CN117344005B - 一种lncRNA及其在制备诊断、筛查或评估冠状动脉粥样硬化的产品中的应用 - Google Patents
一种lncRNA及其在制备诊断、筛查或评估冠状动脉粥样硬化的产品中的应用 Download PDFInfo
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Abstract
本发明公开一种lncRNA及其在制备诊断、筛查或评估冠状动脉粥样硬化的产品中的应用,属于生物医学技术领域。本发明提供的冠状动脉粥样硬化lncRNA标志物,为检测循环中单个核细胞的lncRNA‑ASD表达,具有较好的稳定性,在诊断、筛查或评估冠状动脉粥样硬化方面更加便捷、创伤小,不具有辐射危害。本发明所涉及的标志物在冠状动脉粥样硬化诊断方面具有较好的价值(AUC=0.883,95%CI:0.824‑0.941),当Cutoff值为0.0052时,敏感性83%,特异性为82%,准确度为82.5%,具有较好的临床价值,适宜推广应用。
Description
技术领域
本发明属于生物医学技术领域,特别涉及一种长链非编码RNA(lncRNA),及其在制备诊断、筛查或评估冠状动脉粥样硬化的产品中的应用。
背景技术
心血管疾病(cardiovascular disease,CVD)是目前是全球范围内致死、致残率最高的疾病。动脉粥样硬化(atherosclerosis,AS)是引起冠心病、脑梗死、外周血管病等心脑血管疾病的主要病理基础,是由脂质代谢异常引起的血管慢性炎症性疾病。动脉粥样硬化的主要特征是血管内皮下脂质的堆积、病变粥样斑块的形成,导致冠状动脉腔隙狭窄,甚至因斑块破裂导致动脉血栓,最终导致组织器官缺血或坏死。动脉粥样硬化的症状主要决定于血管病变及受累器官的缺血程度,早期诊断困难;当冠脉狭窄大于75%,可诱发心绞痛、心梗等心血管事件,发生后预后不良,迫切需要我们对动脉粥样硬化进行早期诊断。
早期冠状动脉粥样硬化的主要病变为局部内膜下脂质沉积,粥样斑块形成,通常无明显临床症状,容易被忽略。目前,动脉粥样硬化的诊断主要依靠劲动脉超声、冠脉造影技术、冠脉CT等影像技术,通过测量颈动脉内-中膜厚度及形态学观察有无斑块形成,作为判断早期AS的指标。影像检查需要注射造影剂,具有一定的辐射;费用较为昂贵,不适合用于常规筛查。目前,尚无提示早期斑块的血清学特异性标志物。因此,有必要寻找一种便捷的、有效的动脉粥样硬化早期临床诊断的新型生物标志物。
长链非编码RNA(long noncoding RNA,lncRNA)是指序列长度大于200核苷酸,无编码功能蛋白质能力的非编码RNA。已有充分证据证明,lncRNA可在转录、转录后和表观遗传水平调控基因表达,参与多种细胞过程,对心血管病的进程具有重要调控作用。LncRNAANRIL在动脉粥样硬化病人中表达明显升高;抑制心脏组织中lncRNA MHRT的表达,可加速心肌肥大向心力衰竭的进程;研究者发现,线粒体来源lncRNA LIPCAR在心血管不良事件发生的早期表达较低,晚期表达增高。由于lncRNA通常形成相对稳定的二级结构,因其可以稳定存在于血液及其他各种体液中,较传统的分子标志物具有更高的稳定性和特异性,可以在血液中被检测到,有望成为心血管疾病诊断及预后的新型生物学标志物。寻找对动脉粥样硬化具有较高特异性和敏感性的lncRNA,对于早期诊断动脉粥样硬化具有较好的前景。
发明内容
为了克服现有技术的缺点与不足,本发明的目的在于提供一种冠状动脉粥样硬化生物标志物。特别是lncRNA作为生物标志物在诊断、筛查或评估冠状动脉粥样硬化中的应用。
本发明的目的通过下述技术方案实现:
第一方面,本发明提供了一种冠状动脉粥样硬化lncRNA标志物,所述lncRNA标志物为lncRNA-ASD。LncRNA-ASD是一个全新的、尚未报道过的lncRNA分子。所述lncRNA-ASD的序列为SEQ ID No.1所示。
