagnosis of bladder cancer and prediction of recurrence", INT J CLIN EXP PATHOL, vol. 12, no. 8, pages 2951 - 2958 * |
| QING LIU等: "Suppression of tumor growth and metastasis in Shkbp1 knockout mice circ_0000936 lung", CANCER GENE THER, vol. 29, no. 6, pages 709 - 721 * |
| WENBIAO CHEN等: "Exosomal circSHKBP1 participates in non-small cell lung cancer progression through PKM2-mediated glycolysi", MOL THER ONCOLYTICS, vol. 24, pages 470 - 485 * |
| 刘擎: "Shkbp1对肺癌发生发展的作用及其机制研究", 中国优秀硕士学位论文全文数据库医药卫生科技辑, no. 1, pages 072 - 728 * |
| 王兵等: "环状RNAs在肿瘤防治中的研究进展", 临床肿瘤学杂志, vol. 25, no. 04, pages 362 - 267 * |
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