CN107988368B - 胶质瘤诊断标志物circ 9:33948374|33948587及应用 - Google Patents

胶质瘤诊断标志物circ 9:33948374|33948587及应用 Download PDF

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CN107988368B
CN107988368B CN201711454314.9A CN201711454314A CN107988368B CN 107988368 B CN107988368 B CN 107988368B CN 201711454314 A CN201711454314 A CN 201711454314A CN 107988368 B CN107988368 B CN 107988368B
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李沛瑶
武明花
刘涛
张严
余志斌
刘洋
赵春花
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Xiangya Hospital of Central South University
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Abstract

本发明属于生物技术领域，公开了一种胶质瘤诊断标志物circ 9:33948374|33948587及应用。通过荧光定量PCR技术对胶质瘤患者血清外泌体中circ 9:33948374|33948587的含量进行分析，发现胶质瘤患者血清外泌体中circ 9:33948374|33948587表达水平相比于正常对照组明显降低(p＝0.0007)，ROC曲线分析则显示其对胶质瘤具有较高诊断价值，AUC＝0.962，p＝0.007，敏感度和特异度分别为100％和92.30％。

Description

胶质瘤诊断标志物circ 9:33948374|33948587及应用
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外泌体circRNA标志物、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
脑胶质瘤是中枢神经系统较常见的恶性肿瘤之一，根据WHO可分为四个等级，其中1～2级为低级别，预后较好，3～4级为高级别胶质瘤，其患者预后较差，且术后存活时间不超过五年。尽管目前诊疗技术有了很大的进展，但疗效仍然不尽如人意。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。
circRNA是一类广泛且多样地存在于哺乳动物细胞中、具有调控基因表达作用的内源性非编码RNA分子，具有共价闭合的环形结构，广泛存在于各种细胞中，也是继microRNA(miRNA)后RNA家族的最新研究热点。近年来，随着深度测序技术的广泛应用和生物物理和信息学技术的快速发展，人们发现人类许多外显子的转录本可被非线性地反向剪接或通过基因重排而形成circRNA，且它们在所有剪接转录本中占了相当大的比例。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。目前，由于circRNA具有丰富性、稳定性、高保守性和时空特异性等特点，在肿瘤诊断标志物方面正发挥越来越大的作用。
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circ 9:33948374|33948587，其序列如SEQ NO:1所示。
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circ 9:33948374|33948587的含量。
所述的胶质瘤诊断试剂盒含有检测circ 9:33948374|33948587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circ 9:33948374|33948587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(from serum)，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：circ 9:33948374|33948587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circ 9:33948374|33948587这种血清外泌体中的环状RNA，并发现其对胶质瘤具有较高的诊断价值；通过血清外泌体circRNA标志物和诊断试剂盒的研制和应用，可以使得胶质瘤的诊断更加方便易行，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circ 9:33948374|33948587在正常人血清外泌体与胶质瘤患者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circ 9:33948374|33948587对胶质瘤诊断的特异性，灵敏性。
具体实施方式
一、研究对象
病例组为2016年1月至2017年6月在湖南省肿瘤医院收集的30例胶质瘤血清样本。对照组为同期进行社区疾病筛查的健康个12例，按性别和年龄(±5岁)与病例进行频数匹配。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)血清外泌体的制备：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 IsolationReagent(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，于沉淀中加入200μl Trizol(MRC公司)使沉淀重悬，将悬液移至新的1.5mltube管，补Trizol至1ml。
(2)外泌体中RNA的抽提：将上述重悬液于冰上静止裂解15分钟。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 Trizol混匀，冰上静置20min，4℃，12000rpm离心10min；弃上清，加入75％DEPC水稀释的乙醇1-2ml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。
(3)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RNA10μl，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
第一步逆转录的产物 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。
(4)实时荧光定量PCR：汉恒生物科技(上海)有限公司合成的circ 9:33948374|33948587特异性引物(见序列表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：
Figure BDA0001528965370000031
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)数据分析：：采用2-ΔΔCT表示circ 9:33948374|33948587相对于参照的表达倍数，其中△CT＝CT样本–CT内参，△△CT＝△CT–△CT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(引物见序列表SEQNO:4和5)，ΔCT正常为正常血清外泌体的ΔCT平均值，数据利用软件GraphPad Prism及SPSS 17.0进行分析。
三、研究结果
病例组血清外泌体circ 9:33948374|33948587表达水平较对照组显著下调(p＝0.0007)。具体数据如图1所示。
ROC曲线分析显示，作为生物标记物对胶质瘤具有较高诊断价值(AUC＝0.962，p＝0.007，敏感度和特异度分别为100％和92.3％)。详细结果见图2。
序列表
<110> 中南大学湘雅医院
<120> 胶质瘤诊断标志物circ 9:33948374|33948587及应用
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 214
<212> RNA
<213> 智人(Homo sapiens)
<400> 1
ucauccgucc uuggcucagg auuuggagag cuugcaccac caaaaauggc aaacaucacc 60
agcucccaga uuuuggacca guugaaagcu ccgaguuugg gccaguuuac caccacccca 120
aguacacagc agaauaguac aagucacccu acaacuacua cuucuuggga ccucaagccc 180
ccaacauccc aguccucagu ccucagucau cuug 214
<210> 2
<211> 18
<212> DNA
<213> 未知(Unknown)
<400> 2
acatcccagt cctcagtc 18
<210> 3
<211> 18
<212> DNA
<213> 未知(Unknown)
<400> 3
aactcggagc tttcaact 18
<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circ 9:33948374|33948587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，circ 9:33948374|33948587，其序列如SEQ NO:1所示；胶质瘤患者血清外泌体中circ 9:33948374|33948587表达水平比正常人低。
2.根据权利要求1所述的应用，其特征在于，检测circ 9:33948374|33948587在血清外泌体中表达量的试剂含有检测circ 9:33948374| 33948587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，检测circ 9:33948374|33948587在血清外泌体中表达量的试剂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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Publication number Priority date Publication date Assignee Title
WO2009122444A2 (en) * 2008-03-31 2009-10-08 Council Of Scientific & Industrial Research Method for the diagnosis of higher- and lower-grade astrocytoma using biomarkers and diagnostic kit thereof
CN106435004A (zh) * 2016-12-20 2017-02-22 南京医科大学 血清外泌体miRNAs标志物在地方性砷中毒早期诊断中的应用

Patent Citations (2)

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Publication number Priority date Publication date Assignee Title
WO2009122444A2 (en) * 2008-03-31 2009-10-08 Council Of Scientific & Industrial Research Method for the diagnosis of higher- and lower-grade astrocytoma using biomarkers and diagnostic kit thereof
CN106435004A (zh) * 2016-12-20 2017-02-22 南京医科大学 血清外泌体miRNAs标志物在地方性砷中毒早期诊断中的应用

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Title
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that predicts outcome in malignant glioma patients;ATSUSHI KAWAGUCHI等;《INTERNATIONAL JOURNAL OF ONCOLOGY 》;20111021;第40卷;721-730 *

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