CN110205320B - 一种lncRNA分子linc00998及其在胶质瘤治疗/预后评估中的应用 - Google Patents
一种lncRNA分子linc00998及其在胶质瘤治疗/预后评估中的应用 Download PDFInfo
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Abstract
一种lncRNA分子linc00998及其在胶质瘤治疗/预后评估中的应用。本发明公开了一种lncRNA分子及其在胶质瘤辅助诊断中的应用。该lncRNA分子名称为linc00998,其核苷酸序列如SEQ ID NO.1所示。linc00998在胶质瘤组织中的表达量明显高于正常脑组织,生存分析linc00998高表达组预后更差。提示其可作为胶质瘤分子标志物或分子靶点,应用于胶质瘤患者预后判断或靶向治疗,并有利于进一步阐明胶质瘤的发生发展的分子机制及信号通路等,具有较高的应用前景和理论研究价值。
Description
技术领域
本发明属于分子生物学和肿瘤学领域,具体涉及一种lncRNA分子及其在胶质瘤临床治疗、预后评估或科学研究中的应用。
背景技术
胶质瘤(glioma)是中枢神经系统最常见的原发性恶性肿瘤,约占所有颅内原发肿瘤45%。尽管随着科技的发展,针对胶质瘤的治疗出现了手术治疗、放疗、化疗、基因治疗、生物治疗、中医中药、免疫治疗等多种治疗手段,但患者的预后仍然较差。近年来,胶质瘤的发病率逐渐升高,但患者预后却没有明显改善。据统计,间变型胶质瘤经传统手术及术后辅助标准放化疗的中位生存期仅2-5年,而胶质母细胞瘤(glioblastoma,GBM)的中位生存期仅12-14个月。目前针对胶质瘤预后判断的分子主要包括IDH1、1p19q、TERT突变等。由于异质性的存在,上述分子标志物无法完全预测胶质瘤患者预后。因此,进一步寻找胶质瘤相关分子标志物,以进一步明确其发病机制,并开发出可应用于临床早期诊断、治疗和预后评估的试剂或产品,对改善胶质瘤患者的预后具有重要意义。
长链非编码RNA(Long Noncoding RNA,lncRNA),是一类转录本长度超过200nt的不编码蛋白的RNA分子。在基因水平,转录水平,转录后水平,翻译水平,翻译后水平等多个层面,lncRNA通过介导信号转导、作为分子阻断剂、与蛋白结合、提供中心平台等方式来发挥分子功能,并与肿瘤的发生、发展密切相关。linc00998位于7q31.1,有研究表明,linc00998在重度抑郁症患者中表达下调。(Ye N et al.Intergenic variants maypredispose to major depression disorder through regulation of long non-codingRNA expression[J].Gene,2017,601:21-26.)。然而,linc00998在胶质瘤患者中的表达情况及对患者预后的影响尚无文献报道。
发明内容
为了解决现有技术的不足,本发明的目的在于提供linc00998分子在胶质瘤治疗或预后评估中应用。
本发明的另一个目的在于提供linc00998分子在制备胶质瘤治疗药物中的应用。
本发明所采取的技术方案是:
一种在胶质瘤组织中高表达的lncRNA分子,名称为linc00998,其核苷酸序列如SEQ ID NO.1所示。
lncRNA分子linc00998在胶质瘤治疗/预后评估中的应用。
lncRNA分子linc00998在制备胶质瘤治疗/预后评估试剂或试剂盒中的应用。
用于检测lncRNA分子linc00998的试剂在制备胶质瘤治疗/预后评估试剂或试剂盒中的应用。
所述用于检测lncRNA分子linc00998的试剂包括用于扩增所述lncRNA分子的特异性引物对。
所述用于扩增lncRNA分子linc00998的特异性引物对,序列如下所示:
F:5’-TGCTTTTGGTGCTGCCTGTT-3’(SEQ ID NO.4);
