CN107937528A - 胶质瘤预后标志物hsa_circ_0125365及应用 - Google Patents

胶质瘤预后标志物hsa_circ_0125365及应用 Download PDF

Info

Publication number
CN107937528A
CN107937528A CN201711453665.8A CN201711453665A CN107937528A CN 107937528 A CN107937528 A CN 107937528A CN 201711453665 A CN201711453665 A CN 201711453665A CN 107937528 A CN107937528 A CN 107937528A
Authority
CN
China
Prior art keywords
circ
glioma
hsa
samples
circrna
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN201711453665.8A
Other languages
English (en)
Other versions
CN107937528B (zh
Inventor
武明花
冯紫阳
***
刘长红
刘涛
佘晓玲
孙英男
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Xiangya Hospital of Central South University
Original Assignee
Xiangya Hospital of Central South University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Xiangya Hospital of Central South University filed Critical Xiangya Hospital of Central South University
Priority to CN201711453665.8A priority Critical patent/CN107937528B/zh
Publication of CN107937528A publication Critical patent/CN107937528A/zh
Application granted granted Critical
Publication of CN107937528B publication Critical patent/CN107937528B/zh
Expired - Fee Related legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00Oligonucleotides characterized by their use
    • C12Q2600/118Prognosis of disease development
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00Oligonucleotides characterized by their use
    • C12Q2600/158Expression markers
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00Oligonucleotides characterized by their use
    • C12Q2600/178Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Analytical Chemistry (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Hospice & Palliative Care (AREA)
  • Biophysics (AREA)
  • Oncology (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

本发明属于生物技术领域,公开了一种胶质瘤预后标志物hsa_circ_0125365及应用。即检测胶质瘤组织来源的circRNA hsa_circ_0125365表达量的试剂用于制备胶质瘤患者的预后制剂。通过研究证实胶质瘤中circRNA hsa_circ_0125365表达量较高的患者,拥有更高的术后生存率。通过检测胶质瘤患者胶质瘤组织中circRNA hsa_circ_0125365的表达水平,从而对胶质瘤患者做出预后判断。

