CN111733226B - 一种检测克罗恩病的环状rna标志物及应用 - Google Patents
一种检测克罗恩病的环状rna标志物及应用 Download PDFInfo
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Abstract
本发明在芯片筛查到的CD患者155个上调的circRNA分子中，hsa_circRNA_009084在诊断敏感度和特异性评价时表现优异。本发明用于CD诊断的hsa_circRNA_009084未见国内外有相关文献报道和专利申请。本发明技术中血液中hsa_circRNA_009084分子检测仅需RT‑PCR方法，简单易操作，临床应用推广可行性大；hsa_circRNA_009084可应用于CD诊断和疗效的快速判断。本发明提供了一种检测克罗恩病的环状RNA标志物，环状标志物为hsa_circRNA_009084。本发明提供了一种检测克罗恩病的环状RNA标志物的应用，环状标志物为hsa_circRNA_009084作为克罗恩病检测方法中的标志物。
Description
技术领域
本发明属于分子生物医学领域，特别涉及克罗恩病。
背景技术
近年来,炎症性肠病(Inflammatory bowel disease，IBD)，包括克罗恩病(Crohn's disease，CD)和溃疡性结肠炎(ulcerative colitis， UC)在发展中国家的发病率日益上升。
环状RNA(Circular RNAs，circRNAs)由信使RNA前体(pre-mRNAs)反向剪接而来，可能包含内含子序列、外显子序列，或同时包含内含子和外显子序列。circRNA多数是可行使调控功能的非编码RNA(non-coding RNAs，ncRNAs)，可通过吸附microRNA 或结合到其它分子上，在转录水平或转录后水平调控基因表达。 circRNA因闭合环状结构不受外切酶影响，半衰期长，可稳定存在于细胞，具有一定组织特异性、时序特异性及疾病特异性，是理想的潜在的疾病分子生物标记物。越来越多的研究表明circRNA分子在多种疾病，如结直肠癌(colorectal cancer，CRC)、乳腺癌(breast cancer)、类风关(rheumatoid arthritis，RA)和先兆子痫(pre-eclampsia)的发生发展中起作用，可作为疾病诊疗的分子生物学标记物。
CD可累及全消化道，表现为非连续性病变。UC主要累及直肠和结肠，呈连续性弥漫性分布。为鉴别诊断CD和UC，需综合考虑临床症状、内镜检查、影像技术及组织活检结果等，目前没有特异性强的血清生物学标志。目前对IBD诊断主要依赖于临床症状、内镜、病理、影像技术和生化特征等，且常用的生化指标如粪便钙卫蛋白、血沉、白细胞、血清抗微生物抗体等特异性差、灵敏度不高。对CD 和UC的鉴别诊断依赖于内镜检查。若能减少传统的侵入性检查，将特异性circRNA用于评估疾病的严重程度和复发情况，将具有重要临床意义。
circRNA是否可作为IBD临床诊疗的分子生物学标志物？目前国内外相关研究报道非常少，东南大学附属中大医院陈洪教授等[1] 的研究表明:在环状RNA芯片检测的基础上，通过实时定量PCR 检测发现活动期CD患者病变组织中hsa circ 0023397明显低于健康对照组,miR-106b明显高于健康对照组，活动期CD患者肠黏膜组织中可能存在着由于hsacirc 0023397下调引起miR-106b过度表达,继而导致ATG16L1表达下调并降低CD患者自噬功能。上海交大附属仁济医院的冉志华教授等对环状RNA组织芯片的研究也发现：CD患者组织样本中163个上调的circRNA靶向435miRNAs，及55个下调的circRNA靶向207个miRNA。生信分析预测 hsa circRNA 102685可能参与凋亡、Toll样受体及p53信号通路 [2]。
发明人前期已收集CD患者和健康对照人群的外周血，分离外周血单个核细胞，通过Arraystar的Human circRNA Arrays筛查到CD 患者外周血单个核细胞中155个上调和229个下调的circRNA分子，实时荧光定量PCR法大样本量验证了4个上调的circRNA分子在CD病患者临床诊断中的应用价值，用miRDB_V5.0和TaregetScan7.1 分析预测circRNA最可能结合的5个microRNA分子的种子序列。前期结果发表在Medicine[3]。
现有用于CD的血液分子生物标志物用于灵敏度和特异性均不高；尚未见成熟的试剂盒应用circRNA分子于CD诊断和疗效判断。
[1]沈朵,陈洪,李红霞,等.hsa circ RNA0023397和miR-106b在活动性克罗恩病肠黏膜组织中的表达及意义[J].东南大学学报:医学版.2018,37(1):22-27.