R:5’-AGCCTCTAAAGCGCAAAGGT-3’(SEQ ID NO.5)。
lncRNA分子linc00998作为胶质瘤治疗靶点的应用。
一种用于胶质瘤预后评估的试剂盒,其中包括:
(1)用于扩增lncRNA分子linc00998的特异性引物对;
(2)标准DNA模板;
(3)PCR反应液。
一种检测lncRNA的方法,包括如下步骤:
(1)提取样品总RNA;
(2)制备样品cDNA
(3)定量扩增lncRNA;
所述lncRNA为linc00998。
本发明的有益效果是:本发明首次发现与胶质瘤相关的分子标志物linc00998,其可作为胶质瘤分子标志物或靶点,应用于胶质瘤临床预后判断或靶向治疗,并有利于进一步阐明胶质瘤发病的分子机制,极具应用前景和理论价值。
附图说明
图1为基因芯片检测发现linc00998在脑胶质瘤组织中表达上调。
图2qRT-PCR检测linc00998在169例脑胶质瘤组织及30例正常组织中表达升高散点图。
图3linc00998低表达组脑胶质瘤患者和高表达组脑胶质瘤患者的生存曲线图。
具体实施方式
下面结合实施例对本发明作进一步的说明,但并不局限于此。以下实施例中所采用的分子生物学实验技术包括PCR扩增、质粒提取、质粒转化、DNA片段连接、酶切、凝胶电泳等,如无特殊说明,通常按照常规方法操作,具体可参见《分子克隆实验指南》(第三版)(Sambrook J,Russell DW,Janssen K,Argentine J.黄培堂等译,2002,北京:科学出版社),或按照制造厂商所建议的条件。
实施例1胶质瘤相关lncRNA分子的筛选。
1、病例采集。
收集于2008年10月-2017年10月就诊于中国医科大学附属盛京医院行手术治疗,且术后病理证实为胶质瘤的病例169例。排除行辅助放化疗病例及复发胶质瘤病例及肿瘤活检病例。另外收集30例因外伤行手术治疗的病例作为对照。排除合并其他肿瘤病人,其中男91例,女78例。样本临床资料详见表1-1,试验中标本采集均已取得病人知情同意,并从中国医科大学附属盛京医院伦理委员会得到认可授权。随机选择其中3例胶质瘤组织及3例正常组织用于芯片研究,其余病例标本用于进一步验证。
表1-1 收集169例胶质瘤病例的相关临床参数
2、样本采集。
1)首先准备好用于保存组织的冻存管,且用黑色油性记号笔在冻存管上标明样本编号、收集日期等信息;
2)术中切取胶质瘤组织(具有5年以上操作手术经验的神经外科医师操作);
3)将切除的胶质瘤组织标本均分成2份,其中1份送病理,另1份放入备好的冻存管中并拧紧,迅速投入液氮罐中冻存;
4)填写标本登记单,写明样本编号、分组、来源患者名称、病案号、样本收集日期及样本处理过程等情况;
5)待病理性质确诊后取出标本开展下一步研究。
3、总RNA样品制备、纯化及质检
1)TRIZOL法提取RNA样品;
2)采用RNeasy柱对总RNA样品进行纯化;
3)RNA定量和质量控制(采用Nanodrop ND 1000紫外可见分光光度计确定样品总RNA浓度和质量)。
Nanodrop分光光度计对3例胶质瘤及3例正常组织总RNA测定结果如表1-2所示。
表1-2 Nanodrop分光光度计对胶质瘤及正常脑组织总RNA测定
*对于分光光度计中,O.D.A260/A280比值应接近2.0为纯的RNA(1.8和2.1之间的比值是可接受的)。在O.D.A260/A230比值应大于1.8。
4、检测胶质瘤样本和正常组织样本中lncRNA表达差异。
利用二代测序技术,对三例胶质瘤组织及三例正常脑组织中的lncRNA表达情况进行检测,选择胶质瘤中表达上升最明显的lncRNA分子。经序列比对,发现,发现linc00998分子在胶质瘤组织中表达明显上调(图1所示)。
实施例2大样本验证linc00998在胶质瘤组织中表达上调。
1、提取组织中RNA及质检。
对收集的169例胶质瘤组织及30例正常组织使用TRIZOL法提取RNA;采用RNeasy柱对总RNA样品进行纯化;采用Nanodrop ND 1000紫外可见分光光度计确定样品总RNA浓度和质量。