通过高通量测序,对冠状动脉粥样硬化患者及冠脉正常患者外周血单个核细胞进行lncRNA差异分析,发现lncRNA-ASD在冠状动脉粥样硬化患者中表达显著升高(图1、图2)。通过qRT-PCR在更大的冠状动脉粥样硬化患者及冠脉正常患者样本群外周血单个核细胞进行lncRNA-ASD表达验证,发现lncRNA-ASD在冠状动脉粥样硬化患者中表达明显升高,差异具有统计学意义(图3);ROC曲线显示lncRNA-ASD在冠状动脉粥样硬化的诊断中具有可靠的临床价值(图4)。进一步的,通过在胸痛患者样本群外周血单个核细胞进行lncRNA-ASD验证,发现lncRNA-ASD诊断冠状动脉粥样硬化的准确度较高。
第二方面,本发明提供了一种lncRNA-ASD在制备如下任一项产品中的应用:
(1)在制备诊断冠状动脉粥样硬化的产品中的应用;
(2)在制备筛查冠状动脉粥样硬化的产品中的应用;
(3)在制备评估冠状动脉粥样硬化风险的产品中的应用。
进一步的,所述产品包括检测lncRNA-ASD表达水平的试剂;
更进一步的,所述产品包括检测lncRNA-ASD表达水平的荧光定量PCR试剂;
更进一步的,所述检测lncRNA-ASD表达水平的荧光定量PCR试剂为特异性检测lncRNA-ASD表达水平的引物对,所述引物对包含正向引物(5’-CTGGCTGATGGAGGCTTGT-3’)及反向引物(5’-CCACCACTTCTTGAACCGCT-3’)。
再进一步的,所述产品为芯片、制剂、试纸条或试剂盒。
进一步的,所述产品的检测样本为外周静脉血;更进一步的,所述样本为外周血单个核细胞;再进一步的,所述样本为从外周静脉血分离得到的外周血单个核细胞。
第三方面,本发明提供了一种试剂盒,所述试剂盒包括检测lncRNA-ASD表达水平的试剂;且所述试剂盒为应用于如下任一项应用的试剂盒:
(1)用于冠状动脉粥样硬化诊断的试剂盒;
(2)用于冠状动脉粥样硬化筛查的试剂盒;
(3)用于冠状动脉粥样硬化风险评估的试剂盒;
优选地,所述试剂盒包括特异性检测lncRNA-ASD表达水平的引物对。
所述引物对包含正向引物(5’-CTGGCTGATGGAGGCTTGT-3’)及反向引物(5’-CCACCACTTCTTGAACCGCT-3’)。
进一步地,所述试剂盒还包括特异性检测GAPDH表达水平的引物对、荧光染料。
优选的,所述荧光染料为SYBR Green荧光染料或TB Green荧光染料。
优选的,所述的特异性检测GAPDH表达水平的引物对包括GAPDH正向引物(5’-GAACGGGAAGCTCACTGG-3’)和GAPDH反向引物(5’-GCCTGCTTCACCACCTTCT-3’)。
本发明相对于现有技术,具有如下的优点及有益效果:
本发明提供的冠状动脉粥样硬化lncRNA标志物,为检测循环中单个核细胞的lncRNA-ASD表达,具有较好的稳定性,在诊断、筛查或评估冠状动脉粥样硬化方面更加便捷、创伤小,不具有辐射危害。本发明所涉及的标志物在冠状动脉粥样硬化诊断方面具有较好的价值(AUC=0.883,95%CI:0.824-0.941),当Cutoff值为0.0052时,敏感性83%,特异性为82%,准确度为82.5%,具有较好的临床价值,适宜推广应用。
附图说明
为了更清楚地说明本发明实施例的技术方案,下面将对实施例中所需要使用的附图作简单地介绍,应当理解,以下附图仅示出了本发明的某些实施例,因此不应被看作是对范围的限定,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他相关的附图。
图1冠状动脉粥样硬化患者和冠脉正常患者差异表达lncRNA火山图。
图2冠状动脉粥样硬化患者和冠脉正常患者差异表达lncRNA聚类热图。
图3冠状动脉粥样硬化患者和冠脉正常患者lncRNA-ASD表达情况。
