CN116622866A - 布鲁氏菌病的外泌体miRNA标志物及应用 - Google Patents
布鲁氏菌病的外泌体miRNA标志物及应用 Download PDFInfo
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Abstract
本发明属于生物检测技术领域,具体公开了布鲁氏菌病的外泌体miRNA标志物,包括hsa‑miR‑11400、hsa‑miR‑199a‑5p或hsa‑miR‑148a‑5p中的一种或两种以上;hsa‑miR‑11400的核苷酸序列为SEQ ID NO:3,hsa‑miR‑199a‑5p的核苷酸序列为SEQ ID NO:5,hsa‑miR‑148a‑5p的核苷酸序列为SEQ ID NO:7。本发明还公开了上述用于检测上述布鲁氏菌病的外泌体miRNA标志物的试剂在制备检测试剂盒中的应用,以及用于检测布鲁氏菌病的外泌体miRNA标志物的试剂在制备检测试剂盒中的应用。本发明公开的在布鲁氏菌病中具有诊断潜力,为布鲁氏菌病提供潜在的诊断生物标志物。
Description
技术领域
本发明属于生物检测技术领域,尤其涉及布鲁氏菌病的外泌体miRNA标志物及应用。
背景技术
布鲁氏菌病(Brucellosis,简称“布病”)是由布鲁氏菌属细菌侵入机体,引起人畜共患的传染性变态反应性疾病,多因接触患病的牛、羊等牲畜及其制品感染,发病隐匿、诊断延迟和致残率高,对患者的健康尤其危险,不具有典型的临床症状并易形成慢性感染性疾病。目前已经有12种布鲁氏菌被报道,其中引起人类感染的最常见的布鲁氏菌包括羊种、牛种和猪种,其中羊种布鲁氏菌毒力最强。
随着畜牧业的迅速发展,我国牲畜饲养量逐渐增加,畜牧产品的流通速度加快,使布鲁氏菌病扩散速度增快,流行范围也不断扩大,布鲁氏菌病感染已成为我国重要的公共卫生问题之一。
微小RNA(microRNA,miRNA)是一类约为22个核苷酸大小的非编码单链RNA,对多种生物功能具有重要的调节作用,可作为疾病的分期、分级、疾病预测以及临床干预措施监测等生物标志物。目前,布鲁氏菌病患者的精确临床诊断面临着许多挑战。血清miRNAs的表达水平往往与机体状态存在密切关系,miRNAs有望成为布鲁氏菌病精确诊断和预后判断的良好标志物。近年来,国内外研究学者已发表了许多有关miRNAs作为生物标志的研究报道。但是相比于在肿瘤中已知的重要作用,miRNAs在感染性疾病尤其是细菌感染所致疾病中的作用研究还较少。
发明内容
本发明的目的在于提供布鲁氏菌病的外泌体miRNA标志物及应用,以解决上述技术问题。
本发明的目的之一在于提供布鲁氏菌病的外泌体miRNA标志物,包括hsa-miR-11400、hsa-miR-199a-5p或hsa-miR-148a-5p中的一种或两种以上;hsa-miR-11400的核苷酸序列为SEQ ID NO:3,hsa-miR-199a-5p的核苷酸序列为SEQ ID NO:5,hsa-miR-148a-5p的核苷酸序列为SEQ ID NO:7。
本发明目的之二在于提供上述用于检测上述布鲁氏菌病的外泌体miRNA标志物的试剂在制备检测试剂盒中的应用。
本发明的有益效果:本发明提供的外泌体hsa-miR-11400、hsa-miR-199a-5p和hsa-miR-148a-5p具有布鲁氏菌病诊断潜力,可以作为布鲁氏菌病精确诊断和预后判断的生物标志物。
附图说明
图1为血清外泌体的鉴定;A:血清来源外泌体中标记蛋白CD9和TSG101的蛋白质印迹;B:使用Zetasizer纳米zs90粒度仪测量的外泌体直径;C:从血清中纯化的外泌体的电子显微照片(比例尺500nm);
图2为差异表达的血清外泌体miRNA的热图;
图3为布鲁氏菌病患者中五种外泌体miRNA的相对表达;A:hsa-miR-194-5p的表达水平;B:hsa-miR-11400的表达水平;C:hsa-miR-148a-5p的表达水平;D:hsa-miR-199a-5p的表达水平;E:hsa-miR-23b-3p的表达水平;
