Influences of the lncRNA TUG1‐miRNA‐34a‐5p network on fibroblast‐like synoviocytes (FLSs) dysfunction in rheumatoid arthritis through targeting the lactate dehydrogenase A (LDHA)

BackgroundRheumatoid arthritis (RA) is a systemic and chronic inflammatory disease. The cellular glucose metabolism of fibroblast‐like synoviocytes (FLSs) of RA has been revealed to be essential to the pathogenesis and development of RA. To date, the precise roles and molecular mechanisms of long noncoding RNA TUG1 in RA have not been elucidated.MethodsTUG1 and miR‐34a‐5p were detected by qRT‐PCR. Interactions between lncRNA‐miRNA and miRNA‐mRNA were validated by RNA pull‐down assay and luciferase assay. The glucose metabolism was evaluated by glucose uptake and extracellular acidification rate (ECAR). Cell viability was determined by MTT assay and Annexin V assay.ResultsTUG1 expression was significantly upregulated in synovial fibroblast‐like synoviocytes (FLSs) compared with normal FLSs. Functional assays uncovered that silence of TUG1 suppressed FLSs‐RA invasion, migration, glucose metabolism, and increased apoptosis. Bioinformatics analysis indicated that TUG1 interacted with miR‐34a‐5p. RNA pull‐down assay and luciferase assay validated that TUG1 sponged miR‐34a‐5p in FLSs‐RA. Overexpression of miR‐34a‐5p effectively inhibited glucose metabolism of FLSs‐RA. Furthermore, the glucose metabolism of FLSs‐RA was significantly elevated compared with normal FLSs. The glucose metabolism enzyme, LDHA, was directly targeted by miR‐34a‐5p in FLSs. Rescue experiments validated that the miR‐34a‐5p‐inhibited glucose metabolism of FLSs‐RA was through targeting LDHA. Finally, we showed restoration of miR‐34a‐5p in TUG1‐overexpressing FLSs‐RA successfully overcame the TUG1‐promoted glucose metabolism and apoptosis resistance via targeting LDHA.ConclusionThe present study uncovered critical roles and molecular mechanisms underlying the TUG1‐mediated glucose metabolism and apoptosis of FLSs‐RA through modulating the miR‐34a‐5p‐LDHA pathway in fibroblast‐like synoviocytes of rheumatoid arthritis.     

卵泡抑素样蛋白1在类风湿关节炎中的作用

目的研究卵泡抑素样蛋白1(FSTL1)与类风湿关节炎(RA)活动度的相关性。方法 20例RA患者纳入观察组,20例非RA患者纳入对照组。用酶联免疫吸附试验(ELISA)法测定20例RA患者治疗前后的血清FSTL1水平;魏氏法测定患者治疗前红细胞沉降率(ESR),免疫透射比浊测定C反应蛋白(CRP)、类风湿因子(RF)水平,抗环瓜氨酸肽(CCP)抗体酶联免疫试剂盒检测抗CCP抗体,并进行疾病活动指数28(DAS28)评分。用免疫印迹法测定两组被试成纤维样滑膜细胞(FLSs)中FSTL1的水平。结果观察组患者血清FSTL1水平与ESR、CRP、RF、抗CCP抗体及DAS28呈较好的相关性(P<0.05);观察组关节组织FSTL1蛋白相对表达量明显高于对照组(P<0.05);观察组治疗后,血清FSTL1水平明显低于治疗前(P<0.05)。结论 FSTL1可作为RA患者疾病活动度的新炎性标志物。     

