CN107937530A - 胶质瘤预后标志物hsa_circ_0125361及应用 - Google Patents

胶质瘤预后标志物hsa_circ_0125361及应用 Download PDF

Info

Publication number
CN107937530A
CN107937530A CN201711453686.XA CN201711453686A CN107937530A CN 107937530 A CN107937530 A CN 107937530A CN 201711453686 A CN201711453686 A CN 201711453686A CN 107937530 A CN107937530 A CN 107937530A
Authority
CN
China
Prior art keywords
glioma
circ
hsa
samples
primer
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
CN201711453686.XA
Other languages
English (en)
Other versions
CN107937530B (zh
Inventor
武明花
许梓豪
李沛瑶
余志斌
张严
刘强
陈琼
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 CN201711453686.XA priority Critical patent/CN107937530B/zh
Publication of CN107937530A publication Critical patent/CN107937530A/zh
Application granted granted Critical
Publication of CN107937530B publication Critical patent/CN107937530B/zh
Expired - Fee Related legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING 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
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/118—Prognosis of disease development
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/178—Oligonucleotides 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

本发明公开了一种胶质瘤预后标志物hsa_circ_0125361及应用，即检测胶质瘤来源的circRNA hsa_circ_0125361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_0125361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_0125361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_0125361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_0125361，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_0125361的含量。
所述的胶质瘤预后试剂盒，含有检测hsa_circ_0125361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_0125361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_0125361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_0125361所用随机引物；
(3)将cDNA实时定量PCR所用试剂，包括circRNA hsa_circ_0125361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。
申请人通过荧光定量PCR对20例胶质瘤组织与8例正常脑组织进行分析发现，hsa_circ_0125361在两者中的表达差异明显(P＝0.0054)，后对其中12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_0125361与患者的生存率相关(P＝0.011)，见图2，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检测hsa_circ_0125361在脑胶质瘤患者胶质瘤组织和在非胶质瘤患者脑组织中的表达改变；
图2为胶质瘤中hsa_circ_0125361表达量与脑胶质瘤患者预后的关系。
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circRNA hsa_circ_0125361用于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 50ml
5.无酶水10ml
6. 1μM随机逆转录引物50μl
7. 5×逆转录缓冲液200ml
8. 10mM三磷酸碱基脱氧核苷酸100μl
9. 40U/μl RNA酶抑制剂500μl
10. 200U/μl MMLV逆转录酶50μl
11.Premix Ex Taq 50μl
12. 10μM circRNA hsa_circ_0125361实时荧光定量PCR特异性引物30μl
circRNA hsa_circ_0125361正向引物：5'-CCAAGCAGCTCACTACGATA-3'，
circRNA hsa_circ_0125361反向引物：5'-CAAGCAGGTAGGAGATTCCA-3'；
13. 10μM GAPDH特异性引物30μl
正向引物为5′-ATCATCAGCAATGCCTCCT-3′，
反向引物为5′-CATCACGCCACAGTTTCC-3′。
实施例2：circRNA hsa_circ_0125361在胶质瘤组织的表达量与预后的关系
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，研磨成液体状后移至tube管，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管中，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。由实验员每天记录冰箱温度。
3、circRNA hsa_circ_0125361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 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、广州吉赛生物工程有限公司合成的hsa_circ_0125361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指标的测定：本实验数据采用相对定量的分析方法，GAPDH作为内参基因，其中△CT＝CT样本–CT内参，△△CT＝△CT–△CT对照(取正常样本△CT的平均值为△CT对照)，数据利用软件GraphPad Prism进行分析。使用SPSS 17.0进行Kaplan-Meier生存分析发现，与胶质瘤组织中circRNA hsa_circ_0125361低表达(低于平均值)的患者相比，胶质瘤组织中circRNA hsa_circ_0125361高表达(高于平均值)的患者存活率更高，差异有显著性(P＝0.011)，见图2。
序列表
<110> 中南大学湘雅医院
<120> 胶质瘤预后标志物hsa_circ_0125361及应用
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1858
<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 gcuuugagau acuacuacca aaggggaauu cuugcaaagg 600
uugaaggaca gaggcuugua uaucaguuca aggauaugcc gaaaaacaua guggucauag 660
augaugacaa aagugaaacc uguaaugaag auuuagcagg aacuacugau gaaaaaucau 720
uagaacgagu gucacugucu gcagaaaguc uccugaaagc agcauccucu guucgcagug 780
gaaaaaauuc auccccuaua aacugcucca gagcagagaa ggguguagcu agaguuguga 840
auaucacuuc cccugggcac gaugcuucau ccaggucucc uacuaccacu gcaucugugu 900
cagcaacagc agcuccaagg acaguucgug uggcaaugca gguaccuguu guaaugacau 960
cauuggguca gaaaauuuca acuguggcag uucagucagu uaaugcaggu gcaccauuaa 1020
uaaccagcac uaguccaaca acagcgaccu cuccaaaggu agucauucag acaaucccua 1080
cugugaugcc agcuucuacu gaaaauggag acaaaaucac caugcagccu gccaaaauua 1140
uuaccauccc agcuacacag cuugcacagu gucaacugca gacaaaguca aaucugacug 1200
gaucaggaag cauuaacauu guuggaaccc cauuggcugu gagagcacuu accccuguuu 1260
caauagccca ugguacaccu guaaugagac uaucaaugcc uacucagcag gcaucuggcc 1320
agacuccucc ucgaguuauc agugcaguca uaaaggggcc agagguuaaa ucggaagcag 1380
uggcaaaaaa gcaagaacau gaugugaaaa cuuugcagcu aguagaagaa aaaccagcag 1440
auggaaauaa gacagugacc cacguagugg uugucagugc gccuucagcu auugcccuuc 1500
cuguaacuau gaaaacagaa ggacuaguga caugugagaa auaaaauagc agcuccacca 1560
uggacuucag gcuguuagug gcaguacuga cauaaacauu ugcaagggaa gucaucaaga 1620
aaagucaaag aagacuuuaa aacauuuuua augcauauac aaaaacaauc agacuuacug 1680
gaaauaaauu accuauccca uguuucagug ggaaaugaac uacauauuga gaugcugaca 1740
gaaaacugcc ucuuacagua ggaaacaacu gaacccauca auaagaaaaa ggaucgaaag 1800
ggaccaagca gcucacuacg auaucaaguu acacuaagac uuggaacacu aacauucu 1858
<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_0125361，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_0125361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_0125361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_0125361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。
CN201711453686.XA 2017-12-28 2017-12-28 胶质瘤预后标志物hsa_circ_0125361及应用 Expired - Fee Related CN107937530B (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201711453686.XA CN107937530B (zh) 2017-12-28 2017-12-28 胶质瘤预后标志物hsa_circ_0125361及应用