图4为通过ROC曲线分析确定外泌体miRNA的诊断能力;A:hsa-miR-11400;B:hsa-miR-199a-5p;C:hsa-miR-148a-5p;D:hsa-miR-11400和hsa-miR-199a-5p;E:hsa-miR-11400和hsa-miR-148a-5p;F:hsa-miR-148a-5p和hsa-miR-199a-5p;G:hsa-miR-11400,hsa-miR-148a-5p和hsa-miR-199a-5p;
图5为基因本体论(GO)和京都基因和基因组百科全书(KEGG)对血清衍生的外泌体miRNAs(hsa-miR-11400、hsa-miR-199a-5p和hsa-miR-148a-5p)的免疫相关基因的分析;A:对明显富集的靶基因的GO分析;在生物过程(BP)、细胞成分(CC)和分子功能(MF)术语中,列出了前5个显著富集的项目;P值、富集因子和miRNA靶点数量被用来确定GO的富集程度;B:通过KEGG进行miRNA靶点分析的途径富集度分析。Allenrichment分数被评估为P值的负对数(-Log p);
图6为miRNA-免疫相关基因调控网络。方块代表基因,椭圆代表miRNAs;
图7为根据GSE69597数据确定18个免疫基因的mRNA表达;A:NFYA;B:CYLD;C:TRAF3;D:CDH1;E:PLXNA2;F:LEPR;G:TGFB2;H:GNRHR;I:IL-17RA;J:RORA;K:CD48;L:PRKCA;M:TNF;N:ACTG1;O:PTGDR;P:CD22;Q:ACVR1B;R:CBL。
具体实施方式
下面通过具体实施方式进一步详细说明:
实施例1筛选布鲁氏菌病的外泌体miRNA标志物
以下实验通过了伦理审批,并获得了所有参与者的知情同意。实验主要分为布鲁氏菌病组和健康对照组,参与者的信息参见表1。
布鲁氏菌病组入选标准:
根据中华人民共和国卫生行业标准《布鲁氏菌病诊断》(WS269-2019),临床诊断为原发性布鲁氏菌症;年龄30-50岁。
布鲁氏菌可以从任何病理物质培养物中分离出来,如患者的血液、骨髓、其他体液和排泄物初筛中玫瑰红-孟加拉平板试验(RBPT)阳性,血清凝集试验(SAT),滴度≥1:100或病程持续一年以上仍有症状,滴度为1:50参与者没有并发糖尿病、艾滋病和其他自身免疫疾病。
排除标准:已接受药物治疗的布鲁氏菌病患者;患有传染病、基础疾病和肿瘤相关疾病者。
表1参与者信息及临床特征
以下实验的第一步和第二步仅涉及3名布鲁氏菌病患者和3名健康对照者外周血用于高通量测序。第三步验证血清外泌体miRNA时,每组增加22名参与者,共涉及25名布鲁氏菌病患者和25名健康对照者。
以下实验的所有数据均通过GraphPad Prism软件Ver.8或R版本4.1.3进行统计分析。数据表示为平均值±标准差。基于方差的同质性,使用双尾Student’s T检验或Mann-Whitney U检验对两组进行比较。使用ROC曲线评估血清外泌体miRNA对布鲁氏菌病的诊断能力。P<0.05被认为具有统计学意义。
第一步:外泌体的分离和表征
为了分离外泌体,3名布鲁氏菌病患者和3名健康者的血清样本预先通过0.22μM过滤器过滤。按照制造商的说明,使用QIAGEN exoRNeasy Maxi Kit试剂盒(Qiagen,Hilden,德国)分离外泌体。分离的外泌体用PBS稀释后,使用Zetasizer纳米zs90粒度仪测量外泌体的直径。再通过透射电子显微镜(TEM)观察外泌体的微观结构。
为了验证外泌体是从布鲁氏菌病和健康对照组的血清中分离出来的,使用Western blotting蛋白质印迹法分析评估外泌体特异性蛋白(TSG101和CD9)。
