CN107937543B - 胶质瘤诊断标志物circ10:72715111|72715902及应用 - Google Patents
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Abstract
本发明属于生物技术领域，公开了胶质瘤诊断标志物circ10:72715111|72715902及应用。在本发明中，首次发现胶质瘤患者血清外泌体中circ10:72715111|72715902的表达水平相比于对照组明显升高(p＝0.0322)，ROC分析则显示其对胶质瘤具有较高诊断价值(AUC＝0.882，P<0.005，灵敏度和特异性分别为79.2％和100％)。因此通过检测胶质瘤患者血清外泌体中circ10:72715111|72715902的表达水平，可对胶质瘤患者做出早期、快速的无创性诊断。
Description
技术领域
本发明属于生物技术领域，涉及一种用于胶质瘤诊断的血清circRNA标志物circ10:72715111|72715902、以及检测该标志物的试剂用于制备胶质瘤诊断制剂的应用、还有试剂盒。
背景技术
胶质瘤又叫胶质细胞瘤或神经胶质瘤,其发病率高,恶性程度高，治疗复杂且预后差,是对人类健康造成巨大威胁的一类疾病，其中成年人多形性胶质母细胞瘤(glioblastoma multiforme,GBM)中位发病年龄约为65岁，中位生存期仅为14个多月，5年病死率在全身肿瘤中仅次于胰腺癌和肺癌,位列第3位。在神经系统肿瘤中，原发性中枢神经系统(central nervous system,CNS)肿瘤的发生率约为5:100 000～10:100 000，其中胶质瘤就占40％，而恶性胶质瘤约占胶质瘤的50％。恶性胶质瘤给患者带了巨大痛苦，给社会造成巨大经济负担,一直以来都是肿瘤研究的热点。因此，寻找胶质瘤诊断标志物对患者进行诊断分析，尽早确诊病情，并相应地选择合理的后续治疗方案，提高生存率，是神经科学领域亟待解决的研究任务。
环状RNA(Circular RNA,circRNA)是一类具有闭合环状结构的内源性非编码RNA(noncoding RNA,ncRNA)，主要由前体RNA(pre-mRNA)通过可变剪切加工产生，circRNA广泛存在于所有真核生物中，并且非常稳定。目前，circRNA的研究已经成为了RNA研究领域的新热点，研究发现circRNA在转录本中占有相当大的比例，有的表达丰度甚至显著高于其他转录本。同时，circRNA对基因的表达有重要调控作用，在生物的发育进程中发挥了重要的生物学功能，如充当miRNA海绵、作为内源性RNA以及生物标记物，在疾病的诊断与治疗中也发挥重要作用。研究发现circRNA在一些疾病的发生中扮演了重要角色，包括动脉硬化、神经系统紊乱、糖尿病和癌症的发生。近年来逐渐发现外泌体中也含有大量的CircRNA，可能发挥重要作用。外泌体是指包含了复杂RNA和蛋白质的小膜泡(30-150nm)，现今，其特指直径在40-100nm的盘状囊泡。外泌体是细胞与细胞间通讯的重要的分子，参与诸多生理与病理过程。外泌体所携带的RNA统称为外泌体来源RNA，其中circRNA具有丰富性、稳定性、高保守性和时空特异性等特点，有望作为液体活检分子标志物，在精准医学发展上有着光明的前景。
发明内容
本发明的第一个目的是：提供一种用于胶质瘤诊断的血清外泌体circRNA标志物。
主要内容包括：一种用于胶质瘤诊断的血清外泌体circRNA标志物circ10:72715111|72715902，其序列如SEQ NO:1所示。该circRNA位于人类的第10号染色体上，全长792bp。
本发明的第二个目的是，提供检测所述的circRNA标志物在血清外泌体中表达量的试剂在制备胶质瘤诊断制剂中的应用。
本发明的第三个目的是，提供一种胶质瘤诊断试剂盒，其能够测定血清外泌体中的circ10:72715111|72715902的含量。
所述的胶质瘤诊断试剂盒，含有检测circ10:72715111|72715902含量的PCR引物。优选引物的序列如SEQ NO:2和3所示。
所述的胶质瘤诊断试剂盒，除circ10:72715111|72715902的引物外，还含有从血清中提取外泌体、由外泌体中提取RNA并进行逆转录及荧光定量PCR的所有试剂。
包括：
(1)提取血清外泌体所需试剂：Total Exosome Isolation Reagent(fromserum)，可由Invitrogen公司购得，货号4478360；
(2)提取外泌体RNA所需试剂：Trizol试剂、三氯甲烷、异丙醇、75％乙醇、无酶水；
