CN116144759A - LncRNA-ENST00000581911在制备甲状腺相关眼病诊断试剂中的应用 - Google Patents
LncRNA-ENST00000581911在制备甲状腺相关眼病诊断试剂中的应用 Download PDFInfo
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Abstract
本发明公开了LncRNA‑ENST00000581911在制备甲状腺相关眼病(TAO)诊断试剂中的应用。通过高通量测序和定量PCR验证发现lncRNA‑ENST00000581911与TAO发生呈现显著地相关性，可以用于临床TAO病人的筛查。本发明还提供了一种TAO诊断的lncRNA‑ENST00000581911试剂盒。该试剂盒主要包括RNA抽提体系、反转录反应体系和PCR反应体系。通过实时荧光定量PCR技术，检测眼外肌中lncRNA‑ENST00000581911的相对表达量，用于TAO的早期诊断。该方法具有创伤性小、灵敏度高和操作简单的特点。
Description
技术领域
本发明属于医学领域和分子诊断领域，具体涉及lncRNA-ENST00000581911在制备甲状腺相关眼病(TAO)诊断试剂中的应用，以及一种辅助TAO早期诊断的lncRNA诊断试剂盒，及其在辅助TAO早期诊断中的应用和检测方法。
背景技术
甲状腺相关眼病(TAO)是一种累及眼外肌、眼眶脂肪和眼结缔组织的自身免疫性疾病，发病机制十分复杂，在遗传因素和环境因素的共同作用下，同时涉及多种细胞、细胞因子以及免疫因子，导致免疫功能紊乱。TAO现已成为成人眼球突出的最常见原因，并有逐年升高的趋势。严重者可导致暴露性角膜炎、容貌受损、压迫性视神经病变、视力下降，甚至造成失明。
目前，临床针对TAO仅限于对症治疗，如激素冲击疗法、眼眶放疗、手术治疗(复视矫正术、眼睑退缩矫正术和眼眶减压术)等，缺乏行而有效的治疗手段，且激素冲击等免疫抑制疗法缺乏针对性且受副作用限制，治疗后易出现反复复发，因此，亟待对TAO的发生进行早期诊断，寻找出新型无创的、高灵敏度和特异性的生物标记物。
长链非编码RNA(lncRNA)是一类转录本长度超过200nt不具备蛋白编码功能的RNA分子，可以从多个层面包括表观遗传调控、转录调控以及转录后调控等调控基因的表达。LncRNA可以调节许多细胞生物学过程，包括细胞增殖、细胞分化和细胞凋亡等，而且lncRNA表达异常与众多人类疾病发生发展密切相关，包括肿瘤、心血管疾病、神经系统疾病和免疫系统疾病等。近年来研究发现lncRNA RUNX1-IT1的表达水平改变参与了弥漫性甲状腺毒症(Graves病)的发生和发展(Huang FJ,Liu YL,Wang J et al.LncRNA RUNX1-IT1affectsthe differentiation of Th1 cells by regulating NrCAM transcription in Graves'disease.Cell Cycle.2022May；21(9):921-933.)，中国人群lncRNA-GAS5基因多态性和lncRNA-GAS5表达水平与发生系统性红斑狼疮风险密切相关(Liu CH,Lu YL,Huang HT etal.Association of LncRNA-GAS5 gene polymorphisms and PBMC LncRNA-GAS5 levelwith risk of systemic lupus erythematosus in Chinese population.J Cell MolMed.2021Apr；25(7):3548-3559.),LncRNA LOC100912373的表达改变与影响了类风湿关节炎的发生(Jiang H,Fan C,Lu YQ et al.Astragaloside regulates lncRNALOC100912373and the miR-17-5p/PDK1 axis to inhibit the proliferation offibroblast-like synoviocytes in rats with rheumatoid arthritis.Int J MolMed.2021Jul；48(1):130.)。
