类风湿关节炎湿热痹阻证患者外周血单核细胞中circRNA 0003353的变化及其对炎症反应的影响

Correlation between circRNA0003353 in Peripheral Blood Mononuclear Cells and Immune Inflammation in Rheumatoid Arthritis Patients with Damp Heat Obstruction Syndrome

目的观察类风湿关节炎(rheumatoid arthritis,RA)湿热痹阻证患者外周血单核细胞(peripheral blood mononuclear cells,PBMCs)中circRNA 0003353的表达,探究其对成纤维样滑膜细胞(fibroblast-like synoviocytes,FLS)炎症反应的影响。方法收集本院RA湿热痹阻证患者55例、正常人30例PBMCs及血清,观察circRNA 0003353表达及其与临床指标的相关性。构建circRNA 0003353过表达质粒和小干扰RNA,转染至RA-FLS中;RT-qPCR检测circRNA 0003353表达;酶联免疫吸附法(ELISA)检测白细胞介素(interleukin,IL)-10、IL-17的表达;Western blot检测Janus激酶2(Janus kinase 2,JAK2)、磷酸化(p)-JAK2、信号转导和转录激活因子3(signal transducers and activators of transcription 3,STAT3)、pSTAT3蛋白的表达;CCK-8法检测细胞活力;Transwell实验检测细胞迁移能力。结果(1)与正常对照组相比,circRNA0003353在RA湿热痹阻证患者PBMCs中表达升高(P<0.05)。(2)Pearson相关性分析表明,RA湿热痹阻证患者circRNA0003353与红细胞沉降率(erythrocyte sedimentation rate,ESR)、类风湿因子(rheumatoid factor,RF)、核因子κB受体活化因子配体(receptor activator of nuclear factor-κB ligand,RANKL)、DAS28呈正相关,circRNA 0003353与IL-10呈负相关(P<0.05)。(3)关联规则结果表明,circRNA 0003353的升高与ESR、IL-17、CRP、免疫球蛋白(Ig)G升高存在显著相关。(4)Logistic回归分析结果表明,circRNA 0003353是RANKL、CRP、ESR的危险因素。(5)RT-qPCR结果显示,与pcDNA3.1-NC组相比,pcDNA3.1-circRNA 0003353组circRNA 0003353表达升高(P<0.05),与si-NC组相比,si-circRNA 0003353组circRNA 0003353表达降低(P<0.05)。(6)ELISA和Western blot结果显示,与pcDNA3.1-NC组相比,pcDNA3.1-circRNA0003353组IL-10表达降低、IL-17表达升高,p-JAK2/JAK2、p-STAT3/STAT3比值升高(P<0.05);与si-NC组相比,si-circRNA0003353组IL-10表达升高、IL-17、JAK2表达降低,p-JAK2/JAK2、p-STAT3/STAT3比值降低(P<0.05)。(7)CCK-8和Transwell实验结果表明,与pcDNA3.1-NC组相比,pcDNA3.1-circRNA 0003353组RA-FLS细胞活力和迁移能力升高(P<0.05);与si-NC组相比,si-circRNA 0003353组RA-FLS细胞活力和迁移能力降低(P<0.05)。结论circRNA 0003353在RA湿热痹阻证患者中表达上调,通过激活JAK2/STAT3信号通路,促进炎症反应,从而参与RA的发病机制。

Objective To investigate the expression of circRNA 0003353 in the peripheral blood mononuclear cells(PBMCs)of rheumatoid arthritis(RA)patients with dampness heat obstruction syndrome and to examine its effect on inflammatory response of fibroblast-like synoviocytes(FLS).Methods The PBMCs and serum samples of 55 RA patients with dampness heat obstruction syndrome and 30 healthy volunteers were collected.The expression of circRNA 0003353 and its correlation with clinical indexes were examined.The circRNA 0003353 overexpression plasmid and siRNA were constructed and transfected into RA-FLS cell line.RT-qPCR was used to determine the expression of circRNA 0003353 mRNA.The expressions of interleukin(IL)-4,IL-10 and IL-17 were examined by ELISA.The expressions of Janus kinase 2(JAK2),p-JAK2,signal transducers and activators of transcription 3(STAT3)and p-STAT3were exmained by Western blot.CCK-8 assay was used to assess cell viability.Cell migration was assessed with Transwell migration assay.Results (1)Compared with that of the normal group,the expression of circRNA 003353 in the PBMCs of RA patients with damp heat obstruction syndrome was significantly increased(P<0.05).(2)Pearson correlation analysis showed that circRNA 0003353 was positively correlated with erythrocyte sedimentation rate(ESR),rheumatoid factor(RF),receptor activator of nuclear factor-κB ligand(RANKL)and DAS28,and circRNA 0003353 was negatively correlated with IL-10(P<0.05).(3)The findings on the association patterns showed that the increase in circRNA 0003353was significantly correlated with the increase of ESR,IL-17,CRP and immunoglobulin(Ig)G.(4)Logistic regression analysis showed that circRNA 0003353 was a risk factor for RANKL,CRP and ESR.(5)RT-qPCR results showed that the expression of circRNA 003353 mRNA in pcDNA3.1-circRNA 0003353 group was significantly higher than that in pcDNA3.1-NC group(P<0.05),and that the expression of circRNA 003353 mRNA in si-circRNA 0003353 group was significantly lower than that in si-NC group(P<0.05).(6)ELISA and Western blot results showed that,compared with those of pcDNA3.1-NC group,the expression of IL-10 in pcDNA3.1-circRNA 0003353 group significantly decreased,the expression of IL-17 increased,and p-JAK2/JAK2 and p-STAT3/STAT3 ratios significantly increased(P<0.05).Compared with those of si-NC group,the expression of IL-10 in si-circRNA 0003353 group significantly increased,the expression of IL-17 and JAK2 decreased,and p-JAK2/JAK2 and p-STAT3/STAT3 ratios significantly decreased(P<0.05).(7)The results of CCK-8 and Transwell assays showed that the viability and migration of RA-FLS in pcDNA3.1-circRNA 0003353 group were higher than those in pcDNA3.1-NC group(P<0.05).Compared with those of si-NC group,the viability and migration ability of RA-FLS in si-circRNA 0003353 group decreased(P<0.05).Conclusion The expression of circRNA0003353 is up-regulated in RA patients with damp heat obstruction syndrome,and it is involved in the pathogenesis of RA by activating the JAK2/STAT3 signaling pathway and promoting the inflammatory response.