(3)逆转录所需试剂：随机引物(Random Primer)、无酶水、5×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；
(4)荧光定量PCR所需试剂：circ10:72715111|72715902上下游引物、GAPDH内参上下游引物、SYBR染料、无酶水。
本发明的有益效果在于：
首次发现胶质瘤患者的血清外泌体中circ10:72715111|72715902相比正常血清外泌体照组显著上调(p＝0.0322)。ROC曲线分析显示circ10:72715111|72715902作为生物标记物对胶质瘤具有较高诊断价值(AUC＝0.882，灵敏性和特异性分别为79.2％和100％)。通过该环状RNA在胶质瘤诊断分析中的应用，可以使得胶质瘤的诊断更加方便准确，为临床医生快速准确掌握患者病情，为提高临床治疗效果奠定基础，并为发现具有潜在治疗价值的新型小分子药物靶标提供帮助。
附图说明
图1为实时荧光定量PCR分析circ10:72715111|72715902在胶质瘤血清与正常血清外泌体中的表达差异；
图2为ROC曲线分析血清外泌体来源的circ10:72715111|72715902对胶质瘤早期诊断的特异性，灵敏性。
具体实施方式
以下结合实施方式旨在进一步说明本发明，而非限制本发明。
一、研究对象
24例胶质瘤患者的血清样本由湘雅医院提供，6例正常血清样本为同期进行社区疾病筛查的健康个体。用于研究的样本为同期收取，采样、分装、保存条件均一致。
二、研究方法
1.胶质瘤/正常血清外泌体中RNA的抽提
取血清200μl于2000g常温离心30分钟，用微量移液器抽取上层清液至新的600μl离心管，加入40μl外泌体提取试剂(Total Exosome Isolation Reagent(from serum)，货号4478360，Invitrogen公司)轻轻上下颠倒摇匀，4℃孵育45分钟。孵育结束后10000g常温离心10分钟，弃掉上清液，所得沉淀即为血清中的外泌体。于沉淀中加入200μl Trizol(MRC公司)使沉淀重悬，将悬液移至新的1.5ml tube管，补Trizol至1ml。冰上裂解15min。裂解结束后4℃，12000rpm离心10min，上清液移至新的tube管。加氯仿200μl于Tube中，用手震荡15-30s，冰上放置5min，4℃，12000rpm离心15min；小心取上层水相入新tube中，加入预冷的异丙醇0.5ml混匀，冰上静置大于20min，4℃，12000rpm离心10min；弃上清，加入75％DEPC水稀释的乙醇1ml混匀，4℃，7500rpm离心5min，尽量弃上清，室温干燥5-10min，加入无酶水10μl溶解RNA。-80℃保存。
2.cDNA制备
按照逆转录试剂盒(Thermo公司)说明书进行逆转录反应。反应总体积为20μl(总RNA 10μl，Random primer 1μl,无酶水1μl，5×Reaction Buffer 4μl，RI 1μl，RT 1μl和10mM dNTP 2μl)。
| 成分 | 剂量/管 |
| 随机逆转录引物(1μM) | 1μl |
| RNA样本 | 10μl |
| 无酶水 | To 12μl |
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(40U/μl) | 1μl |
| MMLV逆转录酶(200U/μl) | 1μl |
| 第一步逆转录产物 | 12μl |
| 总体积 | 20μl |
逆转录第二步程序：25℃5分钟，42℃60分钟，70℃5分钟。
3.实时荧光定量PCR
采用汉恒生物科技(上海)有限公司合成的特异性引物(引物序列见SEQ NO：2和3)进行实时定量PCR：先将逆转录产物稀释10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。
4.数据分析：采用2-ΔΔCT表示胶质瘤血清外泌体的circ10:72715111|72715902相对于正常血清外泌体的表达倍数，其中△CT＝CT样本–CT内参，ΔΔCT＝ΔCT胶质瘤–ΔCT正常。本实验数据采用相对定量的分析方法，GAPDH作为内参基因(引物序列见SEQ NO：4和5)，数据利用软件GraphPad Prism及SPSS 17.0进行分析。
三、研究结果
胶质瘤患者的血清外泌体中circ10:72715111|72715902相比正常血清外泌体照组显著上调(p＝0.0322)。具体数据如图1所示。ROC曲线分析显示circ10:72715111|72715902作为生物标记物对胶质瘤具有较高诊断价值(AUC＝0.882，灵敏性和特异性分别为79.2％和100％)，详细结果见图2。
