CN112961914A - miR-3074-5p作为类风湿关节炎标志物的应用及其试剂盒 - Google Patents
miR-3074-5p作为类风湿关节炎标志物的应用及其试剂盒 Download PDFInfo
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Abstract
本发明涉及miR‑3074‑5p的新用途，miR‑3074‑5p可作为microRNA分子标志物在制备类风湿关节炎的诊断试剂或试剂盒中进行应用。在miR‑3074‑5p抑制表达组中TNFSF14的mRNA表达水平明显高于对照组，通过计算受试者工作特征曲线(ROC)和斯皮尔曼相关性检验评估miRNA在RA中的诊断价值，确认其在RA诊断中的潜在作用。miR‑3074‑5p可以被认为是一个新的类风湿关节炎的检测或治疗靶点，为RA的诊断和治疗提供新应用方向。miR‑3074‑5p作为类风湿关节炎的检测或治疗靶点时，其AUC＝1，被认为是及其可信的检测指标。
Description
技术领域
本发明属于生物分子技术领域，涉及miR-3074-5p作为类风湿关节炎标志物的应用及其试剂盒。
背景技术
类风湿关节炎(Rheumatoid arthritis,RA)是一种常见的以滑膜炎、滑膜增生、骨软骨破坏为特征的慢性炎症疾病，最终可导致软骨或骨损伤，甚至是残疾。RA在女性中的发病率高于男性，遗传因素、环境因素和生活方式是风湿性关节炎的高危因素，如吸烟、饮酒、饮食不良等，但其发病机制尚不清楚。RA的早期诊断对于积极治疗关节或器官的不可逆损伤以及通过广泛使用常规药物和抗风湿性关节炎药物(DMARDs)控制慢性炎症至关重要。然而，部分类风湿因子和环瓜氨酸抗体均为阴性的RA患者可能会被误诊，延误可能导致临床预后恶化。因此，探讨RA的病因病机对RA的早期预防、早期诊断和有效干预具有重要价值。
小分子核糖核酸(microRNA，miRNA)是一种18-25nt长的内源性非编码RNA，广泛分布在多个真核生物，通过调节mRNA的降解或翻译抑制来调节一个或多个基因的表达。miRNA的合成和功能的分子机制已被广泛研究。随着分子生物学的发展，miRNA可以影响免疫系统的各个方面，从造血到效应功能，但其在RA中的作用机制尚未明确。
TNFSF14(tumor necrosis factor superfamily 14),又名LIGHT[lymphotoxin-like,exhibits inducible expression,and competes with HSV glycoprotein D(gD)for HVEM,a receptor expressed by T lymphocytes],或HVEM-L(Herpesvirus entrymediator-Ligand),是Mauri等于1998年在对单纯疱疹病毒活化T细胞的研究中发现的TNF超家族的第14个成员。LIGHT是一个多功能、多效应分子，在体内的异常表达参与了机体自身免疫，与一些自身免疫性疾病有关。
发明内容
有鉴于此，本发明的目的在于提供一种miR-3074-5p作为microRNA分子标志物在制备类风湿关节炎的诊断试剂或试剂盒中的应用，以及检测miR-3074-5p表达水平的引物的相关应用，还提供一种检测类风湿关节炎的诊断试剂盒。
为达到上述目的，本发明提供如下技术方案：
1.miR-3074-5p作为microRNA分子标志物在制备类风湿关节炎的诊断试剂或试剂盒中的应用。
2.检测miR-3074-5p表达水平的引物在制备类风湿关节炎的诊断试剂或试剂盒中的应用。
进一步，所述miR-3074-5p表达水平为外周血单个核细胞中的miR-3074-5p表达水平。
进一步，检测miR-3074-5p表达水平的引物的序列为SEQ ID NO:6。
3.一种检测类风湿关节炎的诊断试剂盒，所述诊断试剂盒包括检测miR-3074-5p表达水平的引物。
进一步，检测miR-3074-5p表达水平的引物的序列为SEQ ID NO:6。
进一步，试剂盒还包括反转录试剂部分和荧光定量检测试剂部分。