目前，尚无lncRNA-ENST00000581911与相关疾病的研究。
发明内容
本发明目的在于提供了LncRNA-ENST00000581911的新的应用，可作为诊断试剂用于TAO的早期诊断。
本发明的目的可通过以下技术方案实现:
核苷酸序列如SEQ ID NO:1所示的LncRNA-ENST00000581911在制备诊断TAO试剂盒中的应用，特别是TAO的早期诊断。
所述诊断试剂包括诊断试剂盒、诊断试纸、诊断芯片。检测样本为眼外肌组织。
本发明的另一个目的在于提供一种TAO诊断的LncRNA-ENST00000581911检测试剂盒，试剂盒包括RNA抽提体系、RNA反转录反应体系和PCR反应体系，其中PCR反应体系含有特异性扩增SEQ ID NO:1基因序列的引物序列。上述检测试剂盒中RNA抽提体系包括总RNA提取试剂、RNA反转录反应体系包括反转录酶、反转录体系缓冲液和RNA酶抑制剂；PCR反应体系包括扩增系统和引物系统，所述扩增系统由SYBR Premix Ex TaqTM试剂组成；所述引物系统包括RNA反转录随机引物和LncRNA-ENST00000581911特异性的qRT-PCR的引物，其中，RNA反转录随机引物为GAPDH和/或Beta-actin的定量PCR引物序列，GAPDH定量PCR引物序列，上游引物序列如SEQ ID NO:2所示，下游引物序列如SEQ ID NO:3所示；LncRNA-ENST00000581911特异性的qRT-PCR的上游引物如SEQ ID NO:4所示，下游引物SEQ ID NO:5所示；Beta-actin定量PCR引物序列，上游引物序列如SEQ ID NO:6所示，下游引物序列如SEQ ID NO:7所示。
上述的检测试剂盒，包括：
(a)抽提体系
1)Trizol reagent，1管，2000μL/管；
2)氯仿，1管，500μL/管；
3)无水乙醇，1管，8000μL/管；
4)DEPC ddH2O，1管，1000μL/管；
5)ddH2O，1管，2000μL/管；
6)异丙醇，8000μL/管；
(b)反转录体系
1)总RNA反转录引物(包括Oligo dT和Random 6mers)，1管，浓度:50μM，50μL/管；
2)反转录酶(200U/μL)50μL；
3)dNTP Mixture(10mM each)50μL；
4)反转录buffer 50μL；
(c)PCR体系
1)SYBR Premix Ex Taq酶；
2)buffer 100μL；
3)LncRNA-ENST00000581911特异性qRT-PCR的上游引物，1管，10μM，100μL/管；
LncRNA-ENST00000581911特异性qRT-PCR的下游引物，1管，10μM，100μL/管；
GAPDH定量PCR上游引物，1管，10μM，100μL/管；
GAPDH定量PCR下游引物，1管，10μM，100μL/管；
和/或，Beta-actin定量PCR上游引物，1管，10μM，100μL/管；
Beta-actin定量PCR下游引物，1管，10μM，100μL/管；
4)dNTP Mixture(10mM each)50μL。
本发明确定了LncRNA-ENST00000581911表达量的改变，与TAO的发生存在显著地相关性。LncRNA-ENST00000581911可以作为TAO早期诊断的生物标记物，定期评估TAO的进展。
本发明采用美国Aglient公司microarray lncRNA表达谱芯片筛选出TAO患者的眼外肌组织和已故的正常捐献者眼外肌组织中lncRNAs的差异表达谱，并联合应用定量PCR方法验证芯片分析结果。最后，选用LncRNA-ENST00000581911作为生物标记物，用于辅助TAO的早期诊断。
本发明中确定LncRNA-ENST00000581911作为TAO早期诊断的生物标记物包括如下步骤:
第一步：样本准备：眼科斜视手术后的TAO患者的眼外肌组织(实验组，n＝5)和正常捐献者眼外肌组织标本(对照组，n＝5)，RNA采用TRIzol(Invitrogen)试剂提取，并保存于-80℃备用；