双向电泳和质谱技术在类风湿关节炎滑膜细胞差异蛋白分析中的应用及意义

目的 采用2-DE和质谱技术分析RA患者和创伤件关节炎患者关节滑膜组织中FLS蛋白质组学的变化,寻找RA关节滑膜特异性自身抗原.方法 采用2-DE分离6例创伤性关节炎患者及3例RA患者关节滑膜组织中FLS总蛋白质,凝胶经银染显色和图像数据分析识别差异蛋白质点,并用MALDI-TOF-MS对筛选出的差异蛋白质点进行鉴定.结果 RA患者与创伤性关节炎患者中的FLS蛋白质通过2-DE检测,分别检出1 633、1 603个蛋白质点;对2组相互匹配的蛋白质点进行差异分析后,其中蛋白质差异量大于3倍的差异点共有92个,从中选取在RA患者FLS中表达上调的33个差异蛋白质点进行MALDI-TOF-MS 鉴定分析,并与IPI和Swiss-Prot数据库进行比对,共鉴定出包括肿瘤型丙酮酸激酶、α-烯醇化酶、二硫键异构酶A3 前体、谷胱甘肽转移酶、泛素结合酶E2K、血小板活化因子乙酰水解酶、二氢嘧啶酶样2、腺苷酸酶、转醛醇酶、乙酰基辅酶A异构酶、26S蛋白调节亚单位S6a、膜联蛋白A11、过氧化物还原酶1、人色氨酰tRNA合成酶、核纤层蛋白、肌间蛋白、核基质蛋白、肌动蛋白、T-复合物多肽1、葡萄糖调节蛋白75、αB-晶体蛋白、葡萄糖调节蛋白78、未知蛋白:如PSMFA和SELENBP1等30个与RA发病有关的蛋白.结论 RA患者与创伤性关节炎患者FLS间存在差异表达的蛋白质,这些差异蛋白有可能是构成RA关节滑膜的相关自身抗原,并可能在诱导RA自身免疫反应中发挥一定作用.     

The cyclooxygenase-2/thromboxane A2 pathway: a bridge from rheumatoid arthritis to lung cancer?

Patients with rheumatoid arthritis (RA) appear to be at a higher risk of lung cancer (LC). Although the connection between RA and LC has been an active area of research for many years, the molecular pathogenesis of the disease process remains unclear. The cyclooxygenase (COX)-2/thromboxane A2 (TxA2) pathway has been shown to play a potential role in LC development through an auto-regulatory feedback loop. An increased level of TxA2 has been found in RA patients, and intriguingly, the positive feedback loop for the COX-2/TxA2 pathway was shown to have a potential function in RA fibroblast-like synoviocytes (RA-FLS). Thus, the molecular basis of LC development in patients with RA has been at least in partly described. It is possible that COX-2-derived TxA2 could be monitored for the early detection of LC in RA patients, and targeting this molecular pathway may decrease the risk of LC in patients with RA.     

佐剂诱导关节炎大鼠模型的建立及成纤维样滑膜细胞的分离

目的建立类风湿性关节炎(RA)动物模型;从炎性关节滑膜中分离成纤维样滑膜细胞(FLS)。方法应用热灭活结核杆菌H37Ra菌株与矿物油混合制备改良的佐剂,尾根部皮内注射Lewis大鼠诱导关节炎;剪取成功诱导关节炎大鼠的病变踝关节,从中剥离滑膜组织,充分剪碎后采用胶原酶消化法分离成纤维样滑膜细胞。结果成功诱导了大鼠关节炎,发病率为100%,发病时间有规律,组织学表现与RA相似;成功在体外培养了FLS并对其进行了鉴定,掌握了其生长形态和特征。结论成功制备RA动物模型并获得FLS,为今后RA发病机制探索和药物评价提供了良好的体内动物模型和体外细胞模型。     