图4lncRNA-ASD在冠状动脉粥样硬化中的临床诊断价值图。
具体实施方式
下面结合实施例及附图对本发明作进一步详细的描述,但本发明的实施方式不限于此。
为了便于理解本发明,下面结合优选实施例和附图对本发明做更全面的说明。需要明白,下面所具体描述的内容是说明性的而非限制性的,不应以此限制本发明的保护范围。
冠状动脉粥样硬化患者的诊断符合美国心脏协会(AHA)和美国心脏病学会(ACC)制定的关于冠状动脉粥样硬化的诊断标准。排除标准:心肌病、心肌炎、风湿性心脏病、心脏移植、各种急、慢性感染性疾病、冠状动脉以外的血栓性疾病、自身免疫性疾病、恶性肿瘤、近期重大外伤或手术史、严重的肝及肾损伤、病情危重(心源性休克及心功能Ⅳ级)以及使用主动脉球囊反搏、不同意参与研究的患者。
冠脉正常者为行冠状动脉造影检测排除冠状动脉粥样硬化的患者。冠脉正常者与冠状动脉粥样硬化患者通过年龄、性别、高血压、糖尿病、高脂血症、吸烟等变量进行匹配。所有患者均签署知情同意书。
实施例1:高通量测序筛选冠状动脉粥样硬化患者和冠脉正常患者中差异表达lncRNA
1.研究对象
收集梅州市人民医院急诊入院诊断为冠状动脉粥样硬化患者(10例)和冠脉正常者外周血(10例),基线资料如表1所示,两组患者临床基线资料间的差异均无统计学意义,P>0.05。
表1RNA测序患者临床基线资料
2.采血方法
所有患者均于入院当时采集外周静脉血3mL。
3.外周血单个核细胞分离和总RNA提取
3.1外周血单个核细胞(PBMC)分离
收集10例冠状动脉粥样硬化患者及10例冠脉正常患者外周血。
①分离细胞:外周静脉血室温静置30min后,3000rpm,室温离心15min,去除上层血浆;
②稀释细胞:在分离血浆后的2mL体积的血细胞中加入2mL PBS,轻柔混合,形成细胞悬液;
③梯度离心:于15mL离心管中加入4mL淋巴细胞分离液,动作轻柔地将细胞悬液缓慢加入淋巴细胞分离液表面(注意勿与淋巴细胞分离液混合),1500rpm,4℃离心30min。
④洗涤细胞:使用3mL吸管吸取交界层白膜,移入另一离心管,使用PBS洗涤两次,最后一次将细胞悬液移入1.5mLEP管中,备用。
3.2总RNA提取
①细胞裂解:在PBMC中加入1mL Trizol,充分震荡3min,混匀,在室温下静置10min;加入200μL氯仿,颠倒混匀,室温静置3min,13000rpm,4℃离心15min;
②RNA沉淀:小心吸取上清水相约0.5mL,移入1.5mL EP管,加入0.5mL异丙醇,颠倒混匀,室温静置10min,13000rpm,4℃离心10min;
③RNA漂洗及溶解:弃上清,管底可见白色物质,加入1mL 75%的预冷乙醇,洗涤沉淀,13000rpm,4℃离心5min;吸去乙醇,室温干燥10分钟,根据沉淀大小加入30-100μL56℃预热的DEPC水,溶解RNA;
④RNA质控:取3μL RNA样本,于1.5%琼脂糖凝胶中电泳;取l.5μL RNA样品于紫外分光光度计(IMPLEN-NP80)检测浓度和纯度,以A260/280在1.8-2.0视为RNA样本合格。
4.lncRNA测序
4.1总RNA定性和定量:①使用1%琼脂糖凝胶电泳对RNA样品进行定性,以排除可能的污染和降解;②使用紫外分光光度计检测RNA纯度和浓度;③最后使用Bioanalyzer2100***的RNANano 6000Assay Kit测定RNA的完整性和数量。
