CN103966337B - 血清Exosomes来源的长链非编码RNA PRKAG2-AS1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)PRKAG2-AS1的应用方法,即血清Exosomes来源的LncRNA?PRKAG2-AS1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?PRKAG2-AS1表达水平下降。利用本发明的制剂对胶质瘤早期诊断的特异性可达85.7%,灵敏度达到88.2%。通过检测胶质瘤患者血清Exosomes中LncRNA?PRKAG2-AS1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LncRNAPRKAG2-AS1在制备胶质瘤患者预后试剂中应用方法。
背景技术
胶质瘤约占颅内肿瘤的40.49%。脑肿瘤中胶质细胞瘤发病率最高,大脑半球发生的胶质瘤约占全部胶质瘤的51.4%。由于大多数胶质瘤呈浸润性生长且与周围脑组织边界不清,即使是最现代的神经外科技术也难以做到病理学上的全切除,因而胶质瘤术后复发率非常高,且随着手术和复发次数的增加,其恶性程度有增加的趋势。目前而言胶质瘤的诊断和治疗方法虽处于不断改进阶段,但是胶质瘤患者生存率并没有明显提高。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,而且一经诊断,绝大多数均为中晚期,远远不能适应对胶质瘤进行高危人群筛查和早期诊断的需求。因此,寻找无创伤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来源于多囊泡体,是由活细胞分泌而来的大小介于30-100nm的含有RNA、脂质和蛋白质的微囊泡。Exosomes广泛存在于血清,尿液,细胞培养上清,唾液等各种体液中,可在体液中来回穿梭,在细胞之间运送遗传物质和蛋白质。研究发现,在顺铂诱导急性肾损伤的大鼠模型的尿液Exosome中发现Fetuin-A表达上调,由此尿液Exosome中Fetuin-A的检测可作为诊断标志物;在膀胱癌患者尿液来源的Exosomes中还发现了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;肿瘤细胞来源的Exosomes中miRNA的表达谱可作为卵巢癌的诊断标记物。因此Exosomes可用于肿瘤的早期诊断,也可作为靶向药物的载体进行疾病治疗。Exosomes具有在血清中稳定存在的特性,含有特异性的物质且可保护RNA免受RNA酶的作用,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。利用Exosomes来源的LncRNAPRKAG2-AS1来诊断脑胶质瘤不仅减少了患者的痛苦和不便,而且提高了检验结果的灵敏性,特异性。以上显示出血清Exosomes来源的LncRNAPRKAG2-AS1在胶质瘤早期诊断和筛查中的巨大潜力。
LncRNAPRKAG2-AS1(proteinkinase,AMP-activated,gamma2non-catalyticsubunit)定位于染色体7q36.1,GeneBank登录号:NR_038926。有研究证实PRKAG2可在TP53的信号通路中发挥作用,并且PRKAG2与癌症患者的存活率密切相关。
发明内容
本发明的目的在于提供一种血清Exosomes来源的LncRNAPRKAG2-AS1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LncRNAPRKAG2-AS1可用于胶质瘤患者的诊断,Exosomes来源的LncRNAPRKAG2-AS1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RNAPRKAG2-AS1的应用方法,所述的血清Exosomes来源的长链非编码RNAPRKAG2-AS1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RNAPRKAG2-AS1的序列见SEQNO: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具体包括实时荧光定量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:
LncRNAPRKAG2-AS1正向引物:5'-CAGTTCTCATCAAATAGGGTGT-3',
LncRNAPRKAG2-AS1反向引物:5'-TATTGTCCCACTGAATGCTC-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LncRNAPRKAG2-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LncRNAPRKAG2-AS1所用随机引物;(3)将cDNA实时定量PCR所用试剂,包括LncRNAPRKAG2-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;
LncRNAPRKAG2-AS1实时荧光定量PCR特异性引物:
正向引物:5'-CAGTTCTCATCAAATAGGGTGT-3',
反向引物:5'-TATTGTCCCACTGAATGCTC-3'。
U6snRNA内参特异性PCR引物:
正向引物为5′-ATTGGAACGATACAGAGAAGATT-3′,
反向引物为5′-GGAACGCTTCACGAATTTG-3′。
申请人通过癌症基因图集TCGA数据库分析发现LncRNAPRKAG2-AS1在正常人与胶质母细胞瘤中的表达存在明显差异(P=0.0156)(图1)。通过荧光定量分析发现LncRNAPRKAG2-AS1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=0.0005)(图2),且在胶质瘤组织中表达下调,进一步在血清中分离Exosomes用于LncRNAPRKAG2-AS1的检测,发现LncRNAPRKAG2-AS1的表达与正常人的表达存在明显的差异(P=0.0306)(图3),且与组织中的检测结果相一致。此方法可以检测各人群中LncRNAPRKAG2-AS1的表达水平,从而预测胶质瘤的患病风险,筛查易感人群,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4),可达85.7%,灵敏度可达88.2%,只需在Exosomes提取RNA即可检测LncRNAPRKAG2-AS1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为癌症基因图集TCGA数据库中LncRNAPRKAG2-AS1在胶质母细胞瘤与正常脑组织中的表达差异;