将适量的RIPA裂解物(RIPA lysate)添加到分离的外泌体中以提取蛋白质。通过biosharp BCA蛋白浓度测定试剂盒(biosharp,中国,货号BL521A)检测样品的蛋白质浓度为2680.07ng/uL。
将等量的蛋白质样品加入到12% SDS聚丙烯酰胺凝胶中进行电泳。电泳后,将条带转移到PVDF膜上,并用脱脂奶粉密封1.5h;在4℃下将该膜与一抗抗兔多克隆CD9抗体(1:500,NBP2-67310,Novus Biologicals)和一抗抗小鼠多克隆TSG101抗体(1:5000,NB200-112,Novus生物制品)孵育过夜。然后用二级抗体(1:5000,ZB-2305或ZB-2301,中山金桥生物技术)孵育1h。通过增强化学发光法(ECL)获得图像。
图1为血清外泌体的鉴定结果;A:血清来源外泌体中标记蛋白CD9和TSG101的蛋白质印迹;B:使用Zetasizer纳米zs90粒度仪测量的外泌体直径;C:从血清中纯化的外泌体的电子显微照片(比例尺500nm)。
从图1A可以看出,TSG101和CD9在血清样本中表达,从而确定了外泌体的存在。
从图1B可以看出,从血清中提取的外泌体的直径小于100nm。
从图1C可以看出,外泌体的形态通过TEM进一步观察,证明从血清样本中提取了外泌体。
第二步、从外泌体中提取RNA、高通量测序,以及分析已知miRNA和新鉴定的miRNA的表达水平
根据miRcute Plus miRNA qPCR试剂盒(TIANGEN)提取总RNA。RNA的浓度和纯度由Nanodrop2000(Thermo Scientific,USA)测定,检测OD260/OD280的比值为1.96。OD260代表核酸的吸光度,OD280代表蛋白质的吸光度,OD260/OD280的比值在1.8-2.0之间认为RNA较纯,RNA样品被认为是高质量样品。当比值低于1.8时,考虑有蛋白质或酚污染。当比值高于2.0时,表明可能有异硫氰酸残存。
使用高通量测序技术进行小RNA测序。通过高通量测序获得原始图像数据文件,并通过碱基判读技术用将其转换为原始序列数据。使用Cutadapt软件从原始序列读数中删除适配器序列,Bowtie软件去除rRNA、tRNA、miscRNA、snRNA和snoRNA;在miRBase 21.0数据库中(http://mirbase.org/)再次搜索过滤的序列以确认新的miRNA。
使用DEGseq软件分析已知miRNA和新鉴定的miRNA的表达水平。计算出的miRNA表达指数为百万分之一(TPM)。将P值设置为<0.05,将折叠变化(FC)设置为≤0.5或≥2,以识别布鲁氏菌病患者和健康对照者之间血清外泌体中的差异表达miRNAs(DEMs)。
共在3名布鲁氏菌病患者和3名对照者的血清外泌体中鉴定出2006种miRNA。两组之间共有41个成熟的miRNA存在显著差异(P<0.05)。布鲁氏菌病组和对照组之间存在不同的miRNA表达模式。其中,27个miRNA下调,14个miRNA上调。
图2为差异表达的血清外泌体miRNA的热图。热图显示了布鲁氏菌病和对照血清外泌体样品中miRNA的差异表达(P<0.05)。与对照组相比,布鲁氏菌病患者样本中27个miRNA表达下调,14个表达上调。与对照组相比,布鲁氏菌病患者中hsa-miR-11400、hsa-miR-199a-5p和hsa-miR-148a-5p的外泌体表达显著增加。
第三步、qRT-PCR
从25名布鲁氏菌病患者和25名健康对照者中提取血清外泌体,并进一步提取外泌体的总RNA。使用miRcute Plus miRNA第一链cDNA试剂盒(KR211,TIANGEN)将总RNA逆转录成cDNA。使用miRcute Plus miRNA qPCR试剂盒(FP411,TIANGEN)进行定量RT-PCR。所有miRNA引物均购自上海Sangon。U6被设定为内部控制,Ct值被归一化。