4.2文库制备:①lncRNA-seq RNA文库采用rRNA消链法制备。使用rRNA去除试剂盒从总RNA中去除核糖体RNA;②将RNA片段化成250~300bp的片段,利用片段化RNA和dNTP(dATP、dTTP、dCTP和dGTP)逆转录第一链cDNA;③利用RNase H降解RNA,利用DNA聚合酶I和dNTP(dATP、dUTP、dCTP和dGTP)合成第二链cDNA,通过核酸外切酶/聚合酶活性将双链cDNA的剩余部分转化为钝端。在DNA片段3’端腺苷化后,将测序接头连接到cDNA上;④为了优选长度为250~300bp的cDNA片段,利用AMPure XP***对文库片段进行纯化;⑤使用尿嘧啶-n-糖基化酶对尿苷进行酶切,通过PCR扩增cDNA;⑥用荧光仪测量文库浓度,调整至1ng/uL,使用Agilent 2100生物分析仪检查获得的文库的***物大小,使用qPCR检测cDNA文库的准确浓度(***物的大小和文库的浓度相同,则进行测序)。
4.3测序:将不同样品进行文库制备和pooling,进行Illumina测序,使用PE150测序对lncRNA-seq进行测序(12G原始数据)。
4.4原始数据的质量控制:①FASTQ格式的原始数据通过内部perl脚本进行处理,去除接头序列、poly-N reads和低质量reads,以获得干净的数据;②计算Q20、Q30和GC含量,用于检查清洗数据的质量。
4.5映射和组装:每个样品的Clean reads首先用HISAT2软件映射到人类参考基因组(版本:human.GRCH38/hg38)。Reads比对结果被转移到StringTie程序中进行转录本组装。
4.6lncRNA鉴定:使用Cuffmerge软件,对各样品拼接得到的转录本进行合并,并去掉其中链方向不确定的转录本,得到本次测序完整的转录组信息。对合并的转录本集合进行lncRNA的筛选,具体如下:①去除FPKM<0.5的低表达转录本;②去除<200bp和<2外显子的短转录本;③利用CNCI、Pfam和CPC2数据库去除具有蛋白质编码能力的转录本;④使用Cuffcompare去除标记基因1kb侧区内的转录本;⑤新的lncRNA按照HGNC(HUGO基因命名委员会)的规则命名,将新lncRNA与已知lncRNA和mRNA的特征进行比较。
4.7lncRNA定量和差异表达分析:①使用StringTie软件对转录本和基因进行定量,获得转录本每百万映射Reads(Reads Per Kilobase of transcript Per Millionmapped Reads,RPKM);②用Cuffdiff或edgeR进行差异表达分析;③使用Benjamini和Hochberg控制错误发现率的方法调整所得的p值。将log2(Fold Change)|>2和padj<0.05的基因归类为差异表达lncRNA。
图1为差异表达lncRNA火山图,图2为差异表达lncRNA聚类热图。结合测序结果与文献调研结果,筛选了差异表达显著上调的lncRNA-ASD。lncRNA-ASD基因序列如下:SEQ IDNo.1所示。具体为:
TTGTGGTGTGCACTTGTTGGAGCGCGGCCCATCTGGGGCACTGCAGCCGCTCTGACAAATGAGGCTGCCCGGGCAGAGGCCCCAACGTGCTGGCCGCCTGGAAGGCAAGTCAAATGATTTGAGCTAAGTGAAGAAATTGCCGAGCTGGCTGATGGAGGCTTGTGTTCCCTGGGTCCCATCCGCCTTCCCAGGCTGTGCTTCATCAGCTCCGTGAGTCATGTGGGGCAGCGGTTCAAGAAGTGGTGGCTGTGACGAGAGACTTCATGCAAGAAGGCCTTTCCAAAGTGTCATCCAGCAAAGTGTCCCAAGAGCAGACCCTCAACCAGGAGTCTAGGGCAAGTAGTTTATCTCCAAGGAGAGGAGAGAAGAAGGGATGTGGGACAGGGAAAAGAAGAAACCAATAAAAGGGGTTTGAGGGAGGAAGCAGAAGGTAGAAAGGTGAAGCTGGAGATGACCATGATGTAGAAAAC。
实施例2:冠状动脉粥样硬化患者及冠脉正常患者外周血PMBC中lncRNA-ASD表达情况。