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201711453686.XA CN107937530B (zh) 2017-12-28 2017-12-28 胶质瘤预后标志物hsa_circ_0125361及应用

Publications (2)

Publication Number Publication Date
CN107937530A true CN107937530A (zh) 2018-04-20
CN107937530B CN107937530B (zh) 2020-06-30

Family

ID=61940581

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201711453686.XA Expired - Fee Related CN107937530B (zh) 2017-12-28 2017-12-28 胶质瘤预后标志物hsa_circ_0125361及应用

Country Status (1)

Country Link
CN (1) CN107937530B (zh)

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》 *

Also Published As

Publication number Publication date
CN107937530B (zh) 2020-06-30

Similar Documents

Publication Publication Date Title
CN107475258B (zh) 一种RNA circEPSTI1及其在三阴乳腺癌上的应用
CN102876676A (zh) 一种与胰腺癌相关的血清/血浆miRNA标志物及其应用
CN101792793A (zh) miR-182作为胶质瘤发生分子标志物的应用及其检测试剂盒
CN107586844A (zh) 胶质瘤预后标志物Circ9:135881633|135883078的应用
CN107586848A (zh) 胶质瘤预后标志物circ8:127890589|127890998及应用
CN107619869A (zh) 胶质瘤诊断及预后标志物circ16:85633914|85634132及应用
CN107937528A (zh) 胶质瘤预后标志物hsa_circ_0125365及应用
JPWO2019117270A1 (ja) 頭頸部がんの検出を補助する方法
CN107937530A (zh) 胶质瘤预后标志物hsa_circ_0125361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
CN107557441B (zh) 胶质瘤诊断标志物Circ2:23823258|23823569及应用
CN107937536A (zh) 胶质瘤预后标志物circ19:47362476|47362693及应用
CN107937540B (zh) 胶质瘤诊断标志物circ17:47618350|47619164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
CN105969875B (zh) 用于检测微量组织中pik3ca基因突变的引物组合及其应用
CN107604072A (zh) 胶质瘤预后标志物circ15:98707562|98708107的应用
CN104059987A (zh) 骨肉瘤诊断试剂盒及cpe基因在其制备中的用途
CN107937539A (zh) 胶质瘤预后标志物hsa_circ_0135404及应用
CN107619868A (zh) 胶质瘤预后标志物Circ3:129880309|129880559的应用
CN109295059A (zh) 一种共价环状闭合的单链rna及其应用
CN107937533A (zh) 胶质瘤预后标志物circ7:42148226|42148468及应用
CN107937529A (zh) 胶质瘤诊断标志物hsa_circ_0135404及应用
CN107937538A (zh) 胶质瘤诊断标志物circ1:201817088|201817285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