本实验基于测序结果,选择了前5个上调最大的DEMs进行验证,并在表2中列出了实验中用到的miRNA引物。
表2研究中使用的microRNA引物
如表4所示,布鲁氏菌病组中hsa-miR-194-5p的表达水平降低(图3A,P<0.01);布鲁氏菌病组与对照组相比,外泌体hsa-miR-11400、hsa-miR-148a-5p和hsa-miR-199a-5p的相对表达水平显著增加,与miRNA高通量测序结果一致(图3B,C和D,P<0.01);两组之间的hsa-miR-23b-3p表达水平没有显著差异(图3E,P>0.05)。
表4选择用于验证的microRNA的相对表达水平
图3为与对照组相比,布鲁氏菌病患者中五种外泌体miRNA的相对表达;A:hsa-miR-194-5p的表达水平;B:hsa-miR-11400的表达水平;C:hsa-miR-148a-5p的表达水平;D:hsa-miR-199a-5p的表达水平;E:hsa-miR-23b-3p的表达水平。nsP>0.05,*P<0.05和**P<0.0。
qRT-PCR结果显示,hsa-miR-11400、hsa-miR-199a-5p、hsa-miR-148a-5p在布鲁氏菌病血清外泌体中显着稳定上调。
第四步、ROC曲线评估血清外泌体miRNA对布鲁氏菌病的诊断能力
ROC曲线是受试者工作特征曲线,根据患者和健康者指标检测结果计算的曲线,是反应指标敏感度和特异度连续变量的综合指标,用于判断指标诊断预测效能。
根据上述布鲁氏菌病患者和健康者血清外泌体的表达量制定受试者工作特征曲线(ROC)分析,评估了hsa-miR-11400、hsa-miR-199a-5p和hsa-miR-148a-5p是否可以作为布鲁氏菌病的生物标志物。图4为通过ROC曲线分析外泌体miRNA的诊断预测效能,图中显示每个miRNA的敏感性和特异性及ROC曲线下的面积(AUC);A:hsa-miR-11400;B:hsa-miR-199a-5p;C:hsa-miR-148a-5p;D:hsa-miR-11400联合hsa-miR-199a-5p;E:hsa-miR-11400联合hsa-miR-148a-5p;F:hsa-miR-148a-5p联合hsa-miR-199a-5p;G:hsa-miR-11400联合hsa-miR-148a-5p和hsa-miR-199a-5p。
从表5和图4可以得出,每一个单独的miRNA都可以将布鲁氏菌病感染与健康对照区分开来:
hsa-miR-11400的曲线下面积AUC=0.79(95% Cl:0.66–0.91)(P<0.05);
hsa-iR-199a-5p的曲线下面积AUC=0.81(95% Cl:0.69–0.92)(P<0.05);
hsa-miR-148a-5p的曲线下面积AUC=0.74(95% Cl:0.60–0.88)(P<0.05)。
此外,miRNA的组合通常会提高AUC:
组合hsa-miR-11400和hsa-miR-199a-5p的AUC为0.90(95%Cl:0.80–0.98)(P<0.05);
组合hsa-miR-138a-5p与hsa-miR-1240或hsa-miR-19a-5p,AUC>0.8(P<0.05);
同时组合hsa-miR-11400、hsa-miR-199a-5p和hsa-miR-148a-5p的AUC为0.92(95%Cl:0.84–1.00)(P<0.05)。
表5.对血清外泌体单独和联合治疗布鲁氏菌病的预测价值
第五步、游靶基因的预测
通过miRWalk数据库预测miRNA的靶基因。通过比较三个数据库的结果获得miRNA的最终基因:TargetScan、miRDB和miTarBase。
预测了465个DEMs的靶基因,其中包括25个免疫相关的靶基因,这些免疫基因主要富集于癌症中的蛋白聚糖、NF-kappa B信号通路和IL-17信号通路。