1.研究对象
收集梅州市人民医院急诊入院诊断为冠状动脉粥样硬化患者(60例)及冠脉正常者(60例)外周血,基线资料如表2所示,两组患者临床基线资料间的差异无统计学意义,P>0.05。
表2AS患者及冠脉正常患者临床基线资料
2.采血方法
所有患者均于入院当时采集外周静脉血3mL。
3.PBMC分离及总RNA提取
采用与实施例1相同的方法,即密度梯度离心法从外周血中分离获得PBMC;采用与实施例1相同的方法,即Trizol法提取总RNA。
4.逆转录
使用TAKARAPrimeScriptTMRT Master Mix逆转录试剂盒进行逆转录反应。采用10μL逆转录反应体系进行配制逆转录反应缓冲体系(参照下表3组分进行配制)。
表3逆转录反应体系
混匀后使用PCR仪进行逆转录反应,获得cDNA模板。逆转录反应:37℃15min(反转录反应);85℃5s(反转录酶的失活反应)。在获得的10μL cDNA模板加入90μLdH2O,以稀释cDNA模板。
5.实时荧光定量PCR
5.1引物设计
采用在线引物设计软件,设计lncRNA-ASD及GAPDH引物,引物设计后由生工生物工程公司合成,具体引物序列如下:
lncRNA-ASD正向引物(Forward primer):5’-CTGGCTGATGGAGGCTTGT-3’;
lncRNA-ASD反向引物(Reverse primer):5’-CCACCACTTCTTGAACCGCT-3’;
GAPDH正向引物(Forward primer):5’-GAACGGGAAGCTCACTGG-3’;
GAPDH反向引物(Reverse primer):5’-GCCTGCTTCACCACCTTCT-3’。
5.2荧光定量PCR反应
①反应体系
使用TB GreenTMPremix Ex TaqTMII扩增试剂盒进行PCR反应,采用10μL反应缓冲体系进行配制(参照表4组分进行配制)。
表4荧光定量PCR反应体系
混匀后使用实时荧光定量PCR仪(ABI 7500)进行扩增反应。扩增反应:95℃10min,1个循环(预变性);95℃5s,60℃60s,40个循环(PCR反应)。
②结果分析
采用2-△△Ct法以GAPDH作为内参,进行lncRNA-ASD的相对定量分析。
实验结果
实验结果如图3所示,从图中可以看出,冠状动脉粥样硬化组外周血PBMC中的lncRNA-ASD表达量(0.0205±0.0022)显著高于冠脉正常组(0.0037±0.0004),差异具有统计学意义(P<0.001)。
冠状动脉粥样硬化组和冠脉正常组的ROC曲线如图4所示,从图中可以看出,ROC曲线的AUC值为0.883,95%CI为0.718-0.892,P<0.0001。当Cutoff值为0.0052时,敏感性为83%,特异性为82%,说明lncRNA-ASD作为冠状动脉粥样硬化诊断标志物具有较好的临床诊断价值。
实施例3:对lncRNA-ASD诊断冠状动脉粥样硬化的准确性进行验证。
1.研究对象
收集40例急诊入院的胸痛患者外周静脉血,基线资料如表5所示。急诊入院采集外周血分离PBMC,通过qRT-PCR检测lncRNA-ASD表达,诊断、筛查胸痛患者中罹患动脉粥样硬化的人数,再与胸痛患者后续的冠脉造影检查结果进行比对,对lncRNA-ASD诊断冠状动脉粥样硬化的准确性进行评估。
表5急诊入院胸痛患者基线资料
2.采血方法
所有患者均于急诊收治当时采集外周静脉血3mL。
3.外周血单个核细胞分离和总RNA提取
采用与实施例1相同的方法,即密度梯度离心法从外周血中分离获得PBMC;采用与实施例1相同的方法,即Trizol法提取总RNA。
4.逆转录及qRT-PCR检测lncRNA-ASD诊断冠状动脉粥样硬化的准确性