Nanodrop分光光度计对169例胶质瘤及30例正常组织总RNA测定结果如表1-3所示。
表1-3 Nanodrop分光光度计对胶质瘤及正常脑组织总RNA测定
*对于分光光度计中,O.D.A260/A280比值应接近2.0为纯的RNA(1.8和2.1之间的比值是可接受的)。在O.D.A260/A230比值应大于1.8。
2、引物设计。
依据实时定量荧光PCR引物设计原则,使用Primer5.0软件设计实时定量荧光PCR引物。本研究使用的引物全部由上海生工生物技术有限公司合成,其中GAPDH为内参校准基因。
引物详细序列见表1-4:
表1-4 实时定量荧光PCR引物
3、染料一步法qRT-PCR检测linc00998的表达。
PCR(反应体系如表1-5)。
表1-5 PCR反应体系
反应条件:42℃5min,95℃10S,之后[95℃3S,60℃30S]40个循环。
测定CT值,以GAPDH为内参。
4、qRT-PCR结果与计算。
各样品的目的基因和管家基因分别进行qRT-PCR反应。根据绘制的梯度稀释DNA标准曲线,各样品目的基因和管家基因的浓度结果直接由机器生成。每个样品的目的基因浓度除以其管家基因的浓度,即为此样品此基因的校正后的相对含量。统计软件SPSS 20.0进行数据分析。用,以2-△△Ct表示各样本的表示linc00998的相对表达量。数据符合正态分布时釆用两组独立样本的t检验,不符合正态分布时用两组独立样本的秩和检验,P<0.05时具有统计学意义。
qRT-PCR结果的散点图如图2所示,可以看到:以GAPDH作为参照,胶质瘤组织中的linc00998与正常组织比较,表达上调(P<0.001)。
实施例3。
为了进一步研究linc00998作为分子标志物预测脑胶质瘤患者预后的可能性,发明人对病例进行了随访,分析比较脑胶质瘤患者生存时间与linc00998的关系。
1、病例随访
对入组病例进行随访,每月电话随访患者生存情况,直至患者死亡、随访满5年或随访结束。若患者死于脑胶质瘤及相关并发症,标记为1,否则标记为0。
2、绘制生存曲线。
根据PCR中linc00998的相对表达量进行排序,得到linc00998的中位表达量。将表达量大于等于中位表达量的85例定义为高表达,低于中位表达量的84例定义为低表达。使用K-M法计算高表达及低表达组患者的生存曲线。
如图3所示,linc00998低表达组胶质瘤患者的平均生存时间明显长于linc00998高表达组。
以上实验表明,linc00998在胶质瘤组织中的表达明显上调,且linc00998高表达组患者生存期更短,极有可能成为分子标志物或靶点,在胶质瘤临床早期诊断、预后判断或靶向治疗等方向上具有广阔的应用前景。
SEQUENCE LISTING
<110> 中国医科大学附属盛京医院
<120> 一种lncRNA分子linc00998及其在胶质瘤治疗/预后评估中的应用
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Claims (4)
1.用于检测lncRNA分子linc00998的试剂在制备胶质瘤预后评估试剂或试剂盒中的应用。
2.根据权利要求1所述的应用,其特征在于,所述用于检测lncRNA分子linc00998的试剂包括用于扩增所述lncRNA分子的特异性引物对。
3.根据权利要求2所述的应用,其特征在于,所述用于扩增lncRNA分子linc00998的特异性引物对,序列如下所示:
F:5’- TGCTTTTGGTGCTGCCTGTT -3’(SEQ ID NO .4);
R:5’- AGCCTCTAAAGCGCAAAGGT -3’(SEQ ID NO .5)。
4.根据权利要求1所述的应用,其特征在于,所属试剂盒中包括:
(1)用于扩增lncRNA分子linc00998的特异性引物对;
(2)标准DNA模板;
(3)PCR反应液。
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