CN114277126A - Application of hsa _ miR-486-5p and ETS-1 gene in rheumatoid arthritis diagnosis and treatment reagent - Google Patents

Abstract

Translated fromChinese

本发明涉及hsa_miR‑486‑5p联合ETS‑1基因在类风湿关节炎诊断治疗试剂中的应用。根据提取的总RNA序列，设计并合成特异性PCR引物，利用Rt‑qPCR技术，发现hsa_miR‑486‑5p在类风湿关节炎(RA)患者外周血单个核细胞中的表达显著升高，而ETS‑1显著降低。本发明同时设计并合成特异性过表达hsa‑miR‑486‑5p mimics和小干扰该基因si‑ETS‑1序列，能够通过调控炎性细胞因子，显著抑制滑膜成纤维细胞(FLS)的增殖及TNF‑α所诱导的FLS的炎症反应。本发明所建立的联合检测机制，可以应用于类风湿关节炎辅助诊断试剂、分子靶向治疗试剂或者预后评估试剂等方面。

The invention relates to the application of hsa_miR-486-5p combined with ETS-1 gene in rheumatoid arthritis diagnosis and treatment reagents. According to the extracted total RNA sequence, specific PCR primers were designed and synthesized. Using Rt‑qPCR technology, it was found that the expression of hsa_miR‑486‑5p in peripheral blood mononuclear cells of patients with rheumatoid arthritis (RA) was significantly increased, while ETS ‑1 was significantly lower. The present invention simultaneously designs and synthesizes specific overexpression hsa-miR-486-5p mimics and small interference with the gene si-ETS-1 sequence, which can significantly inhibit the proliferation of synovial fibroblasts (FLS) by regulating inflammatory cytokines and TNF-α-induced inflammatory response in FLS. The joint detection mechanism established by the present invention can be applied to auxiliary diagnostic reagents for rheumatoid arthritis, molecular targeted therapy reagents or prognosis assessment reagents and the like.

Description

Translated fromChinese

hsa_miR-486-5p联合ETS-1基因在类风湿关节炎诊断治疗试 剂中的应用Application of hsa_miR-486-5p combined with ETS-1 gene in the diagnosis and treatment of rheumatoid arthritis

技术领域technical field

本发明属于风湿免疫病分子生物学领域，具体涉及一种hsa_miR-486-5p联合ETS-1基因在类风湿关节炎诊断治疗试剂中的应用。具体而言，本发明涉及微小hsa_miR-486-5p基因及其靶标基因mRNA ETS-1在制备RA辅助诊断试剂、分子靶向治疗试剂或者预后评估试剂中的应用。The invention belongs to the field of molecular biology of rheumatoid immune diseases, in particular to the application of hsa_miR-486-5p combined with ETS-1 gene in rheumatoid arthritis diagnosis and treatment reagents. Specifically, the present invention relates to the application of the tiny hsa_miR-486-5p gene and its target gene mRNA ETS-1 in the preparation of RA auxiliary diagnostic reagents, molecular targeted therapy reagents or prognosis assessment reagents.

背景技术Background technique

类风湿关节炎(RA)是一种累及周围关节为主的多系统性、炎症性的自身免疫性疾病。RA疾病发生发展过程中伴随着机体免疫细胞增殖和凋亡动态的失衡，氧化应激激活及包括众多信号通路在内的多种信号传导通路失衡，导致免疫炎症的发生，最终促进RA疾病的发生发展。RA发病机制目前仍不明确。风湿性疾病主要表现为自身抗原免疫反应异常引起的局部或全身炎症症状，而且大部分疾病病因和发病机制尚不完全清楚，早期诊断困难，治疗效果不佳。因此，寻找RA早期诊断、靶向治疗和预后评估的生物标记物，对RA的治疗及预后具有重大的意义。Rheumatoid arthritis (RA) is a multisystem, inflammatory autoimmune disease mainly involving peripheral joints. The occurrence and development of RA disease is accompanied by the imbalance of immune cell proliferation and apoptosis dynamics, the activation of oxidative stress and the imbalance of various signal transduction pathways including many signaling pathways, which lead to the occurrence of immune inflammation and ultimately promote the occurrence of RA disease. develop. The pathogenesis of RA is still unclear. Rheumatic diseases are mainly manifested as local or systemic inflammatory symptoms caused by abnormal immune responses to autoantigens, and the etiology and pathogenesis of most diseases are not completely clear, early diagnosis is difficult, and the treatment effect is poor. Therefore, finding biomarkers for early diagnosis, targeted therapy and prognosis assessment of RA is of great significance for the treatment and prognosis of RA.

随着近年来高通量基因测序技术的发展，对于RA基因表达谱的研究越来越受到学者关注。微小核糖核酸MicroRNA(miRNA)是一种小的内源性非编码RNA分子，大约由21-25个核苷酸组成。miRNA起转录后负调控的分子，参与生命过程中一系列重要进程，包括发育进程，造血过程，器官形成，凋亡，细胞增殖和肿瘤的发生。这些小的miRNA通常靶向一个或者多个mRNA，通过翻译水平的抑制或断裂靶标mRNAs而调节基因的表达。作为一组新的潜在的疾病诊断和治疗的靶分子，已成为了生物医学研究的前沿热点。生物信息学预测显示，在哺乳动物，miRNA虽然占不到2％蛋白编码基因，却可以调控1/3的mRNAs。miRNA可以调控人类60％的蛋白编码基因，表明其功能强大。作为新的特异性基因调节小分子，miRNA不仅有助于阐明类风湿关节炎的发病机制，在类风湿关节炎的临床诊断和治疗上也无疑将具有重要的应用前景。首先，由于miRNA在不同类风湿关节炎中的特异表达模式，将有可能以miRNA作为类风湿关节炎的新的生物标记或诊断指标；其次，由于单个miRNA具有多个mRNA靶点，而多个miRNA又可协同调控某一共同的通路，可以针对疾病的某一特定通路寻找更为有效的干预方法。由于miRNA数量众多，具有广泛的生物学作用，目前的研究仅是冰山一角。miRNA在类风湿关节炎中还有很多新的作用尚待发现，明确miRNA的靶基因及其对靶基因的作用机制可以帮助人们更加深人地了解类风湿关节炎的发病机制，使其作为新的诊断标志和治疗靶点应用于临床。With the development of high-throughput gene sequencing technology in recent years, the study of RA gene expression profile has attracted more and more attention of scholars. MicroRNA (miRNA) is a small endogenous non-coding RNA molecule, approximately 21-25 nucleotides in length. miRNAs function as negative post-transcriptional molecules and participate in a series of important processes in life, including developmental processes, hematopoiesis, organ formation, apoptosis, cell proliferation and tumorigenesis. These small miRNAs typically target one or more mRNAs and regulate gene expression by repressing or cleaving target mRNAs at the translational level. As a new set of potential target molecules for disease diagnosis and treatment, it has become a frontier hot spot in biomedical research. Bioinformatics predictions show that in mammals, miRNAs can regulate 1/3 of mRNAs, although they account for less than 2% of protein-coding genes. miRNAs can regulate 60% of human protein-coding genes, indicating their powerful functions. As new specific gene regulatory small molecules, miRNAs will not only help to clarify the pathogenesis of rheumatoid arthritis, but also will undoubtedly have important application prospects in the clinical diagnosis and treatment of rheumatoid arthritis. First, due to the specific expression patterns of miRNAs in different rheumatoid arthritis, it will be possible to use miRNAs as new biomarkers or diagnostic indicators for rheumatoid arthritis; secondly, because a single miRNA has multiple mRNA targets, and multiple miRNAs can coordinately regulate a common pathway, and can find more effective intervention methods for a specific pathway of the disease. Due to the large number of miRNAs and their broad biological roles, current research is only the tip of the iceberg. There are still many new roles of miRNAs in rheumatoid arthritis yet to be discovered. Clarifying the target genes of miRNAs and their mechanism of action on target genes can help people to understand the pathogenesis of rheumatoid arthritis more deeply and make it a new The diagnostic markers and therapeutic targets are used in clinical practice.

