CN103993088A - 血清Exosomes来源的长链非编码RNA CASC2的应用方法 - Google Patents
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Abstract
本发明公开了一种血清外泌体(Exosomes)来源的长链非编码RNA(long no-coding RNA,LncRNA)CASC2的应用方法,即血清Exosomes来源的LncRNA CASC2用于制备胶质瘤高危人群的筛查、早期诊断或者胶质瘤患者的预后制剂。通过研究证实在胶质瘤患者血清中分离Exosomes后提取RNA,逆转录并进行实时荧光定量分析发现LncRNA CASC2表达水平下调。本发明利用血清Exosomes来源的LncRNA CASC2对胶质瘤早期诊断的特异性可达85.7%,灵敏度达到97.1%。通过检测胶质瘤患者血清Exosomes中LncRNA CASC2的表达水平,从而对胶质瘤患者做出早期、快速的无创性诊断。
Description
技术领域
本发明属于肿瘤分子生物学领域,具体涉及血清Exosomes来源的LncRNA CASC2在胶质瘤诊断中的应用方法。
背景技术
脑胶质瘤占颅内肿瘤的40.49%,可发生于中枢神经系统的任何部位。除少数星型细胞瘤、毛细胞型细胞瘤可视为良性,预后较好外,多数患者预后较差,平均存活3-5年。然而,传统手术很难准确识别肿瘤边界,对其进行完整的手术切除。目前胶质瘤诊断仍然处于以临床、病理学和影像学信息为基础的经验性阶段,胶质瘤的诊断和治疗总体上并没有明显的进步。目前,对胶质瘤的早期确诊和规范治疗还难以令人满意,因此,寻找特异性的胶质瘤早期诊断标志物对高危人群进行筛查,以便对胶质瘤患者进行早期诊断、早期治疗,提高患者生存率,一直是神经科学领域研究的主要任务。
Exosomes是一类直径为30-150nm的囊泡,含有RNA、脂质和蛋白质等成分。在血液,唾液,尿液,脑脊液和母乳等多种体液中均有存在,可在体液中来回穿梭,在细胞之间运送遗传物质和蛋白质。肿瘤在生长过程中会不断地将Exosomes释放到周围环境中去,同时Exoso mes可以在4℃条件下保存96小时或是-70℃下保存更长时间,而且通过在血清中分离Exosom es进行胶质瘤诊断克服了直接利用血液提取分子进行肿瘤诊断的血液中非检测物质的影响,使检验结果更加真实,精确。这些特点使得Exosomes有助于肿瘤的早期诊断和预后判断。通过研究在膀胱癌患者尿液来源的Exosomes中发现了PCA-3,TMPRSS2两个生物标志物;在黑色素瘤患者血浆Exosomes中肿瘤相关标志物CAV1表达水平明显增加,以此可诊断黑色素瘤,在肺癌模型发现Exosomes中的miRNA可作为肺癌的标志物。利用Exosomes来源的LncRNA CASC2来诊断脑胶质瘤不但能更好的对胶质瘤患者进行诊断,提早治疗,而且使得检验结果更加灵敏,特异。以上显示出血清Exosomes来源的LncRNA CASC2在胶质瘤早期诊断和筛查中的巨大潜力。
发明内容
本发明的目的在于提供一种血清Exosomes来源的LncRNA CASC2(cancer susceptibility candidate2)定位于染色体10q26.11,GeneBank登录号:NR_026939的应用方法,尤其是一种能够用于制备胶质瘤高危人群的筛查、早期诊断或者胶质瘤患者的预后制剂的应用方法。研究证实血清Exosomes来源的LncRNA CASC2可用于胶质瘤患者的诊断,Exosomes来源的LncRNA CASC2表达下调与胶质瘤组织验证结果具有一致性,且检验结果的灵敏性高,特异性好。因此Exosomes可用于胶质瘤高危人群的筛查和早期诊断和预后。
血清Exosomes来源的长链非编码RNA CASC2的应用方法,所述的血清Exosomes来源的长链非编码RNA CASC2用于制备胶质瘤高危人群的筛查、早期诊断或者胶质瘤患者的预后制剂,该长链非编码RNA CASC2的序列见SEQ NO:1。
所述的用于制备胶质瘤高危人群的筛查、早期诊断或者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:
CASC2正向引物5′-TTGACCCTTCCAGCTTCC-3′,
