CN103981269A - 长链非编码rna crym-as1的应用方法 - Google Patents
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Abstract
本发明公开了一种长链非编码RNA(long no-coding RNA,LncRNA)CRYM-AS1的应用方法，即用于制备胶质瘤患者的预后制剂，特别是制备出实时荧光定量分析法预测胶质瘤患者预后的试剂盒。通过研究证实LncRNA CRYM-AS1在胶质瘤组织中表达上调，高表达LncRNA CRYM-AS1的胶质瘤患者比低表达LncRNA CRYM-AS1的胶质瘤患者预后差，因此，将Lnc RNA CRYM-AS1的表达用于胶质瘤患者的预后预测，可以为预测胶质瘤患者的预后提供强有力的分子生物学依据，具有深远的临床意义和推广性。
Description
技术领域
本发明属于肿瘤分子生物学领域，具体涉及LncRNA CRYM-AS1在制备胶质瘤患者预后试剂中的应用方法。
背景技术
胶质瘤起源于外胚层，约占神经系统肿瘤的40.49％，其发生部位，发病时间不受限制，可发生在中枢神经系统的任何部位，发病年龄从婴儿到老人均有。而且胶质瘤呈浸润性生长且与周围脑组织边界不清，即使是最先进的神经外科技术也难以做到病理学上的完全切除,因而胶质瘤术后复发率非常高，胶质瘤患者存活率低，平均存活时间3-5年。因此对胶质瘤患者进行预后判定，以便选择最佳治疗方案，显著提高患者生存率，成为神经外科领域亟待解决的重要课题。
lncRNA是一类转录本长度大于200bp，具有相对较长的核苷酸链的非编码RNA，lncRNA具有类型多、作用模式多和数量多的“三多”特点，因此，它所蕴含的信息十分丰富，其参与表达调控的分子机制也多种多样，不仅可以通过直接与靶基因结合来激活或抑制靶基因的表达，还能通过参与组蛋白修饰或募集转录因子而参与基因的表达调控。近年来，随着对lncRNA的不断认识其在临床诊断、化疗敏感性和预后评价等方面的应用受到研究者的关注。有研究在尿液中发现lncRNA PCA3在前列腺癌中高度过表达，可为前列腺癌的诊断提供新的思路，研究发现HOTAIR在原发乳腺肿瘤中的表达增加，利用HOTAIR的表达水平可以用来有效预测癌转移和死亡。由此可见，lncRNA作为诊断、预后和治疗的分子标志物具有巨大潜力。
发明内容
本发明的目的是提供一种LncRNA CRYM-AS1的应用方法，即LncRNA CRYM-AS1(CRYMantisense RNA1)定位于染色体16p12.2，GeneBank登录号：NR-026675能作为胶质瘤预后的判断标准，用于制备胶质瘤患者的预后制剂，更进一步能够提供一种性价比高，易于推广应用的胶质瘤预后预测试剂盒。
长链非编码RNA CRYM-AS1的应用方法，所述的长链非编码RNA CRYM-AS1用于制备胶质瘤患者的预后制剂，该长链非编码RNA CRYM-AS1的序列见SEQ NO:1。
所述的用于制备胶质瘤患者的预后制剂包括实时荧光定量PCR检测试剂。
所述的实时荧光定量PCR检测试剂包括进行实时荧光定量PCR的特异性引物：
LncRNA CRYM-AS1正向引物：5'-CTGGAAGGCACGTAAGC-3'，
LncRNA CRYM-AS1反向引物5'-GAGGCGGCGATGTATTG-3'。
所述的实时荧光定量PCR检测试剂为试剂盒，
该试剂盒包括：(1)从胶质瘤组织中抽提总RNA所用试剂，包括RNA稳定溶液、Trizol试剂、三氯甲烷、异丙醇、无酶水；(2)以总RNA为模板将LncRNA CRYM-AS1逆转录为cDNA所用试剂，包括逆转录缓冲液、三磷酸碱基脱氧核苷酸、RNA酶抑制剂、MMLV逆转录酶以及LncRNA CRYM-AS1所用随机引物；(3)将cDNA实时定量PCR所用试剂，包括LncRNACRYM-AS1实时荧光定量PCR特异性引物、U6snRNA内参特异性PCR引物、实时荧光定量SYBR染料、无酶水；
LncRNA CRYM-AS1实时荧光定量PCR特异性引物：
LncRNA CRYM-AS1正向引物：5'-CTGGAAGGCACGTAAGC-3'，
LncRNA CRYM-AS1反向引物5'-GAGGCGGCGATGTATTG-3'。
U6snRNA内参特异性PCR引物：
其正向引物为5′-ATTGGAACGATACAGAGAAGATT-3′，
反向引物为5′-GGAACGCTTCACGAATT TG-3′。