发明内容SUMMARY OF THE INVENTION

针对目前RA缺乏诊断标志物，缺乏治疗药物靶向性、针对性，本发明提供了一种hsa_miR-486-5p联合ETS-1基因在类风湿关节炎诊断治疗试剂中的应用。Aiming at the current lack of diagnostic markers for RA and the lack of targeting and pertinence of therapeutic drugs, the present invention provides an application of hsa_miR-486-5p combined with ETS-1 gene in a diagnostic and therapeutic reagent for rheumatoid arthritis.

首先，本发明提供了一对用于检测hsa_miR-486-5p基因的引物对，该引物对核苷酸序列如SEQ ID NO.1和SEQ ID NO.2所示，以及，一对用于检测ETS-1基因的引物对，该引物对核苷酸序列如SEQ ID NO.3和SEQ ID NO.4所示。First, the present invention provides a pair of primers for detecting hsa_miR-486-5p gene, the nucleotide sequences of the primer pairs are shown in SEQ ID NO.1 and SEQ ID NO.2, and a pair for detecting The primer pair of ETS-1 gene, the nucleotide sequence of the primer pair is shown in SEQ ID NO.3 and SEQ ID NO.4.

其次，本发明提供了一种RA检测试剂，该检测试剂包括：5uL 2×SYBR Greenmixture、1uL Forward primer(10uM)、1uL Reverse primer(10uM)、浓度为2.5ng/μl的cDNA 1uL、浓度均为10umol/L的hsa_miR-486-5p基因检测引物对或者ETS-1基因检测引物对各1μl、RNase Free water2uL。Secondly, the present invention provides an RA detection reagent, the detection reagent includes:5uL 2×SYBR Greenmixture, 1uL Forward primer (10uM), 1uL Reverse primer (10uM), 1uL of cDNA with a concentration of 2.5ng/μl, and 1uL of each 10umol/L hsa_miR-486-5p gene detection primer pair or ETS-1 gene detection primer pair each 1μl, RNase Free water 2uL.

第三，本发明提供一种hsa_miR-486-5p、ETS-1基因的检测方法，包括RA外周血单个核细胞(peripheral blood mononuclear cells,PBMCs)提取、TRIzol法提取总RNA、RNA浓度和纯度的测定、cDNA合成及PCR扩增。Third, the present invention provides a detection method for hsa_miR-486-5p and ETS-1 genes, including extraction of RA peripheral blood mononuclear cells (PBMCs), extraction of total RNA, RNA concentration and purity by TRIzol method Assay, cDNA synthesis and PCR amplification.

第四，本发明还提供了一种hsa_miR-486-5p基因过表达在制备RA靶向治疗试剂中的应用，具体而言，即根据hsa_miR-486-5p基因制备特异性过表达质粒序列SEQ ID NO.5和SEQ ID NO.6，用于在滑膜成纤维细胞中过表达hsa_miR-486-5p基因。Fourth, the present invention also provides an application of hsa_miR-486-5p gene overexpression in the preparation of RA targeted therapy reagents, specifically, the specific overexpression plasmid sequence SEQ ID prepared according to the hsa_miR-486-5p gene NO.5 and SEQ ID NO.6 for overexpression of hsa_miR-486-5p gene in synovial fibroblasts.

第五，本发明提供了一种RA诊断试剂，所述试剂包括特异性小干扰序列ETS-1，其质粒核苷酸序列如SEQ ID NO.7和SEQ ID NO.8所示，可用于感染滑膜成纤维细胞，使ETS-1基因在细胞中被干扰。Fifth, the present invention provides a diagnostic reagent for RA, the reagent includes a specific small interference sequence ETS-1, and its plasmid nucleotide sequence is shown in SEQ ID NO.7 and SEQ ID NO.8, which can be used for infection In synovial fibroblasts, the ETS-1 gene is disturbed in cells.

与现有技术相比，本发明的有益效果表现在：Compared with the prior art, the beneficial effects of the present invention are shown in:

本发明发现hsa_miR-486-5p在类风湿关节炎(RA)患者外周血单个核细胞中的表达显著升高，而ETS-1显著降低。hsa_miR-486-5p与RA疾病活动性指标ESR、CRP、RF、CCP、DAS-28呈正相关，ETS-1与RA疾病活动性指标呈负相关。RA患者PBMC中hsa_miR-486-5p、ETS-1诊断RA的ROC曲线表明对RA有一定的诊断价值。本发明同时设计并合成特异性过表达hsa-miR-486-5p mimics和小干扰该基因si-ETS-1序列，能够通过调控炎性细胞因子，显著抑制滑膜成纤维细胞(FLS)的增殖及TNF-α所诱导的FLS的炎症反应，可成为RA的分子靶向治疗工具。因此，本发明所建立的联合检测机制，可以应用于类风湿关节炎辅助诊断试剂、分子靶向治疗试剂或者预后评估试剂等方面。The present invention finds that the expression of hsa_miR-486-5p in the peripheral blood mononuclear cells of rheumatoid arthritis (RA) patients is significantly increased, while the ETS-1 is significantly decreased. hsa_miR-486-5p was positively correlated with RA disease activity indexes ESR, CRP, RF, CCP, DAS-28, and ETS-1 was negatively correlated with RA disease activity indexes. The ROC curves of hsa_miR-486-5p and ETS-1 in PBMCs of RA patients in diagnosing RA indicated that they had certain diagnostic value for RA. The present invention simultaneously designs and synthesizes specific overexpression hsa-miR-486-5p mimics and small interference with the gene si-ETS-1 sequence, which can significantly inhibit the proliferation of synovial fibroblasts (FLS) by regulating inflammatory cytokines And the inflammatory response of FLS induced by TNF-α can become a molecular targeted therapy tool for RA. Therefore, the combined detection mechanism established by the present invention can be applied to the aspects of auxiliary diagnostic reagents for rheumatoid arthritis, molecular targeted therapy reagents or prognostic assessment reagents and the like.

附图说明Description of drawings

图1表示hsa_miR-486-5p在RA患者PBMCs中表达。Figure 1 shows the expression of hsa_miR-486-5p in PBMCs of RA patients.

图2表示ETS-1在RA患者血清中表达。Figure 2 shows the expression of ETS-1 in the serum of RA patients.

图3表示RA患者hsa_miR-486-5p和ETS-1的关联性分析。Figure 3 shows the correlation analysis of hsa_miR-486-5p and ETS-1 in RA patients.

图4表示RA患者PBMC中hsa_miR-486-5p诊断RA的ROC曲线。Figure 4 shows the ROC curve of hsa_miR-486-5p in PBMC of RA patients for the diagnosis of RA.

图5表示RA患者血清中ETS-1诊断RA的ROC曲线。Figure 5 shows the ROC curve of ETS-1 in the serum of RA patients for diagnosing RA.

图6表示hsa_miR-486-5p与RA疾病状态、临床指标相关性分析。Figure 6 shows the correlation analysis of hsa_miR-486-5p with RA disease state and clinical indicators.

图7表示ETS-1与RA疾病状态、临床指标相关性分析。Figure 7 shows the correlation analysis of ETS-1 with RA disease state and clinical indicators.