CASC2反向引物5′-CCATCCGCACATCACAAT-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 CASC2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 CASC2所用随机引物;(3)将cDNA实时定量PCR所用试剂,包括LncRNA CASC2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 CASC2实时荧光定量PCR特异性引物:
正向引物5′-TTGACCCTTCCAGCTTCC-3′,
反向引物5′-CCATCCGCACATCACAAT-3′,
U6snRNA内参特异性PCR引物:
正向引物为5′-ATTGGAACGATACAGAGAAGATT-3′,
正向引物为5′-GGAACGCTTCACGAATT TG-3′。
申请人通过荧光定量分析发现Lnc RNA CASC2在52例胶质瘤组织与12例正常人的脑组织中存在差异性表达(P=0.0240)(图1),且在胶质瘤组织中表达下调,进一步在血清中分离Exosomes用于LncRNA CASC2的检测,发现LncRNA CASC2的表达与正常人的表达存在明显的差异(P=0.0418)(图2),且与组织中的检测结果相一致。此方法可以检测各人群中LncRNA CASC2的表达水平,从而预测胶质瘤患者的患病风险,筛查高危人群,并对胶质瘤患者做出早期、快速的无创性诊断。本发明对胶质瘤早期诊断特异性好(图3),可达85.7%,灵敏度可达97.1%,只需在Exosomes提取RNA即可检测LncRNA CASC2的表达水平,操作简单,稳定性好。不仅可用于胶质瘤的早期诊断而且可以用于胶质瘤患者的大规模筛查和患病风险的预测,为胶质瘤的早期诊断和预测提供了强有力的技术支持,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 CASC2在胶质瘤组织与正常脑组织中的表达差异;
图2为实时荧光定量PCR分析Exosomes来源的LncRNA CASC2在胶质瘤患者与正常人中的表达差异;
图3为Roc分析Exosomes来源的LncRNA CASC2对胶质瘤早期诊断的特异性,灵敏性。
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。
实施例1制备长链非编码RNA CASC2用于胶质瘤高危人群的筛查、早期诊断或者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50ul
7.5×逆转录缓冲液200ml
8.10mM三磷酸碱基脱氧核苷酸100ul
9.40U/μl RNA酶抑制剂500ul
10.200U/μl MMLV逆转录酶50ul
11.Premix Ex Taq50ul
12.10μM LncRNA PRKAG2实时荧光定量PCR特异性引物30ul
CASC2正向引物5′-TTGACCCTTCCAGCTTCC-3′,
CASC2反向引物5′-CCATCCGCACATCACAAT-3′,
13.10μM U6snRNA特异性引物30ul
正向引物为5′-ATTGGAACGATACAGAGAAGATT-3′,
正向引物为5′-GGAACGCTTCACGAATT TG-3′。
实施例2
LncRNA CASC2在胶质瘤组织与正常脑组织中的表达差异的验证
1、收集待测胶质瘤组织放入盛有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 CASC2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:
逆转录第一步条件:65℃5分钟
逆转录第二步程序:25℃5分钟,42℃60分钟,70℃5分钟。
4、上海生工生物工程有限公司合成的CASC2特异性引物进行实时定量PCR:先将逆转录产物稀释5倍,混匀。20μL反应体系如下:
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。
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 CASC2在正常脑组织中的表达相比,52例脑胶质瘤患者中LncRNA CASC2的表达明显下调,差异有显著性(P=0.0240)。
实施例3
血清Exosomes来源的LncRNA CASC2用于胶质瘤诊断的特异性,灵敏性的检测
1、血清中Exosomes的分离
收集待测个体的外周血
1.1外周血血清的分离:采用血液促凝管,采集待测个体血液5ml。采血后1000rpm离心6min,吸取血清于EP管中-80℃保存。