选择性β肾上腺素受体激动剂对人滑膜细胞增殖能力的影响

目的：观察β-肾上腺素受体( AR)激动药对人成纤维样滑膜细胞( FLS)增殖的作用,探讨β-AR信号对人FLS功能的影响。方法组织块培养法培养人FLS;使用肿瘤坏死因子α( TNF-α)作为刺激剂,MTT法分别检测β1-AR选择性激动剂多巴酚丁胺和β2-AR选择性激动剂沙丁胺醇对人FLS增殖作用的影响,并计算增殖抑制率;使用沙丁胺醇+β2-AR选择性拮抗剂ICI 118551观察其对FLS增殖功能的影响;Western blot法检测人FLSβ-AR、G蛋白偶联受体激酶2(GRK2)和β抑制蛋白2(β-arrestin2)胞膜蛋白的表达水平。结果 TNF-α可以显著促进人FLS的增殖;β2-AR激动剂沙丁胺醇能明显抑制FLS的增殖,其拮抗剂ICI 118551能显著拮抗沙丁胺醇的作用;β1-AR选择性激动剂多巴酚丁胺对FLS的增殖无显著影响;TNF-α和沙丁胺醇均能显著降低β2-AR胞膜表达,上调 GRK2、β-arrestin2的胞膜表达。结论β2-AR信号的激活可以抑制FLS增殖;TNF-α和沙丁胺醇可以使GRK2、β-arrestin2的胞膜表达增加,β2-AR胸膜表达下降。     

siRNA沉默PCSK6基因对胶原诱导性关节炎的影响

目的 探讨siRNA沉默PCSK6基因对胶原诱导性关节炎(CIA)成纤维样滑膜细胞(FLS)生物学行为的影响。方法 采用牛Ⅱ型胶原诱导大鼠CIA模型,取膝关节滑膜组织原代培养FLS;采用人工合成的siRNA oligo-433干扰剂、siRNA oligo-688干扰剂、siRNA oligo-2506干扰剂特异性抑制PCSK6在CIA FLS中的表达,阴性siRNA为阴性对照组,瞬时转染24、36 h后,采用RT-qPCR法检测抑制效率,同时检测PCSK6基因沉默后各相关基因的表达;采用MTT、Transwell、细胞划痕、流式细胞术、ELISA等方法检测干扰PCSK6表达后对细胞增殖、侵袭、迁移、细胞周期和相关炎性因子分泌的影响。结果 转染特异性siRNA-PCSK6后,各干扰组与阴性对照组相比,siRNA oligo-2506干扰剂转染24 h时,PCSK6 mRNA水平抑制效果显著(P<0.001),CIA FLS的增殖受到抑制(P<0.001),细胞的侵袭和迁移能力下降(P<0.001),肿瘤坏死因子α(TNF-α)和白细胞介素-1β(IL-1β)分泌降低(P<0.001),G₀/G₁期的细胞数目增多(P<0.05),PCSK6下游基因CXCL9、MMP2、MMP9、NOSTRIN、HIF-1α、MPZL2、IGF2、PADI4表达均明显下降(P<0.05)。结论 PCSK6基因沉默对CIA FLS的生物学行为具有一定作用,为进一步研究CIA发病机制奠定基础。     

白藜芦醇抑制低浓度H2O2处理佐剂性关节炎大鼠成纤维样滑膜细胞增殖的机制研究

目的 探讨抗氧化剂白藜芦醇(Res)对低浓度H2 O2处理的佐剂性关节炎(AA)大鼠成纤维样滑膜细胞(FLS)增殖的抑制作用.方法 弗氏完全佐剂足趾皮下注射SD雄性大鼠,建立AA模型,14 d后股动脉放血处死AA大鼠,取血清检测氧化应激指标,组织块培养法培养大鼠滑膜细胞,CCK-8法观察不同浓度的H2O2对FLS的增殖影响,Western blot法检测氧化应激相关蛋白去乙酰化酶3(SIRT3)、锰超氧化物歧化酶(MnSOD)蛋白的表达.结果 AA模型大鼠血清氧化应激指标升高,体外实验表明,随着Res的浓度增加,低浓度H2O2处理的AA大鼠FLS增殖受抑制,SIRT3、MnSOD蛋白表达降低.结论 Res可减轻AA大鼠体内的氧化应激状态,机制与降低抗氧化应激蛋白SIDT3、MnSOD的表达有关.