图8表示hsa_miR-486-5p过表达状态下miR-486-5p和ETS-1的表达。Figure 8 shows the expression of miR-486-5p and ETS-1 in the overexpression state of hsa_miR-486-5p.

图9表示ETS-1干扰状态下miR-486-5p和ETS-1的表达。Figure 9 shows the expression of miR-486-5p and ETS-1 in the state of ETS-1 interference.

图10表示hsa_miR-486-5p过表达、ETS-1干扰状态下滑膜细胞增殖的变化。Figure 10 shows the changes in the proliferation of synovial cells in the state of hsa_miR-486-5p overexpression and ETS-1 interference.

图11表示hsa_miR-486-5p过表达状态下炎性细胞因子的表达。Figure 11 shows the expression of inflammatory cytokines in the overexpression state of hsa_miR-486-5p.

图12表示ETS-1干扰状态下炎性细胞因子的表达。Figure 12 shows the expression of inflammatory cytokines in the state of ETS-1 interference.

具体实施方式Detailed ways

下面结合具体实施例，进一步阐述本发明，仅用于解释本发明的相关原理及步骤，而不能理解为对本发明的限制。本领域的普通技术人员可以理解，在不脱离本发明的原理和宗旨的情况下可以对这些实施例进行多种变化、修改、替换和变型。The present invention will be further described below with reference to specific embodiments, which are only used to explain the relevant principles and steps of the present invention, and should not be construed as limiting the present invention. It will be understood by those of ordinary skill in the art that various changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the present invention.

下列实施例中未注明具体条件的实验方法，通常是按照常规条件或按照厂商所建议的条件进行实施检测。In the following examples, the experimental methods without specific conditions are usually carried out under conventional conditions or according to the conditions suggested by the manufacturer.

实施例1Example 1

RA患者PBMCs中hsa_miR-486-5p、ETS-1的表达情况Expression of hsa_miR-486-5p and ETS-1 in PBMCs of RA patients

申请人在项目通过伦理委员会审核之后，系统研究人RA新的诊断和治疗靶点，通过抽取RA患者的外周血，提取PBMCs，RT-qPCR法检测hsa_miR-486-5p、ETS-1基因的表达。After the project passed the review of the ethics committee, the applicant systematically studied new diagnostic and therapeutic targets for human RA, extracted PBMCs from the peripheral blood of RA patients, and detected the expression of hsa_miR-486-5p and ETS-1 genes by RT-qPCR .

本实施例共收集RA患者血液样本30例，对照组健康人(HC)血液样本30例，共60例样本(来源于30个RA患者具体信息见附表1，样本特点见附表2)。In this example, a total of 30 blood samples from RA patients and 30 healthy people (HC) blood samples from the control group were collected, for a total of 60 samples (seeAppendix 1 for specific information from 30 RA patients, andAppendix 2 for sample characteristics).

表1RA患者样本信息Table 1 RA patient sample information

表2RA患者样本特点Table 2 Characteristics of samples from RA patients

hsa_miR-486-5p、ETS-1基因的检测方法，包括RA外周血单个核细胞(peripheralblood mononuclear cells,PBMCs)提取、TRIzol法提取总RNA、RNA浓度和纯度的测定、cDNA合成及PCR扩增，其具体步骤如下：The detection methods of hsa_miR-486-5p and ETS-1 genes include extraction of RA peripheral blood mononuclear cells (PBMCs), total RNA extraction by TRIzol method, determination of RNA concentration and purity, cDNA synthesis and PCR amplification, The specific steps are as follows:

1、RA患者PBMCs提取1. Extraction of PBMCs from RA patients

室温条件下，在50mL离心管中加入6mL Ficoll-Paque PLUS；10mL离心管中加入4mL新鲜的抗凝血和等量的PBS，混匀，将稀释过的血液样本小心铺到Ficoll-Paque分离液上面，然后将离心管置于水平式离心机内，19℃400g离心35min；离心后分4层，从上往下依次为：血浆层、单核细胞层、Ficoll-Paque PLUS、红细胞层，吸掉血浆层，将单核细胞层转移到另一离心管中，加入至少3倍体积的PBS，混匀，19℃400g离心15min，弃上清；加入6mL PBS重悬细胞，19℃100g离心10min，弃上清；再加入1mL PBS重悬细胞，然后转移到1.5mL EP管中，19℃100g离心10min，弃上清，置-80℃冰箱保存。At room temperature, add 6 mL of Ficoll-Paque PLUS to a 50 mL centrifuge tube; add 4 mL of fresh anticoagulant and an equal amount of PBS to a 10 mL centrifuge tube, mix well, and carefully spread the diluted blood sample into the Ficoll-Paque separation solution Then place the centrifuge tube in a horizontal centrifuge and centrifuge at 400g at 19°C for 35min; after centrifugation, it is divided into 4 layers, from top to bottom: plasma layer, mononuclear cell layer, Ficoll-Paque PLUS, red blood cell layer, suction Remove the plasma layer, transfer the mononuclear cell layer to another centrifuge tube, add at least 3 volumes of PBS, mix well, centrifuge at 400g at 19°C for 15min, discard the supernatant; add 6mL of PBS to resuspend the cells, and centrifuge at 100g at 19°C for 10min , discard the supernatant; add 1 mL of PBS to resuspend the cells, then transfer to a 1.5 mL EP tube, centrifuge at 100 g for 10 min at 19 °C, discard the supernatant, and store in a -80 °C refrigerator.

2、RNA提取2. RNA extraction

1)收集细胞沉淀，加入1ml TRIzol裂解。1) Collect the cell pellet and add 1 ml of TRIzol to lyse.

2)裂解完全后，加入0.2mL氯仿，剧烈振荡15秒，室温放置5分钟。2) After the lysis is complete, add 0.2 mL of chloroform, shake vigorously for 15 seconds, and place at room temperature for 5 minutes.

3)4℃12000rpm离心10分钟，取上清(约500ul)加入到另一EP管中。3) Centrifuge at 12000rpm at 4°C for 10 minutes, take the supernatant (about 500ul) and add it to another EP tube.

4)加入0.5mL预冷的异丙醇，温和混匀，-20℃孵育30分钟。4) Add 0.5 mL of pre-cooled isopropanol, mix gently, and incubate at -20°C for 30 minutes.

5)4℃12000rpm离心15分钟，弃去上清。5) Centrifuge at 12000rpm at 4°C for 15 minutes, and discard the supernatant.

6)加入1mL预冷的75℅乙醇(无水乙醇用DEPC水稀释)。4℃12000rpm离心5分钟，弃上清。6) Add 1 mL of pre-cooled 75℅ ethanol (anhydrous ethanol is diluted with DEPC water). Centrifuge at 12,000 rpm at 4°C for 5 minutes, and discard the supernatant.

7)重复步骤6)。7) Repeat step 6).

8)室温干燥RNA沉淀，加入20-50μL DEPC水，-80℃保存备用。8) Dry the RNA precipitate at room temperature, add 20-50 μL of DEPC water, and store at -80°C for later use.

3、RNA浓度和纯度测定3. RNA concentration and purity determination

用移液器取RNA样品1μL至缓冲液中，测定其在260nm和280nm处吸光值。根据OD260/OD280比值，估测RNA质量。OD260/OD280比值在1.8-2.0之间，可以满足实验要求。当OD260/OD280<1.8时，溶液中蛋白的污染比较明显；当OD260/OD280>2.2时，说明RNA已经降解。Pipette 1 μL of RNA sample into buffer and measure its absorbance at 260 nm and 280 nm. RNA quality was estimated based on the OD260/OD280 ratio. The OD260/OD280 ratio is between 1.8-2.0, which can meet the experimental requirements. When OD260/OD280<1.8, the protein contamination in the solution is more obvious; when OD260/OD280>2.2, it means that RNA has been degraded.