第二步：差异表达筛选：采用美国Aglient公司的表达谱芯片，分析TAO发生相关的lncRNA；分析具体步骤为：采用标记酶将荧光基团标记lncRNA，得到用于与芯片杂交的荧光探针，在标注条件下使用MAUI杂交仪和芯片杂交；使用GenePix 4000B芯片扫描仪扫描芯片的荧光强度，并将实验结果转换成数值型数据进行保存；采用Genespring GX软件分析找出TAO发生相关的lncRNA分子；筛选出一系列的差异表达lncRNA，然后通过定量PCR验证TAO中差异表达最为显著的10个lncRNA；
第三步：收集TAO新的临床样本：TAO患者(实验组，n＝5)和间歇性外斜视患者(对照组，n＝5)眼外肌组织中的表达差异；
第四步：确定检测靶点：采用定量PCR进一步检测芯片中差异表达的10个lncRNA在TAO眼外肌组织的表达丰度及表达差异情况，结合TRANSFAC和catRAPID数据库，最终确定LncRNA-ENST00000581911作为靶点用于后续的测定；
第五步：进一步扩大临床样本量，通过活检的方法收集TAO患者眼外肌组织，验证靶点LncRNA-ENST00000581911在TAO病人和正常人眼外肌中的表达差异；
第六步：基于体外细胞实验，采用体外培养TAO患者和正常对照组眼外肌来源成纤维细胞，从基本原理方面揭示LncRNA-ENST00000581911通过调节成纤维细胞的增殖和其对眼外肌细胞外基质合成和降解的影响，进而明确LncRNA-ENST00000581911在TAO病理过程中的调控作用。
本发明证明通过定量PCR技术，检测患者眼外肌组织中LncRNA-ENST00000581911的表达来实现TAO的早期诊断是完全可行的，本发明的试剂盒还适用于：根据患者年龄、病史以及局部体征初步怀疑为TAO的患者。
本发明的另一目的在于提供干预lncRNA-ENST00000581911表达的抑制剂在制备治疗甲状腺相关眼病药物中的应用。
所述抑制剂为lncRNA-ENST00000581911的反义寡核苷酸、siRNA、miRNA、shRNA、核酸适配体、转录激活RNA中的一种或几种。
本发明一个具体的示例，所述抑制剂为干预lncRNA-ENST00000581911表达的siRNA，核苷酸序列如下所示：
LncRNA-ENST00000581911siRNA sense:5’GUAGCUGACAAUAUCAUCA 3’(SEQ IDNO:8)。
LncRNA-ENST00000581911siRNA antisense:5’UGAUGAUAUUGUCAGCUAC 3’(SEQ IDNO:9)。
LncRNA-ENST00000581911siRNA可以靶向抑制LncRNA-ENST00000581911表达，因此可以作为药物用于治疗TAO。
本发明的试剂盒首次利用定量PCR方法，通过检测眼外肌中LncRNA-ENST00000581911含量，用于TAO的辅助诊断。本发明提供了新的TAO的检测标记物和治疗靶点，为该疾病的诊断和治疗提供了全新的分子信息和生物学基础，具有较大的实际临床价值。
附图说明
图1为lncRNA芯片分析筛选TAO发生相关的lncRNA并进行验证(图A为箱线图分析芯片的质量；图B为火山图筛选TAO相关的lncRNA；图C为聚类图从整体角度，分析TAO组和正常对照组眼外肌样本lncRNA表达差异；图D为定量PCR验证芯片表达差异最为显著的10个lncRNA(5个上调lncRNA，5个下调lncRNA)。
图2为定量PCR分析表达差异最显著的10个lncRNA在TAO患者(实验组)和间歇性外斜视患者(对照组)眼外肌中的表达差异。
图3为定量PCR分析LncRNA-ENST00000581911在TAO患者和健康人眼外肌中的表达差异。
图4为原位杂交显示的DAPI、ER及LncRNA-ENST00000581911在成纤维细胞中的定位。