[2]Qiao YQ,Cai CW,Shen J,et al.Circular RNA expression alterations incolon tissues of Crohn's disease patients.Mol Med Rep. 2019,19(5):4500-4506.
[3]Juan Yin,Tong Hu,Lijuan Xu,et al.Circular RNA expression profilein peripheral blood mononuclear cells from Crohn's diseasepatients.Medicine.2019,98:26(e16072).
发明内容
发明人在芯片筛查到的CD患者155个上调的circRNA分子中， hsa_circRNA_009084在诊断敏感度和特异性评价时表现优异。本发明用于CD诊断的hsa_circRNA_009084未见国内外有相关文献报道和专利申请。
本发明技术中血液中hsa_circRNA_009084分子检测仅需 RT-PCR方法，简单易操作，临床应用推广可行性大；本发明要求 hsa_circRNA_009084可应用于CD诊断和疗效的快速判断。
为解决上述技术问题，本发明的目的是提供了一种检测克罗恩病的环状RNA标志物，环状标志物为hsa_circRNA_009084。
本发明提供了一种检测克罗恩病的环状RNA标志物的应用，环状标志物为hsa_circRNA_009084作为克罗恩病检测方法中的标志物。
本发明还提供了一种检测克罗恩病的环状RNA标志物的应用， hsa_circRNA_009084作为特异性试剂、芯片、探针、引物、试剂盒或核酸膜条所要检测的标志物。
本发明还提供了一种检测克罗恩病的环状RNA标志物的应用， hsa_circRNA_009084相关受体、信号通路用作克罗恩病治疗药物的靶点。
本发明还提供了一种用于检测克罗恩病的特异性引物，特异性引物为特异性扩增hsa_circRNA_009084的引物：正向SEQ ID No.1：5’ -CTGTGCGCTGATTGGAATGT-3’；反向SEQID No.2：5’- AGAAGAGTGAGGCAAAGCAA-3’。
本发明还提供了一种用于检测克罗恩病的特异性引物的用途，特异性扩增hsa_circRNA_009084的引物用于克罗恩病诊断或治疗使用的探针、试剂和芯片中应用。
本发明还提供了一种用于检测克罗恩病的试剂盒，试剂盒含有 SEQ ID No.1和SEQ ID No.2序列。
本发明还提供了一种检测hsa_circRNA_009084的方法，检测 hsa_circRNA_009084的方法使用SEQ ID No.1和SEQ ID No.2序列。
本发明还提供了一种非诊断用途的检测克罗恩病的方法，检测克罗恩病的方法使用hsa_circRNA_009084作为标志物。
本发明还提供了一种非诊断用途的检测克罗恩病的方法，该方法使用上述特异性扩增hsa_circRNA_009084的引物。
上述说明仅是本发明技术方案的概述，为了能够更清楚了解本发明的技术手段，并可依照说明书的内容予以实施，以下以本发明的较佳实施例并配合附图详细说明如后。
附图说明
图1是本发明中实施例hsa_circRNA_009084在CD(n＝32)、 HC(n＝49)和UC(n＝25)PBMC中表达差异检测；
图2是本发明中实施例hsa_circRNA_009084诊断CD ROC曲线分析；
图3是本发明中实施例hsa_circRNA_009084MRE(microRNA response element)分析；
图4是本发明中实施例hsa_circRNA_009084靶标hsa-miR-136-3p 下游靶基因GO及KEGG pathway分析；
图5是本发明中实施例hsa_circRNA_009084靶标hsa-miR-136-3p 靶向CD病相关的TGF-beta signaling pathway。
具体实施方式
下面结合附图和实施例，对本发明的具体实施方式作进一步详细描述。以下实施例用于说明本发明，但不用来限制本发明的范围。
实施例1CD外周血单个核细胞分离