进一步，反转录试剂部分由2×miRNA RT Reaction Buffer、miRNA RT EnzymeMix、RNase-Free ddH2O组成。
进一步，荧光定量检测试剂部分由2×miRcute Plus miRNA PreMix、10μMReverse Primer、ddH2O组成。
进一步，2×miRcute Plus miRNA PreMix中包含SYBR和ROX染料。
进一步，反转录体系总体积20μl，总RNA为2～5μl，2×miRNA RT Reaction Buffer10μl、miRNA RT Enzyme Mix 2μl，最后RNase-Free ddH2O补至20μl。
进一步，荧光定量检测体系总体积20μl或50μl，20μl体系组成为2×miRcute PlusmiRNA PreMix 10μl、10μM Reverse Primer 0.4μl、Forward Primer 0.4～0.8μl、cDNA1-2μl、余量为ddH2O。
进一步，2×miRcute Plus miRNA PreMix中包含SYBR和ROX染料。
4、miR-3074-5p抑制剂在制备抑制TNFSF14表达量降低的试剂中的应用。
5、miR-3074-5p抑制剂在制备类风湿关节炎的治疗试剂或试剂盒中的应用。
本发明的有益效果在于：本发明通过检测RA外周血单个核细胞(Peripheralblood mononuclear cells,PBMC)中不同基因的mRNA表达，发现TNFSF14 mRNA水平在RA的PBMCs存在显著表达差异，进一步利用生信预测可能与其相互作用的microRNA，再通过大量双荧光素酶报告系统和功能性实验等方法进行验证；最后用RT-PCR检测到miR-3074-5p水平在RA的PBMCs存在表达差异，并在miR-3074-5p不同表达状态下检测TNFSF14的表达水平，结果显示TNFSF14的基因mRNA表达量在miR-3074-5p过表达的细胞系中表达量明显下调，而在miR-3074-5p抑制表达组中TNFSF14的mRNA表达水平明显高于对照组。由此可以得出TNFSF14表达受到miR-3074-5p的调控作用，结合双荧光素酶报告系统可以认为TNFSF14是miR-3074-5p下游的靶基因。再进一步通过ROC曲线评估miR-3074-5p在RA中的临床诊断价值，确认其在RA诊断中的潜在作用。miR-3074-5p与RA PBMCs中的anti-CCP水平呈正相关，TNFSF14表达水平与RA PBMCs中RA-IgG、RF和DAS28呈负相关。这些结果都表明miR-3074-5p通过靶向TNFSF14参与RA的发生、发展过程，可以被认为是一个新的类风湿关节炎的检测或治疗靶点，为RA的诊断和治疗提供新应用方向。miR-3074-5p作为类风湿关节炎的检测或治疗靶点时，其AUC＝1，被认为是及其可信的检测指标。
附图说明
为了使本发明的目的、技术方案和有益效果更加清楚，本发明提供如下附图进行说明：
图1为PBMC中TNFSF14的mRNA水平相对表达量差异比较；
图2为作用于TNFSF14的microRNAs韦恩图；
图3为TNFSF14的microRNAs相互作用的网络图；
图4为miR-3074-5p与TNFSF14基因存在的多个互补结合位点示意图；
图5为各RT-PCR结果，其中，a为用miR-3074-5p mimics转染后，miR-3074-5p的表达水平；b，miR-3074-5p模拟物转染后，TNFSF14的表达水平；c，转染miR-3074-5p抑制剂后，miR-3074-5p的表达水平。d，转染miR-3074-5p抑制剂后，TNFSF14的表达水平。
图6为双荧光素酶活性检测结果；
图7为miR-3074-5p的熔解曲线；
图8为TNFSF14的熔解曲线；
图9为RA和HC的定量RT-PCR结果及比较；
图10为RA和HC组的PBMC中miR-3074-5p的ROC分析；
图11为RA患者中miR-3074-5p、TNFSF14表达与部分临床参数的关系。
具体实施方式
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