4、RT反应4. RT reaction

①、hsa_miR-486-5p RT反应①, hsa_miR-486-5p RT reaction

1)去除基因组DNA反应：在0.2mL EP管中加入1) Removal of genomic DNA reaction: add to 0.2mL EP tube

轻轻混匀、点动离心。Gently mix and centrifuge.

2)PCR仪上42℃加热2min，立即冰浴1min。2) Heat on the PCR machine at 42°C for 2 min, and immediately ice bath for 1 min.

3)在上述EP管中加入3) Add in the above EP tube

4)42℃，15min；85℃，5s。4) 42℃, 15min; 85℃, 5s.

5)取出上述反应液，即为cDNA，-80℃保存备用。5) Take out the above reaction solution, which is cDNA, and store it at -80°C for future use.

②、ETS-1RT反应②, ETS-1RT reaction

1)去除基因组DNA反应：在0.2mL EP管中加入1) Removal of genomic DNA reaction: add to 0.2mL EP tube

轻轻混匀、点动离心。Gently mix and centrifuge.

2)PCR仪上42℃加热2min，立即冰浴1min。2) Heat on the PCR machine at 42°C for 2 min, and immediately ice bath for 1 min.

3)在上述EP管中加入3) Add in the above EP tube

4)37℃，15min；85℃，5s。4) 37℃, 15min; 85℃, 5s.

5)取出上述反应液，即为cDNA，-80℃保存备用。5) Take out the above reaction solution, which is cDNA, and store it at -80°C for future use.

5、荧光定量PCR反应5. Fluorescence quantitative PCR reaction

1)分别取出cDNA作为荧光定量的模板，反应体系如下：1) Take out cDNA as a template for fluorescence quantification, and the reaction system is as follows:

2)反应条件如下：2) reaction conditions are as follows:

3)各检测指标引物3) Each detection index primer

检测hsa_miR-486-5p基因的引物对核苷酸序列如SEQ ID NO.1和SEQ ID NO.2所示，检测ETS-1基因的引物对核苷酸序列如SEQ ID NO.3和SEQ ID NO.4所示。The nucleotide sequences of the primer pairs for detecting the hsa_miR-486-5p gene are shown in SEQ ID NO.1 and SEQ ID NO.2, and the nucleotide sequences for the primer pairs for detecting the ETS-1 gene are shown in SEQ ID NO.3 and SEQ ID NO.3 NO.4 is shown.

实施例2Example 2

hsa_miR-486-5p过表达质粒序列和ETS-1小干扰质粒序列及其应用hsa_miR-486-5p overexpression plasmid sequence and ETS-1 small interference plasmid sequence and its application

本实施例针对hsa_miR-486-5p、ETS-1的全长序列设计(全序列分别为SEQ IDNO.9和SEQ ID NO.10)，并合成其特异性过表达序列hsa-miR-486-5p mimics(核苷酸序列如SEQ ID NO.5和SEQ ID NO.6所示)和si-ETS-1(核苷酸序列如SEQ ID NO.7和SEQ IDNO.8所示)，感染FLS，使hsa_miR-486-5p基因在细胞中过表达，ETS-1基因在细胞小干扰，并进一步将该过表达和小干扰基因载体应用于调控FLS的炎症反应。This example is designed for the full-length sequences of hsa_miR-486-5p and ETS-1 (the full sequences are SEQ ID NO. 9 and SEQ ID NO. 10, respectively), and the specific overexpression sequence hsa-miR-486-5p is synthesized. mimics (nucleotide sequences shown in SEQ ID NO. 5 and SEQ ID NO. 6) and si-ETS-1 (nucleotide sequences shown in SEQ ID NO. 7 and SEQ ID NO. 8) were infected with FLS, The hsa_miR-486-5p gene was overexpressed in the cells, and the ETS-1 gene was interfered in the cells, and the overexpression and small interference gene vector was further applied to regulate the inflammatory response of FLS.

具体病毒包装及细胞感染方法如下：The specific virus packaging and cell infection methods are as follows:

(1)293T细胞在6cm dish中培养至80-90％融合时，倾去培养液，用3mLPBS洗涤细胞两次；(2)加1mL Trypsin-EDTA solution，混匀后，小心吸去胰酶溶液，37℃放置3分钟；(3)再加入2mL含10％FBS的DMEM培养液，吹打使细胞形成单细胞悬液；(4)血球计数板计数，将细胞稀释至3x10⁵细胞/mL；(5)按5x10³细胞/孔的浓度接种96孔板，混匀后于37℃5％CO₂培养24h；(6)转染试剂Lipofectamin2000用于转染过表达质粒，剂量如下，每个剂量设三复孔；(1) When 293T cells were cultured in a 6cm dish to 80-90% confluence, pour off the culture medium and wash the cells twice with 3 mL PBS; (2) Add 1 mL Trypsin-EDTA solution, mix well, and carefully remove the trypsin solution , placed at 37°C for 3 minutes; (3) Add 2 mL of DMEM medium containing 10% FBS, pipetting to make the cells form a single cell suspension; (4) Counting on a hemocytometer, dilute the cells to 3×10⁵ cells/mL; ( 5) Inoculate a 96-well plate at a concentration of 5×^{10 3} cells/well, and incubate at 37°C with 5% CO₂ for 24 hours after mixing; (6) The transfection reagent Lipofectamin 2000 is used to transfect the overexpression plasmid, and the dose is as follows, each dose is set triple hole;

Lipo(uL)Lipo(uL)0.20.20.30.30.40.4质粒(ug)Plasmid (ug)0.20.20.30.30.50.5

(7)在1.5mL EP管中加入75μL(25μL/well*3well)无血清DMEM，再加入根据上述表格算出的不同剂量的质粒，混匀，取另一1.5mL EP管，加入75μL(25μL/well*3well)无血清DMEM，加入根据上述表格算出的相应剂量的Lipofectamin2000，混匀，室温放置5分钟后将两组管混合，室温放置20分钟，吸去96孔板中的培养液，每孔加入50μL无血清的DMEM培养液；(8)将转染混合物逐滴加入96孔板中，混匀后，在培养箱中温育5小时；(9)转染质粒组，吸弃转染液，更换为完全培养基，在培养箱孵育24h后观测；(10)48h后，RT-qPCR检测293T的感染效率；(11)48h后，收集293T细胞上清液至15ml离心管中，并于4℃，1500rpm离心5min，弃掉细胞沉淀，0.45μm滤器过滤后转移至1.5ml离心管，-80℃冰箱冻存；(12)在六孔板中接种待感染重组慢病毒的FLS，密度为5～8×10⁵/孔培养；(13)吸尽培养液，加入1mL重组慢病毒液，及8μLpolybrene(终浓度1μg/μL)，放入培养箱培养；(14)4h后半量换液；(15)24h后，全量换液；(16)48h后，RT-qPCR检测293T的感染效率。(7) Add 75 μL (25 μL/well*3 well) of serum-free DMEM to a 1.5 mL EP tube, then add the different doses of plasmids calculated according to the above table, mix well, take another 1.5 mL EP tube, add 75 μL (25 μL/ well*3well) serum-free DMEM, add the corresponding dose of Lipofectamin2000 calculated according to the above table, mix well, place at room temperature for 5 minutes, mix the two groups of tubes, place at room temperature for 20 minutes, suck out the culture medium in the 96-well plate, each well Add 50 μL of serum-free DMEM medium; (8) Add the transfection mixture dropwise to the 96-well plate, mix well, and incubate in the incubator for 5 hours; (9) For the transfection plasmid group, aspirate and discard the transfection solution, Replaced with complete medium and observed after 24h incubation in the incubator; (10) After 48h, RT-qPCR was used to detect the infection efficiency of 293T; (11) After 48h, collect the 293T cell supernatant into a 15ml centrifuge tube, and put it in 4 ℃, centrifuge at 1500 rpm for 5 min, discard the cell pellet, filter with a 0.45 μm filter, transfer to a 1.5 ml centrifuge tube, and freeze at -80 ℃; (12) Inoculate the FLS to be infected with the recombinant lentivirus in a six-well plate at a density of 5 ～8×10⁵ /well culture; (13) Aspirate the culture medium, add 1 mL of recombinant lentivirus solution, and 8 μL polybrene (final concentration 1 μg/μL), put it into the incubator for culture; (14) After 4 hours, half of the medium is changed; ( 15) After 24 hours, the medium was changed in full; (16) After 48 hours, the infection efficiency of 293T was detected by RT-qPCR.