用EDTA-K2抗凝管收集克罗恩病患者和健康对照者外周血，每例采血6mL。样本采集时所有参与者均3个月内未使用免疫抑制剂和口服抗生素，无其它慢性病。克罗恩病的患者均经内镜确诊，排除溃疡性结肠炎、白塞氏病、肠应激综合征等。健康对照者经本院体检中心体检无胃肠道疾病和其它慢性病，采样前各指标正常。样本采集后迅速送中心实验室进行外周血单个核细胞分离，2小时内完成分离。采用Ficoll法分离外周血单个核细胞，加1ml trizol (Invitrogen-15596026)，冻存于-80度冰箱,用于后续realtime-PCR验证。
Ficoll法分离外周血单个核细胞具体步骤如下：
1.1EDTA-K抗凝剂(紫头管)收集3管(约4.5ml)外周血。
1.2离心4000rpm/min，10min，收取上层血浆，再次离心 5000rpm/min，10min，去细胞碎片，取上清分装至1.5mlEP管(无菌无酶)，过液氮后冻存至-80度冰箱。
1.3用生理盐水1:1稀释血细胞，取2ml血细胞加入2ml生理盐水，用移液枪轻轻吹打均匀。
1.4加入3ml Ficoll(GE 17-1440-02)至15ml离心管，用移液枪将稀释后的血细胞缓缓加入15ml离心管的上层，避免破坏Ficoll层。
1.5离心400g，30min。
1.6吸取中间Ficoll层，白细胞主要分布在Ficoll层上部。
1.7加生理盐水6ml，轻轻吹打均匀。
1.8 95g，离心15分钟。
1.9轻轻倒去上清，加生理盐水6ml，轻轻吹打均匀。
1.10 95g，离心15分钟。
1.11加1ml trizol(Invitrogen-15596026)，冻存于-80度冰箱。
实施例2总RNA提取
2.1离心收集细胞，每5-10×106细胞加入1ml TRIzol，反复吸打。加TRIzol之前不要洗涤细胞以免mRNA降解。
2.2将样品在室温(15-30℃)放置5分钟，使核酸蛋白复合物完全分离。
2.3每使用1ml TRIzol加入0.2ml氯仿，剧烈振荡15秒，室温放置3分钟。
2.4 2-8℃10000×g离心15分钟。样品分为三层：底层为黄色有机相，上层为无色水相和一个中间层。RNA主要在水相中，水相体积约为所用TRIzol试剂的60％。
2.5把水相转移到新管中，如要分离DNA和蛋白质可保留有机相，进一步操作见后。用异丙醇沉淀水相中的RNA。每使用1ml TRIzol 加入0.5ml异丙醇，室温放置10分钟。
2.6 2-8℃10000×g离心10分钟，离心前看不出RNA沉淀，离心后在管侧和管底出现胶状沉淀。移去上清。
2.7用75％乙醇洗涤RNA沉淀。每使用1ml TRIzol至少加1ml 75％乙醇。2-8℃不超过7500×g离心5分钟，弃上清。
2.8室温放置干燥或真空抽干RNA沉淀，大约晾5-10分钟即可。不要真空离心干燥，过于干燥会导致RNA的溶解性大大降低。加入 25-200μl无RNase的水或0.5％SDS，用枪头吸打几次，55-60℃放置 10分钟使RNA溶解。
实施例3反转录及hsa_circRNA_009084qPCR定量检测
用PrimeScriptTMMaster Mix(TaKaRa,Shiga,Japan)试剂盒将RNA 反转录成cDNA。以β-actin为内参，用TB GreenTMPremix Ex TaqTMII (TliRNaseH Plus；TaKaRa,Shiga,Japan)试剂盒染料法qPCR检测克罗恩病患者组和对照组样本中目标circRNA分子相对表达水平。
实施例4 qPCR定量检测结果分析
2-ΔΔCt法分析克罗恩病患者组和对照组样本中目标circRNA分子相对表达量。组间差异采用Graphpad prism 7.0软件student’s t-test (两组)或one-way ANOVA(三组)统计分析(图1)。circRNAs 分子临床诊断价值采用Graphpad prism 7.0软件ROC法统计分析(图 2)。
如图1所示：克罗恩病患者(CD,n＝49)、溃疡性结肠炎患者(UC,n＝25) 和健康人群(HC，n＝34)PBMC中hsa_circRNA_009084qPCR定量检测结果。
如图2所示：hsa_circRNA_009084用于检测CD的ROC分析(cutoff 值＝0.0002275时，Sensitivity％＝83.67，Specificity％＝73.53)
实施例5生信分析
TargetScan和MiRanda在线预测hsa_circRNA_009084microRNA responseelement(图3)。使用miRWalk、TargetScan和miRTARbase 数据库在线分析hsa_circRNA_009084靶标hsa-miR-136-3p下游靶基因及其交集(图4)。再用DAVID对hsa-miR-136-3p下靶标进行基因富集分析及KEGG通路分析(图5)。