通过DEMs-免疫基因网络构建,发现免疫基因可能主要受到hsa-miR-199a-5p和hsa-miR-148a-5p的调节。在这些免疫基因中,PLXNA2、IL17RA、PRKCA、CD22、ACVR1B和CBL的表达与GSE69597数据集中一致。
miRWalk预测了总共465个hsa-miR-11400、hsa-miR-199a-5p和hsa-miR-148a-5p的靶基因。此外,表6中列出了这些上调DEMs的靶基因数量。
表6显著上调的DEMs的潜在目标基因
上调的DEMs | 数量 |
hsa-miR-11400 | 26 |
hsa-miR-199a-5p | 174 |
hsa-miR-148a-5p | 265 |
靶基因总数量 | 465 |
第六步、筛选免疫基因,并对25个免疫基因进行分析
使用immport数据库检索miRNA下游靶基因中的免疫相关基因集。
通过imimport数据库,本发明从DEMs的靶基因中筛选了25个免疫相关基因。
为了解推定靶基因的生物学功能和涉及的信号通路,使用GO(www.geneontology.org)和KEGG(www.genome.jp/KEGG)数据库对每个基因进行注释。
使用“clusterProfiler”包进行GO富集分析的结果如图5A所示。生物过程(BP)分析表明,免疫基因在造血、细胞连接组织、细胞-细胞连接组织等的正调节中显著富集;细胞成分(CC)显示靶免疫基因在膜区、膜微区和血清膜受体复合物中富集;分子功能(MF)分析表明,靶基因在受体配体活性和细胞因子受体活性方面显著丰富。
此外,KEGG通路分析表明,DEM的免疫基因在癌症蛋白聚糖、NF-kappa B信号通路和IL-17信号通路等中显著富集(图5B)。
图5为基因本体论(GO)和京都基因和基因组百科全书(KEGG)对血清衍生的外泌体miRNAs(hsa-miR-11400、hsa-miR-199a-5p和hsa-miR-148a-5p)的免疫相关基因的分析;A:对明显富集的靶基因的GO分析;在生物过程(BP)、细胞成分(CC)和分子功能(MF)术语中,列出了前5个显著富集的项目;P值、富集因子和miRNA靶点数量被用来确定GO的富集程度;B:通过KEGG进行miRNA靶点分析的途径富集度分析。Allenrichment分数被评估为P值的负对数(-Log p)。
从GEO数据库下载的GSE69597 mRNA数据集来分析免疫基因的表达水平,数据集在GPL16791平台(Illumina HiSeq 2500)中获得,数据集内包含了46例布鲁氏菌病和46例健康者全血。确定布鲁氏菌病和对照全血之间差异表达免疫基因的Students'T检验。
最后,有意义的免疫基因需要满足以下标准:首先,DEMs与其对应下游的靶基因呈相反关系,若DEMs上调,其靶基因则下调,其次,P<0.05有统计学差异。通过大数据筛选出的差异基因DEMs就是目标外泌体,通过目标外泌体基因预测下游的靶基因。一般来说,目标基因和其下游对应的靶基因是呈相反的关系,即目标基因上调,其对应下游的靶基因是下调的。
为了更好地研究布鲁氏菌病血清外泌体中这些DEMs的分子免疫机制,使用Cytoscape软件构建了DEMs免疫基因网络(图6)。图6为miRNA-免疫相关基因调控网络。方块代表基因,椭圆代表miRNAs。
上调的DEMs与免疫基因之间的相互作用如下:hsa-miR-11400与两个免疫基因CD48和PTGDR相互作用(主要是研究目标基因对应下游哪些靶基因。比如hsa-miR-11400对应下游的靶基因有CD48和PTGDR,有助于后期筛选通路)。hsa-miR-148a-5p与10个免疫基因(TNF、CYLD、PLXNA2、FGF5、PTHLH、CSF2RB、GNRHR、LEPR、RORA和CBL)相互作用;hsa-miR-199a-5p与13个免疫基因(NFYA、TRAF3、KNG1、NOS1、ACTG1、TAFA2、CDH1、CD22、KL、TGFB2、ACVR1B、IL-17RA和PRKCA)相互作用。
