MicroRNA-126对类风湿性关节炎滑膜成纤维细胞增殖、侵袭、凋亡的作用及其机制研究

The role of microRNA-126 in the proliferation,invasion and apoptosis of RASFs and its mechanism

目的探究microRNA-126(miR-126)通过靶向Dickkopf-1(DKK1)参与类风湿性关节炎滑膜成纤维细胞(RASFs)增殖、侵袭、凋亡的机制研究。方法选取2018年6月—2019年9月在武汉市第一医院就诊的25例类风湿性关节炎(RA)患者的滑膜组织(RA组),同时收集在该院同期接受膝关节十字韧带断裂重建手术的21例非类风湿关节炎患者的滑膜组织作为健康组,检测其miR-126和DKK1蛋白。利用RA组织构建原代RASFs,通过双萤光素酶报告验证miR-126与DKK1的靶向关系。将RASFs分为对照组、miR-126组、DKK1组、miR-126+DKK1组。采用CCK-8、Transwell及流式细胞术检测过表达miR-126和/或DKK1对RASFs增殖、侵袭和凋亡的影响。结果RA组滑膜组织miR-126相对表达量低于健康组(P<0.05),而DKK1蛋白相对表达量高于健康组(P<0.05)。miR-126 mimic与DKK1 Wt共转染后的相对萤光素酶活性低于miR-126 NC质粒与DKK1 Wt共转染(P<0.05),证明miR-126与DKK1靶向结合。miR-126组RASFs增殖力和侵袭细胞数低于对照组(P<0.05),而凋亡率高于对照组(P<0.05)。DKK1组RASFs增殖力和侵袭细胞数高于对照组(P<0.05),而凋亡率低于对照组(P<0.05)。miR-126+DKK1组增殖力和侵袭细胞数高于miR-126组(P<0.05),而低于DKK1组(P<0.05);miR-126+DKK1组凋亡率低于miR-126组(P<0.05),而高于DKK1组(P<0.05)。结论miR-126在RA滑膜组织中低表达,miR-126可通过靶向DKK1抑制RASFs增殖、侵袭及凋亡。

Objective To explore whether microRNA(miR)-126 participates in the proliferation,invasion and apoptosis of rheumatoid arthritis(RA)synovial fibroblasts(RASFs)by targeting the expression of Dickkopf-1(DKK1).Methods The synovial tissues of 25 RA patients(RA group)who were treated in Wuhan First Hospital from June 2018 to September 2019 were collected,while the synovial tissues of another 21 patients with nonrheumatoid arthritis undergoing the cruciate ligament reconstruction surgery in the hospital during the same period were collected as the healthy group.The expressions of miR-126 and DKK1 protein in the synovial tissues were detected.RASFs were isolated from the RA synovial tissues,and the relationship between miR-126 and DKK1 was determined via dual luciferase reporter assay.The RASFs were divided into the control group,miR-126 group,DKK1 group,and miR-126+DKK1 group.The effects of overexpression of miR-126 and/or DKK1 on the proliferation,invasion and apoptosis of RASFs were tested via CCK-8,transwell assay and flow cytometry,respectively.Results The expression level of miR-126 in the synovial tissues of RA patients was lower than that of the healthy individuals(P<0.05),while the expression level of DKK1 protein was higher in the synovial tissues of RA patients than that of the healthy individuals(P<0.05).When co-transfected with Wt-DKK1 and miR-126 mimic,the relative luciferase activity in the cells was reduced compared to that in the cells co-transfected with Wt-DKK1and miR-126 NC(P<0.05),suggesting the targeted binding of miR-126 to DKK1.The proliferation and invasion abilities of RASFs in the miR-126 group were lower than those in the control group,while the apoptosis rate in the miR-126 group was significantly higher than that in the control group(P<0.05).The proliferation and invasion abilities of RASFs in the DKK1 group were higher than those in the control group,whereas the apoptosis rate in the DKK1 group was lower than that in the control group(P<0.05).The proliferation and invasion abilities of RASFs in the miR-126+DKK1 group were higher than those in the miR-126 group and lower than those in the DKK1group,while the apoptosis rate in the miR-126+DKK1 group was lower than that in the miR-126 group and higher than that in the DKK1 group(P<0.05).Conclusions The miR-126 is lowly expressed in RA synovial tissues,and can inhibit the proliferation,invasion and apoptosis of RASFs by targeting DKK1.