如图3所示：hsa_circRNA_009084MRE(microRNA response element)分析；TargetScan和MiRanda在线预测hsa_circRNA_009084 microRNA response element。
如图4所示：hsa_circRNA_009084靶标hsa-miR-136-3p下游靶基因GO及KEGGpathway分析；4A miRWalk、TargetScan和 miRTARbase数据库在线分析hsa_circRNA_009084靶标 hsa-miR-136-3p下游靶基因及其交集；4B和4C DAVID对hsa-miR-136-3p下靶标进行基因富集分析及KEGG通路分析。
图5所示：hsa_circRNA_009084靶标hsa-miR-136-3p靶向CD 病相关的TGF-betasignaling pathway；
转化生长因子β(TGF-β)在免疫耐受和免疫稳态中起重要作用。慢性炎症和肠道损伤修复等可诱发CD肠纤维化，这是常见于 CD的严重的并发症之一。TGF-β在肠上皮细胞间充质化(EMT)发挥了重要的诱导作用，促进纤维化。
实施例6特异性检测hsa_circRNA_009084的引物序列
| circRNA | Alias | 正向引物 | 反向引物 |
| hsa_circRNA_009084 | hsa_circ_0009084 | CTGTGCGCTGATTGGAATGT | AGAAGAGTGAGGCAAAGCAA |
实施例7
按照上述方法收集门诊CD可疑患者1例的外周血，分离PBMC，抽提RNA，反转录成cDNA。以β-actin为内参，以特异性引物(正向SEQ ID No.1：5’-CTGTGCGCTGATTGGAATGT-3’；反向SEQ ID No.2：5’-AGAAGAGTGAGGCAAAGCAA-3’)扩增可疑患者PBMC 中hsa_circRNA_009084，2-ΔΔCt法分析可疑患者组和对照组样本中目标circRNA分子相对表达量。取hsa_circRNA_009084ROC分析结果 cutoff值＝0.0002275(Sensitivity％＝83.67，Specificity％＝73.53)。
结果：hsa_circRNA_009084在可疑CD患者中的相对表达量为 0.0695889，大于cutoff值＝0.0002275。临床确诊为CD患者。
以上所述仅是本发明的优选实施方式，并不用于限制本发明，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明技术原理的前提下，还可以做出若干改进和变型，这些改进和变型也应视为本发明的保护范围。
序列表
<110> 苏州市立医院
<120> 一种检测克罗恩病的环状RNA标志物及应用
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 1
ctgtgcgctg attggaatgt 20
<210> 2
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 2
agaagagtga ggcaaagcaa 20
Claims (1)
1.用于扩增hsa_circRNA_009084的引物对在制备克罗恩病诊断试剂中的应用，其特征在于，所述引物对核苷酸序列如下所示：
正向SEQ ID No .1：5’-CTGTGCGCTGATTGGAATGT-3’；
反向SEQ ID No.2：5’-AGAAGAGTGAGGCAAAGCAA-3’。
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| Cell-type specific features of circular RNA expression;Salzman, J.et al;《PLoS Genet》;20130905;第9卷(第9期);第11页右栏最后2段、第12页左栏第2段、补充材料表S7 * |
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