细胞转染方法步骤为：The steps of cell transfection method are:

(1)消化正常FLS、RA-FLS细胞，终止消化后离心弃去培养液，用PBS洗2遍，用培养基重悬，按照5*10⁵个细胞/孔接种到6孔板中，放入37℃，5％培养箱中过夜培养。(1) Digest normal FLS and RA-FLS cells, stop the digestion, centrifuge and discard the culture medium, wash twice with PBS, resuspend with medium, inoculate 5*10⁵ cells/well into 6-well plates, and put them in a 6-well plate. Incubate overnight in a 37°C, 5% incubator.

(2)第二天观察细胞融合70-80％后对各组细胞进行转染处理。(2) The cells of each group were transfected after observing 70-80% cell confluence on the second day.

(3)将siRNA冻干粉加125μlDEPC水，稀释后浓度为20uM。(3) Add 125 μl DEPC water to the siRNA lyophilized powder, and the concentration after dilution is 20 uM.

(4)将mimics冻干粉加入125μlDEPC水，稀释后浓度为20uM。(4) Add mimics lyophilized powder to 125 μl DEPC water, and the concentration after dilution is 20 uM.

(5)在250μl无血清培养基中加入浓度为20uM的siRNA 5μl/20uM的mimics 5μl，轻轻混匀；(5) Add 20uM siRNA 5μl/20uM mimics 5μl to 250μl serum-free medium, and mix gently;

(6)使用前轻轻摇匀Lipofectamine 2000，然后取5ul Lipofectamine 2000在250μl无血清培养基中稀释，室温孵育5分钟。(6) Shake Lipofectamine 2000 gently before use, then dilute 5ul Lipofectamine 2000 in 250 μl serum-free medium, and incubate at room temperature for 5 minutes.

(7)将前两步所稀释的siRNA/mimics和Lipofectamine 2000混合(使总体积为500μl)，轻轻混匀，室温放置20分钟。(7) Mix the siRNA/mimics diluted in the previous two steps with Lipofectamine 2000 (to make the total volume 500 μl), mix gently, and leave at room temperature for 20 minutes.

(8)在每孔细胞中加入500μl转染液，轻轻摇匀。37℃培养24小时后检测基因表达，转染4-6小时后更换培养基进行培养。(8) Add 500 μl of transfection solution to each well of cells and shake gently. Gene expression was detected after culturing at 37°C for 24 hours, and the culture medium was replaced 4-6 hours after transfection.

(9)转染24小时后，胰酶消化细胞，PBS清洗2遍，收集细胞沉淀，置-80℃冰箱保存用于后续实验。(9) 24 hours after transfection, cells were digested with trypsin, washed twice with PBS, collected cell pellets, and stored in a -80°C refrigerator for subsequent experiments.

具体观察结果为：The specific observations are:

(1)hsa_miR-486-5p在RA患者PBMCs中表达(1) Expression of hsa_miR-486-5p in PBMCs of RA patients

结果如图1所示，结果发现，与健康对照组相比，RA患者中hsa_miR-486-5p的mRNA表达显著上调，差异具有统计学意义。The results are shown in Figure 1. It was found that the mRNA expression of hsa_miR-486-5p was significantly up-regulated in RA patients compared with healthy controls, and the difference was statistically significant.

(2)ETS-1在RA患者血清中表达(2) Expression of ETS-1 in serum of RA patients

结果如图2所示，结果发现，与健康对照组相比，RA患者中ETS-1表达显著下调，差异具有统计学意义。The results are shown in Figure 2. It was found that the expression of ETS-1 was significantly down-regulated in RA patients compared with healthy controls, and the difference was statistically significant.

(3)RA患者hsa_miR-486-5p和ETS-1的关联性分析(3) Association analysis of hsa_miR-486-5p and ETS-1 in RA patients

结果如图3所示，Spearman相关分析发现，hsa_miR-486-5p与ETS-1呈负相关。The results are shown in Figure 3, and Spearman correlation analysis found that hsa_miR-486-5p was negatively correlated with ETS-1.

(4)RA患者PBMC中hsa_miR-486-5p诊断RA的ROC曲线(4) ROC curve of hsa_miR-486-5p in PBMC of RA patients in the diagnosis of RA

结果如图4所示，通过ROC曲线分析和ROC曲线下面积(AUC)的计算，结果显示出PBMC中hsa_miR-486-5p诊断RA的AUC值为0.839。该结果表明hsa_miR-486-5p对RA有一定的诊断价值。The results are shown in Figure 4, through ROC curve analysis and calculation of the area under the ROC curve (AUC), the results showed that the AUC value of hsa_miR-486-5p in PBMC for diagnosing RA was 0.839. This result indicates that hsa_miR-486-5p has certain diagnostic value for RA.

(5)RA患者血清中ETS-1诊断RA的ROC曲线(5) ROC curve of ETS-1 in serum of RA patients in the diagnosis of RA

结果如图5所示，通过ROC曲线分析和ROC曲线下面积(AUC)的计算，结果显示ETS-1诊断RA的AUC值为0.710。该结果表明ETS-1对RA有一定的诊断价值。The results are shown in Figure 5, through ROC curve analysis and calculation of the area under the ROC curve (AUC), the results show that the AUC value of ETS-1 for diagnosing RA is 0.710. The results indicate that ETS-1 has a certain diagnostic value for RA.

(6)hsa_miR-486-5p与RA疾病状态、临床指标相关性分析(6) Correlation analysis of hsa_miR-486-5p with RA disease state and clinical indicators

结果如图6所示，hsa_miR-486-5p mRNA表达水平与RA疾病活动性指标CRP、ESR、RF、CCP、DAS28呈明显正相关。The results are shown in Figure 6, the hsa_miR-486-5p mRNA expression level was significantly positively correlated with RA disease activity indicators CRP, ESR, RF, CCP, DAS28.

(7)ETS-1与RA疾病状态、临床指标相关性分析(7) Correlation analysis of ETS-1 with RA disease state and clinical indicators

结果如图7所示，ETS-1表达水平与与RA疾病活动性指标CRP、ESR、RF、CCP、DAS28呈明显负相关。The results are shown in Figure 7, the expression level of ETS-1 was significantly negatively correlated with RA disease activity indicators CRP, ESR, RF, CCP, and DAS28.

(8)hsa_miR-486-5p过表达状态下miR-486-5p和ETS-1的表达(8) Expression of miR-486-5p and ETS-1 under the overexpression state of hsa_miR-486-5p

结果如图8所示，与正常组滑膜细胞比较，RA组和miR-486-5p模拟剂组滑膜细胞miR-486-5p表达升高，ETS-1表达降低，且hsa_miR-486-5p过表达状态下，RA滑膜细胞miR-486-5p表达降低，ETS-1表达升高。The results are shown in Figure 8. Compared with the synovial cells of the normal group, the synovial cells of the RA group and the miR-486-5p mimic group had increased expression of miR-486-5p, decreased ETS-1 expression, and hsa_miR-486-5p In the overexpression state, the expression of miR-486-5p in RA synovial cells was decreased, and the expression of ETS-1 was increased.