下述实施例中所使用的实验方法如无特殊说明，均为常规方法。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
以下实施例中的定量试验，均设置三次重复实验，数据为三次重复实验的平均值或平均值±标准差。
主要试剂：
1、RNA提取试剂盒为日本TaKaRa公司、miRNA逆转录试剂盒和荧光定量PCR试剂盒为天根公司。
2、野生型(WT)和突变型(MUT)双荧光素酶载体由广州锐博构建。
3、miR-3074-5p模拟物(mimics)、阴性对照模拟物(NC mimics)、miR-3074-5p抑制剂(inhibitor)和阴性对照抑制剂(NC inhibitor)由上海吉玛公司合成。
4、双荧光素酶检测试剂盒购于北京普洛麦格公司。
5、胎牛血清(FBS)、细胞培养基(RPMI-1640、DMEM)和转染试剂(Lipofectamine3000)均来源为美国Thermo Fisher公司。
主要仪器：
Nanodrop-2000核酸定量仪来源为美国Thermo Fisher公司，高速冷冻离心机来源为德国Eppendorf公司，qRT-PCR仪来源为美国Bio-Rad公司。
实施例1
研究对象：2019年3月至2020年10月在陆军军医大学西南医院招募22例RA患者。RA疾病活动度由疾病活动度评分28(DAS28)来定义。此外，本研究纳入22名健康人(Healthycontrol,HC)作为对照(表1)。研究由西南医院伦理委员会批准并监督，所有参与者均给出了书面知情同意书。人T淋巴细胞白血病细胞，Jurkat(Clone E6-1)和人胚肾细胞293T细胞(HEK-293T)购自中国科学院上海细胞库。
表1研究人群的临床特征
其中RF:rheumatoid factor(类风湿因子)；anti-CCP:anti-cyclocitrullineantibody(抗环瓜氨酸抗体)；TJC:number of tenderness joints(压痛关节数)；SJC:Number of joint swelling(关节肿胀数)；ESR:Erythrocyte sedimentation rate(红细胞沉降率)；CRP:C-reactive protein(C反应蛋白)；DAS28:Condition score of patientswith rheumatoid arthritis(类风湿关节炎患者的病情评分)。所有数据均表示为平均值±SD。N/A(Not applicable)：不适用。
实施例2
本实施例中使用的mRNA和GAPDH的引物由英潍捷基有限公司(上海，中国)设计和合成。用于检测基因mRNA表达水平的引物如下表2所示：
表2
| 名称 | 引物序列 | 序列号 |
| TNFSF14 | 5'-ATACAAGAGCGAAGGTCTCACG-3'(F) | SEQ ID No.1 |
| TNFSF14 | 5'-CTGAGTCTCCCATAACAGCGG-3'(R) | SEQ ID No.2 |
| GAPDH | 5'-GGAGTCCACTGGCGTCTTC-3'(F) | SEQ ID No.3 |
| GAPDH | 5'-GCTGATGATCTTGAGGCTGTTG-3'(R) | SEQ ID No.4 |
用GAPDH水平标准化circRNA的相对表达水平。
图8为TNFSF14的熔解曲线(箭头所指的曲线)，22例RA患者PBMC中TNFSF14的mRNA水平相对表达量为(0.59±0.36)，与22例HC相比(1.41±0.39)，差异有统计学意义(P<0.0001)，具有显著差异，结果如图1所示，结果提示RA患者PBMC中TNFSF14 mRNA的相对表达量显著低于健康人组。
实施例3