1.2血清中Exosomes的分离:每个血清样本500μl中加入100μl的Total Exosome Isolation Reagent,涡旋混匀,4℃反应30分钟。室温下10000g离心10分钟。EP管底部存有Exosomes,用200μlPBS重悬Exosomes。(选择已商品化的Exosomes分离试剂盒)
2、Exosomes中RNA的提取纯化(选择已商品化的Exosomes分离纯化RNA试剂盒)
2.1Exosomes中RNA的提取:加入200μl的2X Denaturing Solution混匀,冰上孵化5分钟,再加入400μl的acid-Phenol:Chloroform,涡旋60秒。室温下12000g离心10分 钟,上清中含有RNA。
2.2RNA的纯化:将300μl上清液吸取到无酶的EP管中,加入375μl的无水乙醇,两者混匀。将混合液加入过滤柱中,10000g离心15秒,将收集管中的混合液倒掉。加入700μl的miRNA Wash Solution1,室温下10000g离心15s,倒掉收集管中的混合液。加入500μl的Wash Solution2/3,室温下10000g离心15秒,重复此步骤。将过滤柱放进收集管中10000g离心1分钟。将过滤柱放入新的收集管中加入35μl的Elution Solution,室温下10000g离心30秒,即可得到纯化的RNA。
3、采用实施例2中步骤3进行逆转录及实时定量的方法检测LncRNA CASC2
4、-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:与正常人LncRNA CASC2的表达相比,20例患者血清Exosomes中LncRNA CASC2的表达差异明显(P=0.0418),在胶质瘤患者中表达下调,该结果与实施例2组织中的检测结果相一致,说明通过血清exosome来源的LncRNA CASC2表达水平的检测,即可判断该患者是否罹患胶质瘤。同时,通过Roc分析发现利用血清Exosomes中LncRNA CASC2用于胶质瘤早期的诊断ROC curve下方的面积(Area Under Roc Curve,AUC)可达0.899,特异性可达85.7%,灵敏度达到97.1%。因此血清Exosomes来源的LncRNA CASC2能较好地用于胶质瘤的早期诊断。
本发明检测方法只需500μl血清便可分离出足够Exosomes用于LncRNA CASC2的表达水平检测,说明该方法具有较好地操作性。
Claims (4)
1.血清Exosomes来源的长链非编码RNA CASC2的应用方法,其特征在于,所述的血清Exosomes来源的长链非编码RNA CASC2用于制备胶质瘤高危人群的筛查、早期诊断或者胶质瘤患者的预后制剂,该长链非编码RNA CASC2的序列见SEQ NO:1。
2.根据权利要求1所述的长链非编码RNA CASC2的应用方法,其特征在于,所述的用于制备胶质瘤高危人群的筛查、早期诊断或者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 CASC2的应用方法,其特征在于,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 CASC2正向引物5′-TTGACCCTTCCAGCTTCC-3′,
LncRNA CASC2反向引物5′-CCATCCGCACATCACAAT-3′。
4.根据权利要求3所述的长链非编码RNA CASC2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 CASC2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 CASC2所用随机引物;(3)将cDNA实时定量PCR所用试剂,包括LncRNA CASC2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 CASC2实时荧光定量PCR特异性引物:
正向引物5′-TTGACCCTTCCAGCTTCC-3′,
反向引物5′-CCATCCGCACATCACAAT-3′,
U6snRNA内参特异性PCR引物:
正向引物为5′-ATTGGAACGATACAGAGAAGATT-3′,
正向引物为5′-GGAACGCTTCACGAATT TG-3′。
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