(9)ETS-1干扰状态下miR-486-5p和ETS-1的表达(9) Expression of miR-486-5p and ETS-1 under ETS-1 interference

结果如图9所示，与正常组滑膜细胞比较，RA组和si-ETS-1模拟剂组滑膜细胞miR-486-5p表达升高，ETS-1表达降低，ETS-1干扰状态下，RA滑膜细胞miR-486-5p表达升高，ETS-1表达降低。The results are shown in Figure 9. Compared with the synovial cells of the normal group, the synovial cells of the RA group and the si-ETS-1 mimic group had increased expression of miR-486-5p, and decreased ETS-1 expression. , the expression of miR-486-5p in RA synovial cells was increased, and the expression of ETS-1 was decreased.

(10)hsa_miR-486-5p过表达、ETS-1干扰状态下滑膜细胞增殖的变化(10) Changes in the proliferation of synovial cells in the overexpression of hsa_miR-486-5p and ETS-1 interference

结果如图10所示，与正常组滑膜细胞比较，RA组和miR-486-5p模拟剂组、si-ETS-1模拟剂组滑膜细胞增殖增多。且hsa_miR-486-5p过表达状态下，RA滑膜细胞增殖减少，ETS-1干扰状态下滑膜细胞增殖增多。The results are shown in FIG. 10 , compared with the synovial cells of the normal group, the synovial cell proliferation in the RA group, the miR-486-5p mimic group, and the si-ETS-1 mimic group increased. Moreover, under the overexpression state of hsa_miR-486-5p, the proliferation of RA synovial cells decreased, and the proliferation of synovial cells increased in the ETS-1 interference state.

(11)hsa_miR-486-5p过表达状态下炎性细胞因子的表达(11) Expression of inflammatory cytokines under the overexpression of hsa_miR-486-5p

结果如图11所示，与正常组滑膜细胞比较，RA组和miR-486-5p模拟剂组滑膜细胞致炎性细胞因子IL-13、IL-23、TNF-α升高，IL-4降低。且hsa_miR-486-5p过表达状态下，RA滑膜细胞致炎性细胞因子IL-13、IL-23、TNF-α降低，IL-4升高。The results are shown in Figure 11. Compared with the synovial cells of the normal group, the synovial cells of the RA group and the miR-486-5p mimic group increased the inflammatory cytokines IL-13, IL-23, and TNF-α, and IL- 4 lower. Moreover, under the overexpression state of hsa_miR-486-5p, the inflammatory cytokines IL-13, IL-23 and TNF-α induced by RA synovial cells were decreased, and IL-4 was increased.

(12)ETS-1干扰状态下炎性细胞因子的表达(12) Expression of inflammatory cytokines under ETS-1 interference

结果如图12所示，与正常组滑膜细胞比较，RA组和si-ETS-1模拟剂组滑膜细胞致炎性细胞因子IL-13、IL-23、TNF-α升高，IL-4降低。且ETS-1干扰状态下，RA滑膜细胞致炎性细胞因子IL-13、IL-23、TNF-α升高，IL-4降低。The results are shown in Figure 12. Compared with the synovial cells of the normal group, the synovial cells of the RA group and the si-ETS-1 mimic group increased the inflammatory cytokines IL-13, IL-23 and TNF-α, and IL- 4 lower. In addition, in the state of ETS-1 interference, the inflammatory cytokines IL-13, IL-23 and TNF-α increased and IL-4 decreased in RA synovial cells.

以上内容仅仅是对本发明的构思所作的举例和说明，所属本技术领域的技术人员对所描述的具体实施例做各种各样的修改或补充或采用类似的方式替代，只要不偏离发明的构思或者超越本权利要求书所定义的范围，均应属于本发明的保护范围。The above contents are only examples and descriptions of the concept of the present invention. Those skilled in the art can make various modifications or supplements to the described specific embodiments or replace them in similar ways, as long as they do not deviate from the concept of the invention. Or beyond the scope defined by the claims, shall belong to the protection scope of the present invention.

序列表sequence listing

<110> 安徽中医药大学第一附属医院（安徽省中医院）<110> The First Affiliated Hospital of Anhui University of Traditional Chinese Medicine (Anhui Provincial Hospital of Traditional Chinese Medicine)

<120> hsa_miR-486-5p联合ETS-1基因在类风湿关节炎诊断治疗试剂中的应用Application of <120> hsa_miR-486-5p combined with ETS-1 gene in the diagnosis and treatment of rheumatoid arthritis

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tatggaatgt gcagatgtcc cactattaac tccaagcagc aaagaaatga tgtctcaagc 360tatggaatgt gcagatgtcc cactattaac tccaagcagc aaagaaatga tgtctcaagc 360

attaaaagct actttcagtg gtttcactaa agaacagcaa cgactgggga tcccaaaaga 420attaaaagct actttcagtg gtttcactaa agaacagcaa cgactgggga tcccaaaaga 420

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gggtaaagac tgctttctcg agctggcccc agactttgtt ggggacatct tatgggaaca 600gggtaaagac tgctttctcg agctggcccc agactttgtt ggggacatct tatgggaaca 600

tctagagatc ctgcagaaag aggatgtgaa accatatcaa gttaatggag tcaacccagc 660tctagagatc ctgcagaaag aggatgtgaa accatatcaa gttaatggag tcaacccagc 660

ctatccagaa tcccgctata cctcggatta cttcattagc tatggtattg agcatgccca 720ctatccagaa tcccgctata cctcggatta cttcattagc tatggtattg agcatgccca 720

gtgtgttcca ccatcggagt tctcagagcc cagcttcatc acagagtcct atcagacgct 780gtgtgttcca ccatcggagt tctcagagcc cagcttcatc acagagtcct atcagacgct 780

ccatcccatc agctcggaag agctcctctc cctcaagtat gagaatgact acccctcggt 840ccatcccatc agctcggaag agctcctctc cctcaagtat gagaatgact acccctcggt 840

cattctccga gaccctctcc agacagacac cttgcagaat gactactttg ctatcaaaca 900cattctccga gaccctctcc agacagacac cttgcagaat gactactttg ctatcaaaca 900

agaagtcgtc accccagaca acatgtgcat ggggaggacc agtcgtggta aactcggggg 960agaagtcgtc accccagaca acatgtgcat ggggaggacc agtcgtggta aactcggggg 960

ccaggactct tttgaaagca tagagagcta cgatagttgt gatcgcctca cccagtcctg 1020ccaggactct tttgaaagca tagagagcta cgatagttgt gatcgcctca cccagtcctg 1020

gagcagccag tcatctttca acagcctgca gcgtgttccc tcctatgaca gcttcgactc 1080gagcagccag tcatctttca acagcctgca gcgtgttccc tcctatgaca gcttcgactc 1080

agaggactat ccggctgccc tgcccaacca caagcccaag ggcaccttca aggactatgt 1140agaggactat ccggctgccc tgcccaacca caagcccaag ggcaccttca aggactatgt 1140

gcgggaccgt gctgacctca ataaggacaa gcctgtcatt cctgctgctg ccctagctgg 1200gcgggaccgt gctgacctca ataaggacaa gcctgtcatt cctgctgctg ccctagctgg 1200