在前期研究工作中，申请人通过DIANA-LncBase软件分析与miR-101竞争性结合MRE序列的LncRNA时，发现了LncRNA CRYM-AS1。申请人在胶质瘤组织中提取RNA,逆转录后进行实时荧光定量PCR分析LncRNA CRYM-AS1的表达，发现：LncRNA CRYM-AS1在胶质瘤中表达上调，与正常人脑组织中的表达差异显著。且LncRNA CRYM-AS1表达水平不同的胶质瘤患者中预后存在明显差异，经Kaplan-Meier生存分析发现LncRNA CRYM-AS1高表达的胶质瘤患者生存率明显低于低表达的患者；多因素Cox比例风险模型的预后分析发现LncRNA CRYM-AS1的相对危险度>1，表明LncRNA CRYM-AS1高表达的患者预后较低表达的患者预后差，所以LncRNA CRYM-AS1的高表达是胶质瘤患者预后风险预测的独立危险因素。据此，申请人提出利用LncRNA CRYM-AS1制备用于胶质瘤患者预后的制剂。
将LncRNA CRYM-AS1用于胶质瘤患者预后的检测方法：(1)收集待测个体术后切除的胶质瘤组织，抽提总RNA；(2)以总RNA为模板将CRYM-AS1逆转录为cDNA；(3)用CRYM-AS1特异性引物进行实时荧光定量PCR扩增，获得相对表达量2ΔΔCT，当2ΔΔCT>1.72时提示CRYM-AS1为高表达。
利用本发明的检测制剂可以检测LncRNA CRYM-AS1在胶质瘤患者中的表达水平，为胶质瘤患者预后预测提供了强有力的分子生物学基础，具有深远的临床意义和推广性。
附图说明
图1为实时荧光定量PCR分析LncRNA CRYM-AS1在胶质瘤组织与正常脑组织中的表达差异；
图2为LncRNA CRYM-AS1与37例胶质瘤患者预后的关系；
具体实施方式
以下结合实施例旨在进一步说明本发明，而非限制本发明。
在前期研究工作中，申请人通过DIANA-LncBase软件分析与miR-101竞争性结合MRE序列的LncRNA时，发现了LncRNA CRYM-AS1。申请人在胶质瘤组织中提取RNA,逆转录后进行实时荧光定量PCR分析LncRNA CRYM-AS1的表达，发现：LncRNA CRYM-AS1在胶质瘤组织中表达上调，与正常人脑组织中的表达差异显著(P＝0.0025)(图1)。且LncRNACRYM-AS1表达水平不同的胶质瘤患者中预后存在明显差异，经Kaplan-Meier生存分析发现LncRNA CRYM-AS1高表达的胶质瘤患者生存率明显低于低表达的患者(P＝0.0012)(图2)，多因素Cox比例风险模型的预后分析发现LncRNA CRYM-AS1的相对危险度>1(RR＝1.017)，表明LncRNA CRYM-AS1高表达的患者预后较低表达的患者预后差，LncRNA CRYM-AS1的高表达是胶质瘤患者预后风险预测的独立危险因素。
实施例1 制备LncRNA CRYM-AS1用于胶质瘤患者预后的试剂盒(50次反应)
1.RNA稳定溶液50ml
2.异丙醇100ml
3.三氯甲烷100ml
4.Trizol50ml
5.无酶水10ml
6.1μM随机逆转录引物50μl
7.5×逆转录缓冲液200ml
8.10mM三磷酸碱基脱氧核苷酸100μl
9.40U/μl RNA酶抑制剂500μl
10.200U/μl MMLV逆转录酶50μl
11.Premix Ex Taq50μl
12.10μM Lnc RNA CRYM-AS1特异性引物30μl
正向引物5'-CTGGAAGGCACGTAAGC-3'，
反向引物5'-GAGGCGGCGATGTATTG-3'。
13.10μM U6snRNA特异性引物30μl
正向引物为5'-ATTGGAACGATACAGAGAAGATT-3'
反向引物为5'-GGAACGCTTCACGAATTTG-3'。
实施例2 胶质瘤组织中Lnc RNA CRYM-AS1的检测
1、胶质瘤组织的保存：收集待测胶质瘤组织存放于盛有RNA稳定溶液的冻存管中，放至-80℃冰箱备用。
2、组织中RNA的抽提：取适量标本于经180℃烘烤6-8h后的研钵中加入液氮研磨标本，研磨至粉末状后于研钵中加入1ml Trizol研钵标本，研磨成液体状后用移至tube管，加氯仿200μl/mlTrizol于Tube中，用手震荡15-30s，冰上放置5min，4℃12000g离心15min；小心取上层水相入新tube中，加入预冷的异丙醇0.5ml/mlTrizol混匀，-20℃冰箱静置20min，4℃12000g离心10min；弃上清，加入75％DEPC水稀释的乙醇1-2ml混匀，4℃7500g离心5min，尽量弃上清，室温干燥5-10min，加入DEPC水10-20μl溶解RNA。分光光度计测RNA的浓度及质量，OD260/280比值在1.8-2.0之间，-80℃保存。
3、LncRNA CRYM-AS1逆转录：使用Thermo公司的逆转录试剂盒。20μL逆转录反应的体系如下：
| 成分 | 剂量/管 |
| 随机逆转录引物(1μM) | 1μl |
| RNA样本 | 2ug |
| 无酶水 | To12μl |
逆转录第一步条件：65℃5分钟