图5为分析LncRNA-ENST00000581911表达干预对TAO相关病理过程的影响(图A为定量PCR分析LncRNA-ENST00000581911siRNA转染对LncRNA-ENST00000581911表达的影响；图B为采用MTT法检测LncRNA-ENST00000581911表达干预对成纤维细胞增殖的影响；图C为采用Western Blot检测LncRNA-ENST00000581911表达干预后，各组成纤维细胞促甲状腺激素受体(TSHR)、结缔组织生长因子(CTGF)、MMP-9、TIMP-1的表达；图D为LncRNA-ENST00000581911表达干预后，ELISA检测各组培养基中透明质酸和胶原蛋白含量。
图6为LncRNA-ENST00000581911表达干预后，定量PCR检测各组成纤维细胞a-SMA、MMP-9、CTGF和TIMP-1的表达。
具体实施方式
以下通过实施例说明本发明的具体步骤，但不受实施例限制。
在本发明中所使用的术语，除非另有说明，一般具有本领域普通技术人员通常理解的含义。
下面结合具体实施例并参照数据进一步详细描述本发明。应理解，这些实施例只是为了举例说明本发明，而非以任何方式限制本发明的范围。
在以下实施例中，未详细描述的各种过程和方法是本领域中公知的常规方法。
实施例1LncRNA-ENST00000581911与TAO的相关性验证，lncRNA芯片分析筛选TAO有关的lncRNA并进行验证。
第一步：样本准备：TAO患者斜视手术过程中收集病变的眼外肌组织(实验组，n＝5)和正常捐献者的眼外肌组织(对照组，n＝5)，总RNA采用TRIzol(Invitrogen)试剂提取，并保存于-80℃冰箱备用。
第二步：差异表达lncRNA筛选：采用美国Aglient公司的lncRNA表达谱芯片，分析TAO发生相关的lncRNA；分析具体步骤为：采用标记酶将荧光基团标记lncRNA，得到用于与芯片杂交的荧光探针，在标注条件下使用MAUI杂交仪和芯片杂交；使用GenePix4000B芯片扫描仪扫描芯片的荧光强度，并将实验结果转换成数值型数据进行保存；各个样本的分布如图1A所示；各个样本的lncRNA整体表达分布如图1B所示，表明TAO和非TAO病理样本之间存在显著的lncRNA表达差异；采用Genespring GX软件分析找出TAO发生相关的lncRNA分子，筛选出一系列的差异表达lncRNA(如图1C所示)。
第三步：采用定量PCR验证芯片中表达差异最显著的10个lncRNA(如图1D所示)；然后收取新的临床样本：TAO患者(实验组)和间歇性外斜视患者(对照组)眼外肌组织，通过定量PCR进一步分析这10个lncRNA在TAO患者眼外肌中表达情况，结果表明芯片验证数据中LncRNA-T277939在TAO患者眼外肌中中表达上调最为显著，但在新的TAO眼外肌样本验证数据中，LncRNA-T277939表达封度很低，且差异表达不是最大，而LncRNA-ENST00000581911在TAO患者和正常对照组眼外肌组织中表达差异最大，且表达封度最高，故而推测LncRNA-ENST00000581911与TAO疾病具有较大的相关性(如图2所示)。
第四步：收集临床样本：TAO患者(实验组)和间歇性外斜视患者(对照组)眼外肌组织，采用Trizol试剂提取总RNA，通过反转录PCR的方法得到总RNA的cDNA；图3所示为定量PCR检测靶点LncRNA-ENST00000581911在TAO患者和对照组眼外肌的表达差异，结果表明LncRNA-ENST00000581911在TAO患者眼外肌中表达上调。
第五步：基于体外细胞实验，从基本原理方面揭示LncRNA-ENST00000581911通过调控成纤维细胞的功能，进而确证其参与TAO病理过程的调控，如图4、图5、图6所示。
实施例2制备本发明的试剂盒LncRNA-ENST0000058191的序列如SEQ ID:NO:1所示，其特异性定量PCR上下游引物以及内参GAPDH和/或Beta-tubulin的定量PCR上下游引物，通过Primer 5设计，由Invitrogen公司负责引物合成，纯度为PAGE级，合成后的引物采用DEPC H2O溶解，总浓度为10μM。
制备包括以下组成成分的试剂盒：
(a)抽提体系
1)Trizol reagent，1管，2000μL/管；