进一步探究TNFSF14参与RA发病可能的机制，我们通过miRDB(http://www.mirdb.org/),miRwalk(http://mirwalk.umm.uni-heidelberg.de/)和TargetScan(http://www.targetscan)预测可能作用于TNFSF14的microRNAs。韦恩图分析发现71个共同交叉的microRNAs(图2)，其中蓝色代表miRDB，黄色代表miRwalk，绿色代表TargetScan。并通过cytoscape作出它们相互作用的网络图(图3)。并通过Targetscan软件分析各microRNAs与TNFSF14基因的多个互补结合位点，图4为miR-3074-5p与TNFSF14基因存在的多个互补结合位点示意图。进一步通过实验研究证明各microRNAs与TNFSF14基因的潜在关系，以发现和证明microRNAs可作为RA诊断分子标志物的潜能。经过重重实验筛选和证实，TNFSF14是miR-3074-5p的潜在靶基因，以下只记载与本发明技术方案相关的内容，其余实验在此不提。
实施例4
为进一步证实TNFSF14是miR-3074-5p的潜在靶基因，我们在细胞系Jurkat中转染miR-3074-5p minics和miR-3074-5p inhibitor，NC minics和NC inhibitor作为阴性对照。miRNA inhibitor为化学合成的经过甲氧基修饰设计的成熟miRNA互补单链。专门针对细胞中特异的靶miRNA的抑制剂，可高效地抑制生物体内源型miRNA的活性。
1、细胞培养与转染：Jurkat细胞用RPMI-1640培养基(10％FBS、1％青霉素链霉素)，37℃5％CO2恒温孵箱培养，第二天换液。将细胞分为转染组(miR-3074-5p mimics、miR-3074-5p inhibitor)，阴性对照组(NC mimics、NC inhibitor)和只加转染试剂的空白对照组。转染用12孔板，每孔5×105个jurkat细胞，使用Opti-MEMTM培养基分别稀释带转染的质粒和Lipofectamine 3000，5min后将两者按1:1比例轻轻充分混匀并在室温下静置15min，每个培养孔加入质粒-脂质体复合物，前后轻轻摇动细胞培养板使复合物与培养板中培养液混匀，最后将细胞放置于细胞培养箱中培养，48h之后收集细胞用于后续RT-PCR实验。
图5为各RT-PCR结果，其中，a为用miR-3074-5p mimics转染后，通过RT-PCR检测miR-3074-5p的表达水平；b，miR-3074-5p模拟物转染后，通过RT-PCR检测TNFSF14的表达水平；c，转染miR-3074-5p抑制剂后，通过RT-PCR检测miR-3074-5p的表达水平。d，转染miR-3074-5p抑制剂后，通过RT-PCR检测TNFSF14的表达水平。各RT-PCR结果显示，miR-3074-5pminics转染jurkat之后，miR-3074-5p mRNA表达增加，TNFSF14 mRNA表达减少，与阴性对照组比较，差异有统计学意义(P<0.05)(图5a，5b)。miR-3074-5p inhibitor转染Jurkat细胞之后，miR-3074-5p mRNA表达减少，TNFSF14 mRNA表达增加，与阴性对照组比较，差异有统计学意义(P<0.05)(图5c，5d)。功能实验综合显示miR-3074-5p可有效调控TNFSF14表达。
2、双荧光素酶报告系统检测荧光素酶活性：将TNFSF14-3'UTR构建到双荧光素酶质粒中，分miR-3074-5p mimics+TNFSF14-3'UTR-WT、NC mimics+TNFSF14-3'UTR-WT、miR-3074-5p mimics+TNFSF14-3'UTR-MUT和NC mimics+TNFSF14-3'UTR-MUT 4组共转染于293T细胞中。
将293T细胞置于DMEM高糖培养基+10％FBS、1％青霉素链霉素，37℃5％CO2恒温孵箱里培养，待293T细胞密度达到50％-70％时，按照以下分组转染细胞：miR-3074-5pmimics+TNFSF14-3'UTR-WT共转染组、NC mimics+TNFSF14-3'UTR-WT共转染组、miR-3074-5p mimics+TNFSF14-3'UTR-MUT共转染组和NC mimics+TNFSF14-3'UTR-MUT共转染组。48h后收集各转染组的293T细胞，弃去细胞培养液后加入200μl被动细胞裂解液，摇床上晃动20min充分裂解细胞后4000rpm/min离心5min，按照双荧光素酶报告基因检测试剂盒说明书吸取上清液测定萤火虫荧光素酶和海肾荧光素酶活性。萤火虫荧光素酶作为内参，计算两者相对荧光强度的比值，以比值作为相对荧光素酶活性的结果。每个转染组独立重复3次实验，对3次测定的实验数据进行统计分析。