图6为miRNA-免疫相关基因调控网络。方块代表基因,椭圆代表miRNAs。
使用GSE69597数据集鉴定了18种免疫基因(CD48、TNF、PTGDR、CYLD、PLXNA2、GNRHR、LEPR、RORA、CBL、NFYA、TRAF3、ACTG1、CDH1、CD22、TGFB2、ACVR1B、IL-17RA和PRKCA)的外周血水平。
图7为根据GSE69597数据确定18个免疫基因的mRNA表达;A:NFYA;B:CYLD;C:TRAF3;D:CDH1;E:PLXNA2;F:LEPR;G:TGFB2;H:GNRHR;I:IL-17RA;J:RORA;K:CD48;L:PRKCA;M:TNF;N:ACTG1;O:PTGDR;P:CD22;Q:ACVR1B;R:CBL。数据以平均值±SD表示。P>0.05,*P<0.05和**P<0.01。对于上调的DEMs(hsa-miR-11400、hsa-miR-199a-5p和hsa-miR-148a-5p),与正常对照组相比,布鲁氏菌病患者外周血中只有PLXNA2、IL17RA、PRKCA、CD22、ACVR1B和CBL的表达持续降低,其余表达增加或差异无统计学意义。因此,hsa-miR-199a-5p-CD22、hsa-miR-9a-5p-IL17RA、hsa-iR-199a-5b-PRKCA、hsa-miR-199a-5p-ACVR1Bhsa-miR-148a-5p-PLXNA2和hsa-miR-138a-5p-CBL被鉴定为布鲁氏菌病的六种潜在调节途径。
综上,本实施例从受试者的外周血中分离出血浆外泌体。通过透射电镜(TEM)、纳米粒追踪分析(NTA)和蛋白质印迹(WB)鉴定外显体的形态和蛋白质标记,证实了外泌体miRNA在布鲁氏菌病中具有诊断潜力,为布鲁氏菌病提供潜在的诊断生物标志物,通过构建了潜在的外泌体miRNA-mRNA调控网络,为布鲁氏菌病的发病机制和治疗提供了新的思路。
实施例2布鲁氏菌病的外泌体miRNA标志物的检测试剂盒
1、试剂盒组份
表7试剂盒组份
管号 | 试剂名称 | 主要组份 | 规格 |
1 | hsa-miR-11400引物 | 引物SEQ ID NO:4 | 10-25μL×1管 |
2 | hsa-miR-199a-5p引物 | 引物SEQ ID NO:6 | 10-25μL×1管 |
3 | hsa-miR-148a-5p引物 | 引物SEQ ID NO:8 | 10-25μL×1管 |
4 | MiliQ-Water | 超纯水 | 1mL×1管 |
2、试剂盒使用方法
(1)提取样本RNA:提取待检样本中的RNA;(2)基因扩增:对待检样本中的RNA进行逆转录和DNA扩增;(3)结果检测:对扩增结果进行检测。
以上详细描述了本发明的较佳具体实施例。应当理解,本领域的普通技术人员无需创造性劳动就可以根据本发明的构思作出诸多修改和变化。因此,凡本技术领域中技术人员依本发明的构思在现有技术的基础上通过逻辑分析、推理或者有限的实验可以得到的技术方案,皆应在由权利要求书所确定的保护范围内。
Claims (2)
1.布鲁氏菌病的外泌体miRNA标志物,其特征在于,包括hsa-miR-11400、hsa-miR-199a-5p或hsa-miR-148a-5p中的一种或两种以上;hsa-miR-11400的核苷酸序列为SEQ IDNO:3,hsa-miR-199a-5p的核苷酸序列为SEQ ID NO:5,hsa-miR-148a-5p的核苷酸序列为SEQ ID NO:7。
2.用于检测权利要求1所述的布鲁氏菌病的外泌体miRNA标志物的试剂在制备检测试剂盒中的应用。
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