2)氯仿，1管，500μL/管；
3)无水乙醇，1管，8000μL/管；
4)DEPC ddH2O，1管，1000μL/管；
5)ddH2O，1管，2000μL/管；
6)异丙醇，8000μL/管；
(b)反转录体系
1)总RNA反转录引物(包括Oligo dT和Random 6mers)，1管，浓度:50μM，50μL/管；
2)反转录酶(200U/μL)50μL；
3)dNTP Mixture(10mM each)50μL；
4)反转录buffer 50μL；
(c)PCR体系
1)SYBR Premix Ex Taq酶；
2)buffer 100μL；
3)LncRNA-ENST00000581911特异性qRT-PCR的上游引物(SEQ ID NO:4)，1管，
10μM，100μL/管；
LncRNA-ENST00000581911特异性qRT-PCR的下游引物(SEQ ID NO:5)，1管，10μM，100μL/管；
GAPDH定量PCR上游引物(SEQ ID NO:2)，1管，10μM，100μL/管；
GAPDH定量PCR下游引物(SEQ ID NO:3)，1管，10μM，100μL/管；
和/或，Beta-actin定量PCR上游引物(SEQ ID NO:6)，1管，10μM，100μL/管；
Beta-actin定量PCR下游引物(SEQ ID NO:7)，1管，10μM，100μL/管；
4)dNTP Mixture(10mM each)50μL。
实施例3LncRNA-ENST00000581911在眼外肌中的表达检测。
眼外肌样本：在术中收集TAO患者(实验组)和间歇性外斜视患者(对照组)眼外肌组织，移入已经消毒1.5mL eppendorf试管中低温保存。
眼外肌样本的RNA提取
在分离的眼外肌样本中，加入TRIzol(Invitrogen)超声震碎仪震碎组织，超声震动强度为30％，4sec×5次，每次震完需立即置冰上降温，然后再震，以免RNA降解，震碎组织后，冰上裂解30min使样品充分裂解(注：如不进行下一步操作，RNA样品可放入-80℃长期保存)。每1ml TRIzol加入200μl氯仿，剧烈振荡混匀后室温放置3-5min使其自然分相。4℃12,000rpm离心15min。样品会分成三层：黄色的有机相，中间层和无色的水相，RNA主要在水相中，把水相转移到新管中。在上清中加入等体积冰冷的异丙醇，室温放置15min。4℃12,000rpm离心10min，弃上清，RNA沉淀于管底。RNA沉淀中加入1ml 75％乙醇(用RNase-free水配制)，温和振荡离心管，悬浮沉淀。每1ml TRIzol加入1ml 75％乙醇。4℃8,000rpm离心5min，弃上清。室温放置晾干后，在沉淀内加入20μl RNase-free水，65℃水浴锅助溶10min以充分溶解RNA，-80℃保存。
RNA质量检测
在260nm和280nm吸光度处，采用紫外分光光度计测定RNA的浓度；RNA溶液的A260/A280的比值即为RNA纯度，比值范围1.8到2.1。同时，结合琼脂糖凝胶电泳检测RNA质量，紫外透射光下观察并拍照。
RNA逆转录获得cDNA样本
采用TaKaRa公司试剂盒PrimeScriptTM RT reagent Kit对所提RNA进行逆转录获得cDNA样本，按照试剂盒提供的体系(20μl)分别加入：
将以上体系置于无Rnase的0.2μl EP管中，按下面程序反转为cDNA：37℃15min，85℃5sec，得到的cDNA放于-20℃冰箱中保存。
实时荧光定量PCR
实时荧光定量PCR反应体系(20μl)，配置如下：SYBR Premix Ex Taq酶10μl，LncRNA-ENST00000581911特异性的定量PCR上游引物、LncRNA-ENST00000581911特异性定量PCR下游引物、GAPDH定量PCR上游引物、GAPDH定量PCR下游引物各0.8μl，和/或Beta-actin定量PCR上游引物、Beta-actin定量PCR下游引物各0.8μl，待测样品cDNA 2μl，dNTP 1μl,ddH2O补足至20μl。