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agccugcuau ugaacacuug guagcuaaca uggggcugag uuuagugagg ugacuguugc 300
acaauagaau auuuuuacua ugguacaccc uaagggauaa uauaucugau cuuagccuga 360
uuagaccaua uugguaaaca accccaauaa aucagaacua ugcuaccuca cacaguuacu 420
cuggucuccu ggaaauuuaa uagggugugg augaccaagg gucugucugc ugguguaaac 480
caacuggauu gcauggaaac agggcagcuc uccuaggaaa gccaauuacc uuggcauuua 540
ucuuaauuaa uuuggcuuaa uauauggaag aaguaauggu ugagguuaau cagguauggg 600
uuucuauguu uaucauuaaa guugcuaaaa ccugguaucu uacuuuugca ggucaucuug 660
uaaauuuugc cuauauaaau uaacacaucc ccuguauguc auaacaacca uguaagggag 720
aguguuuucc ugcucugucu uagguauugg ugaugugacc uguucacgca ggggaaacuu 780
gaacauucgc ag 792
<210> 2
<211> 22
<212> DNA
<213> 未知(Unknown)
<400> 2
tcctgctctg tcttaggtat tg 22
<210> 3
<211> 22
<212> DNA
<213> 未知(Unknown)
<400> 3
gtgaggtaga aacggtatct tg 22
<210> 4
<211> 19
<212> DNA
<213> 未知(Unknown)
<400> 4
atcatcagca atgcctcct 19
<210> 5
<211> 18
<212> DNA
<213> 未知(Unknown)
<400> 5
catcacgcca cagtttcc 18
Claims (4)
1.检测circ10:72715111|72715902在血清外泌体中表达量的试剂在制备胶质瘤诊断制剂中的应用，所述的circ10:72715111|72715902序列如SEQ NO:1所示，所述的circ7:42148226|42148468在胶质瘤患者的血清外泌体中的表达量高于正常人。
2.根据权利要求1所述的应用，其特征在于，所述的检测circ10:72715111|72715902在血清外泌体中表达量的试剂含有检测circ10:72715111| 72715902含量的PCR引物。
3.根据权利要求2所述的应用，其特征在于，引物的序列如SEQ NO:2和3所示。
4.根据权利要求1或2或3所述的应用，其特征在于，所述的检测circ10:72715111|72715902在血清外泌体中表达量的试剂除检测circ10:72715111| 72715902的引物外，还含有从血清中提取外泌体、由外泌体中提取RNA并进行逆转录及荧光定量PCR的所有试剂。
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Non-Patent Citations (3)
| Title |
|---|
| Circular RNAs are abundant，conserved and associated with ALU repeats;Jeck WR et al.;《RNA》;20131231;第19卷(第3期);第141-157页 * |
| Glioblastoma and calcium signaling-analysis of calcium toolbox expression;Fablen Petel et al.;《Int.J.Dev.Biol》;20151231;第59卷;第412页右栏第3-4段和第413页左栏第3段 * |
| Jeck WR et al..Circular RNAs are abundant，conserved and associated with ALU repeats.《RNA》.2013,第19卷(第3期),第141-157页. * |
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