Description

胶质瘤预后标志物hsa_circ_0125365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预后的circRNA标志物、以及检测该标志物的试剂用于制备胶质瘤预后制剂的应用、还有试剂盒。
背景技术
脑胶质瘤是成年人最为频发的脑肿瘤疾病,占颅内肿瘤的40.49%。从确诊开始,脑胶质瘤患者平均生存寿命不超过五年。为了诊治胶质瘤这种与遗传物质相关性极高的恶性疾病,必须在分子生物学水平,从遗传信息表达的方面来探索其发病机理。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,手术后的生存率不容乐观。因此,寻找胶质瘤预后标志物对患者进行预后分析,以及提高胶质瘤患者的术后生活质量,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,并具有丰富性、稳定性、高保守性和时空特异性等特征,有作为诸多疾病分子标志物的潜力。
发明内容
本发明的第一个目的是:提供一种用于胶质瘤患者预后的胶质瘤组织来源的circRNA标志物hsa_circ_0125365,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标志物在胶质瘤组织中表达量的试剂在制备胶质瘤预后制剂中的应用。
本发明的第三个目的是,提供一种胶质瘤预后试剂盒,能够测定胶质瘤组织中的hsa_circ_0125365的含量。
所述的胶质瘤预后试剂盒,含有检测hsa_circ_0125365含量的PCR引物。优选引物的序列如SEQ NO:2和3所示。
所述的胶质瘤预后试剂盒,除hsa_circ_0125365的引物外,还含有从胶质瘤组织中提取RNA并进行逆转录及荧光定量PCR的所有试剂。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hsa_circ_0125365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hsa_circ_0125365所用随机引物;
(3)将cDNA实时定量PCR所用试剂,包括circRNA hsa_circ_0125365实时荧光定量PCR特异性引物、GAPDH内参特异性PCR引物、实时荧光定量SYBR染料、无酶水。
本发明研究证实胶质瘤组织来源的circRNA hsa_circ_0125365可用于胶质瘤患者预后分析,胶质瘤组织来源的circRNA hsa_circ_0125365表达量与患者术后生存率具有相关性。因此,可用于胶质瘤患者的预后分析。
申请人通过荧光定量PCR对26例胶质瘤组织与12例正常脑组织进行分析发现,hsa_circ_0125365在两者中的表达差异明显(P<0.0001),之后对其中15例胶质瘤患者进行生存曲线分析,发现胶质瘤组织来源的circRNA hsa_circ_0125365与患者的生存率相关,含量越高,生存率越高。此法为胶质瘤的预后分析提供了强有力的技术支持,有助于提高胶质瘤患者的术后生活质量,制订术后治疗方案,提高生存率,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hsa_circ_0125365在胶质瘤组织与正常脑组织中表达差异;
图2为ROC曲线分析脑组织来源的hsa_circ_0125365对胶质瘤与正常脑组织差异的特异性、灵敏性;hsa_circ_0125365作为生物标记物对胶质瘤具有较高诊断价值(AUC=0.939,P=0.040,敏感度和特异度分别为90.0%和78.3%)。
图3为生存曲线分析胶质瘤组织来源的circRNA hsa_circ_0125365表达量与患者生存率的相关性。
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:制备检测circRNA hsa_circ_0125365表达量的试剂用于制备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.Trizol(来自Molecular Research Center公司)50ml
5.无酶水10ml
6.1μM随机逆转录引物(Thermo公司)50μl
7.5×逆转录缓冲液(Thermo公司)200ml
8.10mM三磷酸碱基脱氧核苷酸(Thermo公司)100μl
9.40U/μl RNA酶抑制剂(Thermo公司)500μl
10.200U/μl MMLV逆转录酶(Thermo公司)50μl
11.Premix Ex Taq(Thermo公司)50μl
12.10μM circRNA hsa_circ_0125365实时荧光定量PCR特异性引物30μl
circRNA hsa_circ_0125365正向引物:5'-CCAAGCAGCTCACTACGATA-3',
circRNA hsa_circ_0125365反向引物:5'-CAAGCAGGTAGGAGATTCCA-3';
13.10μM GAPDH特异性引物30μl
正向引物为5′-ATCAAGATCATTGCTCCTCCTGAG-3′,
反向引物为5′-CTGCTTGCTGATCCACATCTG-3′。
实施例2:circRNA hsa_circ_0125365在胶质瘤组织的表达量与预后的关系
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,研磨成液体状后移至tube管,加氯仿200μl于Tube中,用手震荡15-30s,冰上放置15min,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DEPC水10-20μl溶解RNA,-80℃保存。
3、circRNA hsa_circ_0125365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
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、汉恒生物科技(上海)有限公司合成的circRNA hsa_circ_0125365特异性引物进行实时定量PCR:先将逆转录产物稀释10倍,混匀。20μl反应体系如下:
PCR的特异性引物:
正向引物:5'-CCAAGCAGCTCACTACGATA-3',
反向引物:5'-CAAGCAGGTAGGAGATTCCA-3'。
GAPDH内参特异性PCR引物:
正向引物为5′-ATCATCAGCAATGCCTCCT-3′,
反向引物为5′-CATCACGCCACAGTTTCC-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、2-ΔΔCT的标定:用2-ΔΔCT表示对于对照组变化的倍数,其中△CT=CT样本–CT内参,△△CT=△CT病例–△CT对照。本实验数据采用相对定量的分析方法,GAPDH作为内参基因,胶质瘤患者△CT的平均值为对照。数据利用软件GraPhPad Prism和SPSS进行分析。申请人通过荧光定量PCR对26例胶质瘤组织与12例正常脑组织进行分析发现,hsa_circ_0125365在两者中的表达差异明显(P<0.0001),见图1。
6、图2的ROC曲线分析脑组织来源的hsa_circ_0125365对胶质瘤与正常脑组织差异的特异性、灵敏性;hsa_circ_0125365作为生物标记物对胶质瘤具有较高诊断价值(AUC=0.939,P=0.040,敏感度和特异度分别为90.0%和78.3%)。
7、对16位患者预后生存曲线分析发现,与胶质瘤组织中circRNA hsa_circ_0125365低表达(低于平均值)的患者相比,胶质瘤组织中circRNA hsa_circ_0125365高表达(高于平均值)的患者存活率更高,差异有显著性(P=0.119),见图3。
以上研究表明,hsa_circ_0125365可作为胶质瘤患者预后的特异性分子标志物。
序列表
<110> 中南大学湘雅医院
<120> 胶质瘤预后标志物hsa_circ_0125365及应用
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 568
<212> RNA
<213> 智人(Homo sapiens)
<400> 1
uggaagcauc aguucacagc aguaaugcac acuguacaga uaagacaauu gaagcugcug 60
aagcccugcu ucauauggaa ucuccuaccu gcuugaggga uucaagaagu ccuguggaag 120
uguuuguucc uccuugugua ucaacuccag aauucaucca ugcugcuaug aggccagaug 180
ucauuacaga aacuguagug gaggugucaa cugaagaguc ugaacccaug gauaccucuc 240
cuauuccaac aucaccagau agccaugaac caaugaaaaa gaaaaaaguu ggccguaaac 300
caaagaccca gcaaucacca auuuccaaug ggucuccuga guuagguaua aagaagaaac 360
caagagaagg aaaaggaaac acaaccuauu ugugggaguu ucuuuuagau cuacuucaag 420
auaaaaauac uugucccagg uauauuaaau ggacucagag agaaaaaggc auauucaagc 480
ugguggauuc aaaggcuguc ucuaagcuuu ggggaaagca uaagaacaaa ccagacauga 540
acuaugaaac caugggacga gcuuugag 568
<210> 2
<211> 20
<212> DNA
<213> 未知(Unknown)
<400> 2
ccaagcagct cactacgata 20
<210> 3
<211> 20
<212> DNA
<213> 未知(Unknown)
<400> 3
caagcaggta ggagattcca 20
<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 (6)