ctacacaggc agtggaccaa tccagctatg gcagtttctt ctggaattac tcactgataa 1260ctacacaggc agtggaccaa tccagctatg gcagtttctt ctggaattac tcactgataa 1260

atcctgtcag tcttttatca gctggacagg agatggctgg gaattcaaac tttctgaccc 1320atcctgtcag tcttttatca gctggacagg agatggctgg gaattcaaac tttctgaccc 1320

agatgaggtg gccaggagat ggggaaagag gaaaaacaaa cctaagatga attatgagaa 1380agatgaggtg gccaggagat ggggaaagag gaaaaacaaa cctaagatga attatgagaa 1380

actgagccgt ggcctacgct actattacga caaaaacatc atccacaaga cagcggggaa 1440actgagccgt ggcctacgct actattacga caaaaacatc atccacaaga cagcggggaa 1440

acgctacgtg taccgctttg tgtgtgacct gcagagcctg ctggggtaca cccctgagga 1500acgctacgtg taccgctttg tgtgtgacct gcagagcctg ctggggtaca cccctgagga 1500

gctgcacgcc atgctggacg tcaagccaga tgccgacgag tgatggcact gaaggggctg 1560gctgcacgcc atgctggacg tcaagccaga tgccgacgag tgatggcact gaaggggctg 1560

gggaaaccct gctgagacct tccaaggaca gccgtgttgg ttggactctg aattttgaat 1620gggaaaccct gctgagacct tccaaggaca gccgtgttgg ttggactctg aattttgaat 1620

tgttattcta ttttttattt tccagaactc attttttacc ttcaggggtg ggagctaagt 1680tgttattcta ttttttattt tccagaactc atttttttacc ttcaggggtg ggagctaagt 1680

cagttgcagc tgtaatcaat tgtgcgcagt tgggaaagga aagccaggac ttgtggggtg 1740cagttgcagc tgtaatcaat tgtgcgcagt tgggaaagga aagccaggac ttgtggggtg 1740

ggtgggacca gaaattcttg agcaaatttt caggagaggg agaagggcct tctcagaagc 1800ggtgggacca gaaattcttg agcaaatttt caggagaggg agaagggcct tctcagaagc 1800

ttgaaggctc tggcttaaca gagaaagaga ctaatgtgtc caatcatttt taaaaatcat 1860ttgaaggctc tggcttaaca gagaaagaga ctaatgtgtc caatcatttt taaaaatcat 1860

ccatgaaaaa gtgtcttgag ttgtggaccc attagcaagt gacattgtca catcagaact 1920ccatgaaaaa gtgtcttgag ttgtggaccc attagcaagt gacattgtca catcagaact 1920

catgaaactg atgtaaggca attaatttgc ttctgttttt aggtctggga gggcaaaaaa 1980catgaaactg atgtaaggca attaatttgc ttctgttttt aggtctggga gggcaaaaaa 1980

gaggtgggtg ggatgaaaca tgttttgggg ggggatgcac tgaaaatctg agaactattt 2040gaggtgggtg ggatgaaaca tgttttgggg ggggatgcac tgaaaatctg agaactattt 2040

acctatcact ctagttttga agcaaagatg gacttcagtg gggaggggcc aaaaccgttg 2100acctatcact ctagttttga agcaaagatg gacttcagtg gggaggggcc aaaaccgttg 2100

ttgtgttaaa atttatttta ttaaattttg tgccagtatt ttttttctta aaaatcgtct 2160ttgtgttaaa atttatttta ttaaattttg tgccagtatt ttttttctta aaaatcgtct 2160

taagctctaa ggtggtctca gtattgcaat atcatgtaag tttgttttta tttgccggct 2220taagctctaa ggtggtctca gtattgcaat atcatgtaag tttgttttta tttgccggct 2220

gaggattctg tcacaatgaa agaaaactgt ttatatagac cccattggaa aagcaaaacg 2280gaggattctg tcacaatgaa agaaaactgt ttatatagac cccattggaa aagcaaaacg 2280

ctctcactga gatcagggat cccaaattca tgggacttat ataagaagga caattaatgc 2340ctctcactga gatcagggat cccaaattca tgggacttat ataagaagga caattaatgc 2340

tgatttgggt acaggggaat tatgtgtgtg aatgtcatct acaattaaaa aaaattagca 2400tgatttgggt acaggggaat tatgtgtgtg aatgtcatct acaattaaaa aaaattagca 2400

catcccttta cttacttgtt atcagtggat tctcggggtt tggacttaat gttgagctaa 2460catcccttta cttacttgtt atcagtggat tctcggggtt tggacttaat gttgagctaa 2460

gaagcattaa gtctttgaac tgaatgtatt ttgcatccct ggttttggac gacagtaaac 2520gaagcattaa gtctttgaac tgaatgtatt ttgcatccct ggttttggac gacagtaaac 2520

gtaggagcac tgttgaagtc ctggaaggga gatcgaagga ggaagattga cttggttctt 2580gtaggagcac tgttgaagtc ctggaaggga gatcgaagga ggaagattga cttggttctt 2580

tcttagtcct atatctgtag catagatgac ttggaataaa agctgtatgc atgggcatta 2640tcttagtcct atatctgtag catagatgac ttggaataaa agctgtatgc atgggcatta 2640

cccctcaggt cctaagaaat aagtcctgaa tgcatgtcgt tccaaactaa cactctgtaa 2700cccctcaggt cctaagaaat aagtcctgaa tgcatgtcgt tccaaactaa cactctgtaa 2700

tttttctttt atgtcttatt ttccaagagt cctccatttt ttgcaccccc tcaccgccaa 2760ttttttctttt atgtcttatt ttccaagagt cctccatttt ttgcaccccc tcaccgccaa 2760

ctctgttatt cagtagagag aagtgtacgg ctttctgatt ggtgagtgaa aaagtaactt 2820ctctgttatt cagtagagag aagtgtacgg ctttctgatt ggtgagtgaa aaagtaactt 2820

gagacacgac ctaagttgaa gagtttagac ttgctgagtt ttagaagtga tggaaattaa 2880gagacacgac ctaagttgaa gagtttagac ttgctgagtt ttagaagtga tggaaattaa 2880

gagagcattt caataaaatg tgacttggct gtctttggaa gagaagtgca aggctttcct 2940gagagcattt caataaaatg tgacttggct gtctttggaa gagaagtgca aggctttcct 2940

ttgaagaatt taaattagtc cggtaggatg tcaggtgaga ctgtgtatgc aaaatgaatg 3000ttgaagaatt taaattagtc cggtaggatg tcaggtgaga ctgtgtatgc aaaatgaatg 3000

gcacaggtga tgccagggcc tcttgcttgg gtctgatgtc ttggcacagg gtaagtgaag 3060gcacaggtga tgccagggcc tcttgcttgg gtctgatgtc ttggcacagg gtaagtgaag 3060

gttaattcca gaagagagga atgacttgaa ggcaaaggaa actaaggaag gaggttcagt 3120gttaattcca gaagagagga atgacttgaa ggcaaaggaa actaaggaag gaggttcagt 3120

gaggaaaata aggttgtcca tgagatttga atagattttt agttccccca aggtttaaat 3180gaggaaaata aggttgtcca tgagatttga atagattttt agttccccca aggtttaaat 3180

acaaacatag tcaagcaagg tagtcatctt tctgctggtt gtgaggggga atctgaaaat 3240acaaacatag tcaagcaagg tagtcatctt tctgctggtt gtgaggggga atctgaaaat 3240

ggagttttag aggaaaagtc aacatctaac tagtgaggaa aagtgcctaa tacaattaga 3300ggagttttag aggaaaagtc aacatctaac tagtgaggaa aagtgcctaa tacaattaga 3300