PCR反应条件：94℃5min,(94℃30sec，60℃30sec，72℃45sec，40个循环)，72℃5min。
数据分析及结果判断
数据分析对同一样本分别进行目标RNA和内参RNA检测，以内参的表达量为基准，对目标RNA进行归一化处理，随后对目标RNA的相对表达量采用本领域通用的delta deltaCt(ΔΔct)法分析。最后，通过比较待测样本和目标样本LncRNA-ENST00000581911的表达差异，判断待测病人的疾病易感性。
Claims (9)
1.LncRNA-ENST00000581911在制备甲状腺相关眼病诊断试剂中的应用，所述lncRNA-ENST00000581911的cDNA核苷酸序列如SEQ ID NO:1所示。
2.如权利要求1所述的应用，其特征在于所述诊断试剂包括诊断试剂盒、诊断试纸、诊断芯片。
3.一种甲状腺相关眼病诊断的lncRNA检测试剂盒，其特征在于试剂盒包括RNA抽提体系、RNA反转录体系和PCR反应体系；其中PCR反应体系含有特异性扩增根据权利要求1所述SEQ ID NO:1核苷酸序列的引物序列。
4.根据权利要求3所述的检测试剂盒，其特征在于所述引物序列为lncRNA-ENST00000581911特异性的荧光实时定量PCR的上下游引物。
5.根据权利要求3所述的检测试剂盒，其特征在于所述RNA抽提体系包括RNA抽提试剂；RNA逆转录反应体系包括反转录酶、反转录体系缓冲液和RNA酶抑制剂；PCR反应体系包括扩增系统和引物系统，所述扩增系统为SYBR Premix Ex TaqTM试剂；所述引物系统包括反转录随机引物和lncRNA-ENST00000581911特异性的qRT-PCR的引物，其中，RNA反转录随机引物为18S rRNA的定量PCR引物序列，上游引物序列如SEQ ID NO:2所示，下游引物序列如SEQID NO:3所示；lncRNA-ENST00000581911特异性的qRT-PCR的上游引物如SEQ ID NO:4所示，下游引物如SEQ ID NO:5所示。
6.根据权利要求5所述的检测试剂盒，其特征在于，所述试剂盒包括:
(a)抽提体系
1)Trizol reagent，1管，2000μL/管；
2)氯仿，1管，500μL/管；
3)无水乙醇，1管，8000μL/管；
4)DEPC ddH2O，1管，1000μL/管；
5)ddH2O，1管，2000μL/管；
6)异丙醇，8000μL/管；
(b)反转录体系
1)总RNA反转录引物(包括Oligo dT和Random 6mers)，1管，浓度:50μM，50μL/管；
2)反转录酶(200U/μL)50μL；
3)dNTP Mixture(10mM each)50μL；
4)反转录buffer 50μL；
(c)PCR体系
1)SYBR Premix Ex Taq酶；
2)buffer 100μL；
3)LncRNA-ENST00000581911特异性qRT-PCR的上游引物，1管，10μM，100μL/管；
LncRNA-ENST00000581911特异性qRT-PCR的下游引物，1管，10μM，100μL/管；
GAPDH定量PCR上游引物，1管，10μM，100μL/管；
GAPDH定量PCR下游引物，1管，10μM，100μL/管；
和/或，Beta-actin定量PCR上游引物，1管，10μM，100μL/管；
Beta-actin定量PCR下游引物，1管，10μM，100μL/管；
4)dNTP Mixture(10mM each)50μL。
7.干预权利要求1所述的lncRNA-ENST00000581911表达的抑制剂在制备治疗甲状腺相关眼病药物中的应用。
8.根据权利要求7所述的应用，其特征在于，所述抑制剂为lncRNA-ENST00000581911的反义寡核苷酸、siRNA、miRNA、shRNA、核酸适配体、转录激活RNA中的一种或几种。
9.根据权利要求8所述的应用，其特征在于所述抑制剂为干预lncRNA-ENST00000581911表达的siRNA，核苷酸序列如SEQ ID NO:8和9所示。
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