1.一种胶质瘤预后标志物hsa_circ_0125365,其序列如SEQ NO:1所示。
2.检测权利要求1所述的标志物在胶质瘤组织中表达量的试剂在制备胶质瘤预后制剂中的应用。
3.一种胶质瘤预后试剂盒,其特征在于,能够测定胶质瘤组织中的hsa_circ_0125365的含量。
4.根据权利要求3所述的胶质瘤预后试剂盒,其特征在于,含有检测hsa_circ_0125365含量的PCR引物。
5.根据权利要求4所述的胶质瘤预后试剂盒,其特征在于,引物的序列如SEQ NO:2和3所示。
6.根据权利要求3或4或5所述的胶质瘤预后试剂盒,其特征在于,除hsa_circ_0125365的引物外,还含有从胶质瘤组织中提取RNA并进行逆转录及荧光定量PCR的所有试剂。
CN201711453665.8A 2017-12-28 2017-12-28 胶质瘤预后标志物hsa_circ_0125365及应用 Expired - Fee Related CN107937528B (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201711453665.8A CN107937528B (zh) 2017-12-28 2017-12-28 胶质瘤预后标志物hsa_circ_0125365及应用

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201711453665.8A CN107937528B (zh) 2017-12-28 2017-12-28 胶质瘤预后标志物hsa_circ_0125365及应用

Publications (2)

Publication Number Publication Date
CN107937528A true CN107937528A (zh) 2018-04-20
CN107937528B CN107937528B (zh) 2020-06-30

Family

ID=61940565

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201711453665.8A Expired - Fee Related CN107937528B (zh) 2017-12-28 2017-12-28 胶质瘤预后标志物hsa_circ_0125365及应用

Country Status (1)

Country Link
CN (1) CN107937528B (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109321657A (zh) * 2018-10-30 2019-02-12 徐州医科大学 一种胶质瘤预后标志物及其应用

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
RYBAK-WOLF ET AL.: "Circular RNAs in the Mammalian Brain Are Highly Abundant, Conserved, and Dynamically Expressed.", 《MOLECULAR CELL》 *
SALZMAN ET AL.: "Cell-Type Specific Features of Circular RNA Expression.", 《PLOS GENETICS》 *
YANG ET AL.: "Identification of circular RNA signature in bladder cancer.", 《JOURNAL OF CANCER》 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109321657A (zh) * 2018-10-30 2019-02-12 徐州医科大学 一种胶质瘤预后标志物及其应用
CN109321657B (zh) * 2018-10-30 2021-07-02 徐州医科大学 一种胶质瘤预后标志物及其应用

Also Published As

Publication number Publication date
CN107937528B (zh) 2020-06-30

Similar Documents

Publication Publication Date Title
CN104694623A (zh) 一种用于肺癌诊断的血浆miRNA标志物及应用
CN108624693A (zh) miR-577在制备肾病诊断标志物中的应用
CN107557472A (zh) 胶质瘤诊断标志物circ9:135881633|135883078及应用
CN107937528A (zh) 胶质瘤预后标志物hsa_circ_0125365及应用
CN107586844A (zh) 胶质瘤预后标志物Circ9:135881633|135883078的应用
CN106755309A (zh) 分子标记物在制备胰腺癌预后评估产品中的应用
CN107586848A (zh) 胶质瘤预后标志物circ8:127890589|127890998及应用
JPWO2019117270A1 (ja) 頭頸部がんの検出を補助する方法
CN107619869A (zh) 胶质瘤诊断及预后标志物circ16:85633914|85634132及应用
CN107988370A (zh) 一种circRNA基因在制备诊断慢性粒细胞性白血病试剂中的应用
CN107586849A (zh) 脑组织来源的circ9:4117768|4118881的应用
CN107937530A (zh) 胶质瘤预后标志物hsa_circ_0125361及应用
CN107604072A (zh) 胶质瘤预后标志物circ15:98707562|98708107的应用
CN107937536A (zh) 胶质瘤预后标志物circ19:47362476|47362693及应用
CN107586846A (zh) 胶质瘤诊断标志物Circ3:129880309|129880559及应用
CN107604076A (zh) 胶质瘤诊断标志物Circ6:4891713|4892379及应用
CN107604074A (zh) 胶质瘤预后标志物circ15:101235082|101235577及应用
CN107937538A (zh) 胶质瘤诊断标志物circ1:201817088|201817285及应用
CN104059987A (zh) 骨肉瘤诊断试剂盒及cpe基因在其制备中的用途
CN107937529A (zh) 胶质瘤诊断标志物hsa_circ_0135404及应用
CN107557441A (zh) 胶质瘤诊断标志物Circ2:23823258|23823569及应用
CN107619868A (zh) 胶质瘤预后标志物Circ3:129880309|129880559的应用
CN109371129A (zh) hsa_circRNA_0000798作为肝癌早期诊断或预后评估生物标记物的应用
CN109295059A (zh) 一种共价环状闭合的单链rna及其应用
CN107937540A (zh) 胶质瘤诊断标志物circ17:47618350|47619164及应用

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant
CF01 Termination of patent right due to non-payment of annual fee

Granted publication date: 20200630