atctccctca ctctatagtt gcccagttga aaggataagg aggaggggtg gcttttatgg 3360atctccctca ctctatagtt gcccagttga aaggataagg aggaggggtg gcttttatgg 3360

acttccatga gagaaggaaa gaaatatttc aggtaagctt ctcagggctg gccctttttg 3420acttccatga gagaaggaaa gaaatatttc aggtaagctt ctcagggctg gccctttttg 3420

ggatttggat gagaaattgg aagtactaac tactttctag catatcttta agaaaattga 3480ggatttggat gagaaattgg aagtactaac tactttctag catatcttta agaaaattga 3480

ttgttattta ctcccagatc ctcttgcaga cccagaatta tcaggaacat agctctgtga 3540ttgttattta ctcccagatc ctcttgcaga cccagaatta tcaggaacat agctctgtga 3540

ttcatgagtg tccccatact gatgaattgg agcatccata tggaaagcaa aggcagaatt 3600ttcatgagtg tccccatact gatgaattgg agcatccata tggaaagcaa aggcagaatt 3600

atcccagctg tattattttg atcttttgga tgcaggtgcc ttaatgaagc tctcaaaata 3660atcccagctg tattattttg atcttttgga tgcaggtgcc ttaatgaagc tctcaaaata 3660

ttttaggagc tgctcaggga gtgttgggtg gaactgtttg gactacattg ttttctctta 3720ttttaggagc tgctcaggga gtgttgggtg gaactgtttg gactacattg ttttctctta 3720

gattatgtga tttttgttgg gcactggcaa aaggtgtgtg tgtgaatgtg tgcatgtgtg 3780gattatgtga tttttgttgg gcactggcaa aaggtgtgtg tgtgaatgtg tgcatgtgtg 3780

tgaatgttgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtttgcagac 3840tgaatgttgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtttgcagac 3840

atgcaaaact gcagctgaaa taatacctta gatttctagg taagtctttc cacatttcaa 3900atgcaaaact gcagctgaaa taatacctta gatttctagg taagtctttc cacatttcaa 3900

taatgggtaa gagtagaacc agggccgggt atcaattatt gcttgctgtt tgcaaccagg 3960taatgggtaa gagtagaacc agggccgggt atcaattatt gcttgctgtt tgcaaccagg 3960

cataaaatca ctttctcaaa tcatccaccg ttcctattaa atttatgccg gaaactctcc 4020cataaaatca ctttctcaaa tcatccaccg ttcctattaa atttatgccg gaaactctcc 4020

ttctgtgagt ataactcctg cagttcctat agcagataag atataagaaa gtgcctccta 4080ttctgtgagt ataactcctg cagttcctat agcagataag atataagaaa gtgcctccta 4080

gtgctcctcc gcccgcttgt ttgctaaaat tccctttctc tctaagtcca ccattttcaa 4140gtgctcctcc gcccgcttgt ttgctaaaat tccctttctc tctaagtcca ccattttcaa 4140

gatttgtaga tagtgtatta gttaagacag ctttgtcgat ctggccagat gttttttctc 4200gatttgtaga tagtgtatta gttaagacag ctttgtcgat ctggccagat gtttttttctc 4200

ctttgtccaa aggccagaga ccatcccagg aagagtggtg ggtggtttat acactggaaa 4260ctttgtccaa aggccagaga ccatcccagg aagagtggtg ggtggtttat acactggaaa 4260

tgttgcgttt atgcttttta aaaacacacg ttaacttcag aggaaggatg ggcaaatctg 4320tgttgcgttt atgcttttta aaaacacacg ttaacttcag aggaaggatg ggcaaatctg 4320

gtctagctgg gtgaaaccct tattttccca gagatgcctt aacctttgtt ggttttggct 4380gtctagctgg gtgaaaccct tattttccca gagatgcctt aacctttgtt ggttttggct 4380

ttagggttca gagtcacttt tgttcccttc tccattctgg agagggactt cccctacata 4440ttagggttca gagtcacttt tgttcccttc tccattctgg agagggactt cccctacata 4440

gagccctgat ttttgtggct gtggggattg gaggtagcat tcaaagatca gatgtgcttt 4500gagccctgat ttttgtggct gtggggattg gaggtagcat tcaaagatca gatgtgcttt 4500

tcctcacttt ggagatgaac actctgggtt ttacagcatt aacctgccta accttcatgg 4560tcctcacttt ggagatgaac actctgggtt ttacagcatt aacctgccta accttcatgg 4560

tgagaaatac accatctctc ttctagtcat gctgtgcatg ccgcttactc tgttggggtc 4620tgagaaatac accatctctc ttctagtcat gctgtgcatg ccgcttactc tgttggggtc 4620

tatataaatt tgttgaactc ttacctacat tccaaagaag tttcaaggaa ccataaatat 4680tatataaatt tgttgaactc ttacctacat tccaaagaag tttcaaggaa ccataaatat 4680

atgtatacat atacatatat aaaatatata tattaaaata aaattatcag gaatactgcc 4740atgtatacat atacatatat aaaatatata tattaaaata aaattatcag gaatactgcc 4740

tcagttattg aacttttttt tttaagaata cttttttttt aagctgagaa gtatagggat 4800tcagttattg aacttttttt tttaagaata cttttttttt aagctgagaa gtatagggat 4800

gaaaaagatg ttatattgtg tttgactatt ttccaacttg tattttcata taatttatat 4860gaaaaagatg ttatattgtg tttgactatt ttccaacttg tattttcata taatttatat 4860

tttttaaaag ctgaaaattt agaagcaaga tgaaaaaaag gaaaagcagg tgctttttaa 4920tttttaaaag ctgaaaattt agaagcaaga tgaaaaaaag gaaaagcagg tgctttttaa 4920

aaatcagaac tgaggtagct tagagatgta gcgatgtaag tgtcgatgtt tttttaaaaa 4980aaatcagaac tgaggtagct tagagatgta gcgatgtaag tgtcgatgtt tttttaaaaa 4980

aaaatgcaaa aaaattctta tggcggagtt ttttgtttgt ttattttagt agctgatgct 5040aaaatgcaaa aaaattctta tggcggagtt ttttgtttgt ttattttagt agctgatgct 5040

ggcacatcat tttgctggag agttttttat atactgtagc ctgatttcat attgtatttt 5100ggcacatcat tttgctggag agttttttat atactgtagc ctgatttcat attgtatttt 5100

aaactgtgtg aaattaaaaa caaagaattt cattcataa 5139aaactgtgtg aaattaaaaa caaagaattt cattcataa 5139

Claims (2)

1. The application of the reagent for detecting the expression quantity of the hsa _ miR-486-5p and ETS-1 genes in the detection of the rheumatoid arthritis is characterized in that the reagent for detecting the expression quantity of the hsa _ miR-486-5p genes comprises a primer pair with nucleotide sequences shown in SEQ ID No.1 and SEQ ID No.2, and the reagent for detecting the expression quantity of the ETS-1 genes comprises a primer pair with nucleotide sequences shown in SEQ ID No.3 and SEQ ID No. 4.

2. An application of hsa _ miR-486-5p combined with ETS-1 gene in a rheumatoid arthritis diagnosis and treatment reagent is characterized in that specific over-expression plasmid sequences SEQ ID NO.5 and SEQ ID NO.6 are prepared according to the hsa _ miR-486-5p gene and used for over-expressing the hsa _ miR-486-5p gene in synovial fibroblasts; the small interference sequence plasmid sequences SEQ ID NO.7 and SEQ ID NO.8 are prepared according to the ETS-1 gene and used for infecting synovial fibroblasts, so that the ETS-1 gene is interfered in the cells.

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