图6为双荧光素酶活性检测结果，其中a为带有miR-3074-5p的TNFSF14 3-UTR上结合位点的突变示意图；b为TNFSF14 3-UTR和miR-3074-5p共转染对293T细胞荧光的影响。检测结果表明，miR-3074-5p mimics+TNFSF14 3'UTR-WT共转染组低于对照组，差异有统计学意义(P<0.05)；突变TNFSF14-3'UTR与miR-3074-5p的结合位点之后，miR-3074-5p mimics+TNFSF14-3'UTR-MUT共转染组与对照组比较，差异无统计学意义(P>0.05)(图6b)。实验结果表明miR-3074-5p通过靶向TNFSF14-3'UTR负向调控TNFSF14的表达。
实施例5
为了验证miR-3074-5p在RA中的表达水平是否与预测的一致，我们在22例RA患者和22例健康对照组中进行了定量RT-PCR。
外周血单个核细胞的制备及总RNA的提取，(TaKaRa，Code No.9108Q)：实验前从每个受试者中取约5ml全血，储存在乙二胺四乙酸(EDTA))抗凝管中。采用室温密度梯度离心法(2000rpm，20min)从供者新鲜血液提取PBMCs。随后，根据说明书用Trizol提取总RNA，nanodrop 2000和1％琼脂糖凝胶用于测定RNA浓度和纯度，将总的RNA保存在-80℃以备进一步实验。
将总RNA反转录为cDNA，选用miRcute增强型miRNA cDNA第一链合成试剂盒(KR211)，反转录体系总体积20μl，具体体系成分见下表3：
表3：反转录体系
| 试剂组分 | 体积 | 终浓度 |
| 总RNA | 2-5μl | 可达2μg |
| 2×miRNA RT Reaction Buffer | 10μl | 1× |
| miRNA RT Enzyme Mix | 2μl | - |
| RNase-Free ddH<sub>2</sub>O | 补至20μl | - |
逆转录miRNA为cDNA：42℃60min加poly A尾和逆转录反应，95℃灭活逆转录酶3min，-20℃保存。
实时定量聚合酶链反应：使用miRcute增强型miRNA荧光定量检测试剂盒(FP411)，定量RT-PCR体系总体积20μl或50μl，具体体系成分见下表4：
表4：
本实施例中使用的microRNA和U6的正向引物由天根生物技术合成(北京，中国)，U6snRNA作为microRNA规范化的内源性对照。通用反向引物在天根逆转录试剂盒中提供。U6Forward Primer为5'-CGCAAGGATGACACGCAAATTC-3'(SEQ ID No.5)，miR-3074-5p的Forward Primer为5’-TTCCTGCTGAACTGAGCCA-3’(SEQ ID No.6)。
将上述cDNA(稀释10倍)为模板，用SEQ ID No.6所示的miR-3074-5p PCR引物进行qRT-PCR反应。qRT-PCR反应条件：95℃预变性30s，95℃变性15s，60℃退火30s，共40个循环。反应结束后根据扩增曲线分析扩增结果，得到了每个样本的Ct值，按照2-ΔΔCt法计算目的基因和内参U6的相对表达量相对表达量。每个样本独立重复三次以上。
图7为miR-3074-5p的熔解曲线，图9为定量PCR检测miR-3074-5p在RA和HC组PBMC中的表达结果及比较；图10为RA和HC组的PBMC中miR-3074-5p的ROC分析。结果表明，22例RA患者PBMC中miR-3074-5p的相对表达量为(1.64±0.34)，与22例HC相比(0.77±0.25)，差异有统计学意义(P<0.001)，RA患者PBMC中miR-3074-5p的相对表达量高于健康人组。进一步，我们使用ROC曲线及曲线下面积(area under the curve，AUC)分析来验证miR-3074-5p作为RA诊断分子标志物的潜能。其中AUC反映了检测效能，AUC越接近1.0，检测方法真实性越高；0.5＜AUC＜0.7检测效能较低；0.7＜AUC＜0.85检测效能一般；0.85＜AUC＜1，检测效能很好；AUC＝1，检测效能完美。ACU的CI值95％。