| 成分 | 剂量/管 |
| 5×逆转录缓冲液 | 4μl |
| 磷酸碱基脱氧核苷酸(10mM) | 2μl |
| RNA酶抑制剂(40U/μl) | 1μl |
| MMLV逆转录酶(200U/μl) | 1μl |
| 第一步PCR的产物 | 12ug |
| 20μl |
逆转录第二步程序：25℃5分钟，42℃60分钟，70℃5分钟。
4、CRYM-AS1特异性引物进行实时定量PCR：CRYM-AS1特异性引物序列由上海生工生物工程有限公司合成。
先将逆转录产物稀释5倍，混匀。20μL反应体系如下：
| 成分 | 剂量/管 |
| SYBR Premix Ex Taq | 10μl |
| CRYM-AS1特异性引物(10μM) | 0.5μl |
| cDNA产物 | 1μl |
| 无酶水 | To20μl |
实时荧光定量PCR反应程序：95℃3分钟,40个循环，95℃10秒，60℃30秒。
LncRNA CRYM-AS1特异性引物序列为：
正向引物5'-CTGGAAGGCACGTAAGC-3'，
反向引物5'-GAGGCGGCGATGTATTG-3'。
5、-2ΔΔCT指标的测定：本实验数据采用相对定量的分析方法，U6作为内参基因，数据利用软件GraphPad Prism进行分析。分析发现，与LncRNA CRYM-AS1在正常脑组织中的表达相比，52例脑胶质瘤患者中LncRNA CRYM-AS1的表达明显上调，差异极显著性(P＝0.0025)。
6、预后判断
通过对实验所采用的52例胶质瘤患者随访统计资料发现，15例患者在随访时由于手机停机或者换号或者其他原因联系不上，最后能联系上的胶质瘤患者或家属为37例，这37例患者或家属接受后续的随访分析。我们详细询问了这些患者或家属首次发病的时间，治疗情况，复发状况及死亡时间等，随访时间为1-46个月。在所选取的胶质瘤患者中，选取荧光定量PCR分析的表达值为参考标准，所得结果降序排列后高于中位数的为LncRNA CRYM-AS1高表达，共20例，其他为LncRNA CRYM-AS1低表达，共17例。经Kaplan-Meier生存分析，LncRNA CRYM-AS1高表达患者的生存期较LncRNA CRYM-AS1低表达的患者短，愈后差。差异有统计学意义(P＝0.0012)。多因素Cox比例风险模型的预后分析发现LncRNACRYM-AS1的相对危险度>1(RR＝1.017)，表明LncRNA CRYM-AS1高表达的患者预后较低表达的患者预后差，LncRNA CRYM-AS1的高表达是胶质瘤患者预后风险预测的独立危险因素。
以上研究表明，LncRNA CRYM-AS1可作为胶质瘤患者预后的特异性分子标志物。
Claims (4)
1.长链非编码RNA CRYM-AS1的应用方法，其特征在于，所述的长链非编码RNACRYM-AS1用于制备胶质瘤患者的预后制剂，该长链非编码RNA CRYM-AS1的序列见SEQNO:1。
2.根据权利要求1所述的长链非编码RNA CRYM-AS1的应用方法，其特征在于，所述的用于制备胶质瘤患者的预后制剂包括实时荧光定量PCR检测试剂。
3.根据权利要求2所述的长链非编码RNA CRYM-AS1的应用方法，其特征在于，所述的实时荧光定量PCR检测试剂包括进行实时荧光定量PCR的特异性引物：
LncRNA CRYM-AS1正向引物：5'-CTGGAAGGCACGTAAGC-3'，
LncRNA CRYM-AS1反向引物5'-GAGGCGGCGATGTATTG-3'。
4.根据权利要求3所述的长链非编码RNA CRYM-AS1的应用方法，其特征在于，所述的实时荧光定量PCR检测试剂为试剂盒，
该试剂盒包括：(1)从胶质瘤组织中抽提总RNA所用试剂，包括RNA稳定溶液、Trizol试剂、三氯甲烷、异丙醇、无酶水；(2)以总RNA为模板将LncRNA CRYM-AS1逆转录为cDNA所用试剂，包括逆转录缓冲液、三磷酸碱基脱氧核苷酸、RNA酶抑制剂、MMLV逆转录酶以及LncRNA CRYM-AS1所用随机引物；(3)将cDNA实时定量PCR所用试剂，包括LncRNACRYM-AS1实时荧光定量PCR特异性引物、U6snRNA内参特异性PCR引物、实时荧光定量SYBR染料、无酶水；
LncRNA CRYM-AS1实时荧光定量PCR特异性引物：
正向引物：5'-CTGGAAGGCACGTAAGC-3'，
反向引物：5'-GAGGCGGCGATGTATTG-3'。
U6snRNA内参特异性PCR引物：
正向引物为5′-ATTGGAACGATACAGAGAAGATT-3′，
反向引物为5′-GGAACGCTTCACGAATT TG-3′。
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