采用与实施例2相同的方法及体系进行逆转录反应及荧光定量PCR反应。采用2-△△Ct法以GAPDH作为内参,进行lncRNA-ASD的相对定量分析。以lncRNA-ASD>0.0052作为诊断界值,与胸痛患者冠脉造影结果进行对比,评估lncRNA-ASD诊断冠状动脉粥样硬化的准确性。
实验结果
实验结果如表6所示,以lncRNA-ASD>0.0052作为诊断界值,检测到40名胸痛患者中有29名为冠状动脉粥样硬化患者,11名为其他疾病引起胸痛症状患者;作为诊断“金标准”的冠状动脉造影结果显示40例胸痛患者中,有30名为冠状动脉粥样硬化患者,10名为其他疾病引起胸痛症状患者。
以lncRNA-ASD>0.0052作为诊断界值与冠脉造影结果相比较,发现以lncRNA-ASD检测作为诊断性试验,检出“真阳性”患者26名,“真阴性”患者7名,其准确度(Acc)为82.5%(真阳性和真阴性在总受检例数中的比例),结果表明lncRNA-ASD诊断冠状动脉粥样硬化的准确性较好。
表6诊断性试验与冠脉造影检查结果符合情况
实施例4:一种用于诊断冠状动脉粥样硬化的荧光定量PCR试剂盒
1.组成成分
lncRNA-ASD及GAPDH引物(10μM,序列如下表所示),SYBR Green荧光染料(2X),ROX参比染料(50X)和ddH2O。
2.反应体系配制方式如下表
3.反应条件
预变性:95℃10min(1个循环);PCR反应:95℃15s,60℃60s(40个循环)。
4.结果分析
采用2-△△Ct法以GAPDH作为内参,进行lncRNA-ASD的相对定量分析。
上述实施例为本发明较佳的实施方式,但本发明的实施方式并不受上述实施例的限制,其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化,均应为等效的置换方式,都包含在本发明的保护范围之内。
Claims (10)
1.一种冠状动脉粥样硬化lncRNA标志物,其特征在于,所述lncRNA标志物为lncRNA-ASD,所述lncRNA-ASD的序列为SEQ ID No.1所示。
2.权利要求1中所述的lncRNA-ASD在制备如下任一项产品中的应用:
(1)诊断冠状动脉粥样硬化的产品;
(2)筛查冠状动脉粥样硬化的产品;
(3)评估冠状动脉粥样硬化风险的产品。
3.根据权利要求2所述的应用,其特征在于:所述产品包括检测lncRNA-ASD表达水平的试剂。
4.根据权利要求2所述的应用,其特征在于:所述产品包括检测lncRNA-ASD表达水平的荧光定量PCR试剂。
5.根据权利要求4所述的应用,其特征在于:所述检测lncRNA-ASD表达水平的荧光定量PCR试剂为特异性检测lncRNA-ASD表达水平的引物对,所述引物对包括正向引物:5’-CTGGCTGATGGAGGCTTGT-3’;及反向引物:5’-CCACCACTTCTTGAACCGCT-3’。
6.根据权利要求2所述的应用,其特征在于:所述产品为芯片、制剂、试纸条或试剂盒。
7.根据权利要求2-6任一项所述的应用,其特征在于:所述产品的检测样本为外周静脉血。
8.一种试剂盒,其特征在于:所述试剂盒包括权利要求5中所述的试剂;且所述试剂盒为用于冠状动脉粥样硬化诊断的试剂盒、或用于冠状动脉粥样硬化筛查的试剂盒、或用于冠状动脉粥样硬化风险评估的试剂盒。
9.根据权利要求8所述的试剂盒,其特征在于:所述试剂盒还包括特异性检测GAPDH表达水平的引物对、荧光染料。
10.根据权利要求9所述的试剂盒,其特征在于:所述荧光染料为SYBR Green荧光染料或TB Green荧光染料;所述的特异性检测GAPDH表达水平的引物对包括GAPDH 正向引物:5’-GAACGGGAAGCTCACTGG-3’和GAPDH反向引物:5’-GCCTGCTTCACCACCTTCT-3’。
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