由曲线下面积(area under The curve,AUC)表明，miR-3074-5p在PBMC中可以区分RA患者与HC(图10)，miR-3074-5p可以作为RA PBMC中的诊断生物标记物。随后，我们分析了RA患者PBMC中miR-3074-5p、TNFSF14的表达与年龄、病程、TNF-α、IL-6、RA-IgG、RA-IgM、RA-IgA、SJC、TJC、ESR、CRP和DAS28等指标的关系。结果显示，miR-3074-5p水平与CCP呈正相关(p<0.001)(图11中a)。TNFSF14水平与RA中RF、RA-IgG、DAS28水平呈负相关，(p<0.001)，(图11中b为TNFSF14水平与RA中RF水平关系)。因此，miR-3074-5p不仅可以作为RA PBMC中的诊断生物标记物进行应用，还至少可以部分反映疾病的严重程度。
以上实验统计学方法：采用双尾Student's t检验和Mann-Whitney's t检验分别评估组间表达的正态分布参数和偏态分布参数。相关分析采用单样本Kolmogorov-Smirnov检验，组间正态分布时采用Pearson相关检验；非正态分布时采用Spearman相关检验分析。采用SPSS22.0统计软件分析，P<0.05为差异有统计学意义。
最后说明的是，以上优选实施例仅用以说明本发明的技术方案而非限制，尽管通过上述优选实施例已经对本发明进行了详细的描述，但本领域技术人员应当理解，可以在形式上和细节上对其作出各种各样的改变，而不偏离本发明权利要求书所限定的范围。
序列表
<110> 安徽省立医院（中国科学技术大学附属第一医院）
中国人民解放军陆军军医大学
<120> miR-3074-5p作为类风湿关节炎标志物的应用及其试剂盒
<160> 6
<170> SIPOSequenceListing 1.0
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<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
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Claims (10)
1.miR-3074-5p作为microRNA分子标志物在制备类风湿关节炎的诊断试剂或试剂盒中的应用。
2.检测miR-3074-5p表达水平的引物在制备类风湿关节炎的诊断试剂或试剂盒中的应用。
3.根据权利要求2所述的应用，其特征在于，检测miR-3074-5p表达水平的引物的序列为SEQ ID NO:6。
4.一种检测类风湿关节炎的诊断试剂盒，其特征在于，所述诊断试剂盒包括检测miR-3074-5p表达水平的引物。
5.根据权利要求5所述的诊断试剂盒，其特征在于，检测miR-3074-5p表达水平的引物的序列为SEQ ID NO:6。
6.根据权利要求4或5所述的诊断试剂盒，其特征在于，试剂盒还包括反转录试剂部分和荧光定量检测试剂部分。
7.根据权利要求6所述的诊断试剂盒，其特征在于，反转录试剂部分由2×miRNA RTReaction Buffer、miRNA RT Enzyme Mix、RNase-Free ddH2O组成。
8.根据权利要求6所述的诊断试剂盒，其特征在于，荧光定量检测试剂部分由2×miRcute Plus miRNA PreMix、10μM Reverse Primer、ddH2O组成。
9.miR-3074-5p抑制剂在制备抑制TNFSF14表达量降低的试剂中的应用。
10.miR-3074-5p抑制剂在制备类风湿关节炎的治疗试剂或试剂盒中的应用。
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