图2为实时荧光定量PCR分析LncRNAPRKAG2-AS1在胶质瘤组织与正常脑组织中的表达差异;
图3为实时荧光定量PCR分析Exosomes来源的LncRNAPRKAG2-AS1在胶质瘤患者与正常人中的表达差异;
图4为Roc分析Exosomes来源的LncRNAPRKAG2-AS1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RNAPRKAG2-AS1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/μlRNA酶抑制剂500ul
10.200U/μlMMLV逆转录酶50ul
11.PremixExTaq50ul
12.10μMLncRNAPRKAG2-AS1实时荧光定量PCR特异性引物30ul
LncRNAPRKAG2-AS1正向引物:5'-CAGTTCTCATCAAATAGGGTGT-3',
LncRNAPRKAG2-AS1反向引物:5'-TATTGTCCCACTGAATGCTC-3';
13.10μMU6snRNA特异性引物30ul
正向引物为5′-ATTGGAACGATACAGAGAAGATT-3′
反向引物为5′-GGAACGCTTCACGAATTTG-3′。
实施例2
LncRNAPRKAG2-AS1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1mlTrizol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、LncRNAPRKAG2-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 | 2μg |
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 |
三磷酸碱基脱氧核苷酸(10mM) | 2μl |
RNA酶抑制剂(40Μ/μl) | 1μl |
MMLV逆转录酶(200Μ/μl) | 1μl |
第一步PCR的产物 | 12μg |
20μl |
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PRKAG2-AS1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:
成分 | 剂量/管 |
SYBR Premix Ex Taq | 10μl |
PRKAG2-AS1特异性引物(10μM) | 0.5μl |
cDNA产物 | 1μl |
无酶水 | To20μl |
PCR的特异性引物:
正向引物:5'-CAGTTCTCATCAAATAGGGTGT-3',
反向引物:5'-TATTGTCCCACTGAATGCTC-3'。
U6snRNA内参特异性PCR引物:
正向引物为5′-ATTGGAACGATACAGAGAAGATT-3′,
反向引物为5′-GGAACGCTTCACGAATTTG-3′。
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GraphPadPrism进行分析。分析发现,与LncRNAPRKAG2-AS1在正常脑组织中的表达相比,52例脑胶质瘤患者中LncRNAPRKAG2-AS1的表达明显下调,差异有显著性(P=0.0005)。
实施例3
血清Exosomes来源的LncRNAPRKAG2-AS1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TotalExosomeIsolationReagent,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2XDenaturingSolution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miRNAWashSolution1,室温下10000g离心15s,倒掉收集管中的混合液。加入500μl的WashSolution2/3,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ElutionSolution,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LncRNAPRKAG2-AS1
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GraphPadPrism进行分析。分析发现:与正常人LncRNAPRKAG2-AS1的表达相比,20例患者血清Exosomes中LncRNAPRKAG2-AS1的表达差异明显(P=0.0306),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LncRNAPRKAG2-AS1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LncRNAPRKAG2-AS1用于胶质瘤早期的诊断ROCcurve下方的面积(AreaUnderRocCurve,AUC)可达0.792,特异性可达85.7%,灵敏度达到88.2%。因此血清Exosomes来源的LncRNAPRKAG2-AS1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LncRNAPRKAG2-AS1的表达水平检测,说明该方法具有较好地操作性。
Claims (3)
1.检测血清Exosomes来源的长链非编码RNAPRKAG2-AS1表达量的试剂在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的制剂中的用途,该长链非编码RNAPRKAG2-AS1的序列如SEQNO:1所示。
2.根据权利要求1所述的用途,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所述的用途,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:
LncRNAPRKAG2-AS1正向引物:5'-CAGTTCTCATCAAATAGGGTGT-3',
LncRNAPRKAG2-AS1反向引物:5'-TATTGTCCCACTGAATGCTC-3'。
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