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circ-SOD2/miR-424-5p分子轴对类风湿关节炎滑膜成纤维细胞增殖与凋亡的调控作用
作者:汪蕾  杨沙  尹小娟  温慧敏 
单位:四川省骨科医院 药学部, 四川 成都 610041
关键词:circ-SOD2 miR-424-5p 类风湿关节炎 滑膜成纤维细胞 增殖 凋亡 
分类号:R593.21
出版年·卷·期(页码):2021·49·第八期(911-918)
摘要:

目的:探讨circ-SOD2/miR-424-5p分子轴对类风湿关节炎滑膜成纤维细胞增殖与凋亡的影响及其可能作用机制。方法:用脂多糖(LPS)诱导的滑膜成纤维细胞建立类风湿关节炎模型(LPS组);实验分组:Con组、LPS+si-NC组、LPS+si-circ-SOD2组、LPS+miR-NC组、LPS+miR-424-5p组、LPS+si-circ-SOD2+anti-miR-NC组、LPS+si-circ-SOD2+anti-miR-424-5p组;采用实时荧光定量PCR法检测circ-SOD2与miR-424-5p的表达量;采用ELISA法检测TNF-α、IL-6的水平;采用MTT实验、平板克隆形成实验、流式细胞术分别检测细胞增殖、克隆形成、凋亡;双荧光素酶报告基因实验检测circ-SOD2与miR-424-5p的靶向关系;采用Western blot法检测Fas、FADD、caspase-8、caspase-3蛋白表达量。结果:与Con组比较,LPS组circ-SOD2的表达水平升高(P<0.05),细胞增殖率与TNF-α、IL-6的水平升高(P<0.05),克隆形成数增多(P<0.05),miR-424-5p的表达水平降低(P<0.05),凋亡率与Fas、FADD、caspase-8、caspase-3蛋白水平降低(P<0.05);与LPS+si-NC组比较,LPS+si-circ-SOD2组细胞增殖率与TNF-α、IL-6的水平降低(P<0.05),克隆形成数减少(P<0.05),miR-424-5p的表达水平升高(P<0.05),凋亡率与Fas、FADD、caspase-8、caspase-3蛋白水平升高(P<0.05);circ-SOD2能够靶向调控miR-424-5p;与LPS+miR-NC组比较,LPS+miR-424-5p组TNF-α、IL-6的水平降低(P<0.05),细胞增殖率降低(P<0.05),克隆形成数减少(P<0.05),凋亡率与Fas、FADD、caspase-8、caspase-3蛋白水平升高(P<0.05),而共转染si-circ-SOD2与anti-miR-424-5p可明显逆转干扰circ-SOD2表达对类风湿关节炎滑膜成纤维细胞炎症、增殖、克隆形成与凋亡的作用。结论:干扰circ-SOD2表达可通过靶向调控miR-424-5p而抑制类风湿关节炎滑膜成纤维细胞炎症反应、增殖、克隆形成及诱导细胞凋亡。

Objective: To explore the effect of circ-SOD2/miR-424-5p molecular axis on the proliferation and apoptosis of rheumatoid arthritis synovial fibroblasts and its possible mechanism.Methods: After primary isolation and culture of rheumatoid arthritis synovial fibroblasts, synovial fibroblasts were induced by lipopolysaccharide (LPS) to establish rheumatoid arthritis model (LPS group). Experimental groups:Con group, LPS+si-NC group, LPS+si-circ-SOD2 group, LPS+miR-NC group, LPS+miR-424-5p group, LPS+si-circ-SOD2+anti-miR-NC group, LPS+si-circ-SOD2+anti-miR-424-5p group. The qRT-PCR method was used to detect the expression of circ-SOD2 and miR-424-5p. ELISA was used to detect the levels of TNF-α and IL-6. MTT experiment, plate clone formation experiment, flow cytometry were used to detect cell proliferation, clone formation, and apoptosis. The dual luciferase reporter gene experiment detects the targeting relationship between circ-SOD2 and miR-424-5p. Western blot was used to detect the protein expression of Fas, FADD, caspase-8, and caspase-3. Results: Compared with the Con group, the expression level of circ-SOD2 in the LPS group was increased (P<0.05), the cell proliferation rate and the levels of TNF-α and IL-6 were increased (P<0.05), and the number of clone formation was increased (P<0.05), while the expression level of miR-424-5p was decreased (P<0.05), the apoptosis rate and the protein levels of Fas, FADD, caspase-8, and caspase-3 were decreased (P<0.05). Compared with the LPS+si-NC group, the cell proliferation rate and the levels of TNF-α and IL-6 in the LPS+si-circ-SOD2 group were reduced (P<0.05), and the number of clones was reduced (P<0.05), while the expression level of miR-424-5p was increased (P<0.05), and the apoptosis rate and protein levels of Fas, FADD, caspase-8 and caspase-3 were increased (P<0.05). circ-SOD2 could target miR-424-5p. Compared with the LPS+miR-NC group, the levels of TNF-α and IL-6 in the LPS+miR-424-5p group were decreased (P<0.05), the cell proliferation rate was decreased (P<0.05), and the number of clone formation was decreased (P<0.05), and the apoptosis rate and protein levels of Fas, FADD, caspase-8 and caspase-3 were increased (P<0.05). The co-transfection of si-circ-SOD2 and anti-miR-424-5p could significantly reverse the effect of interference with circ-SOD2 expression on inflammation, proliferation, cloning and apoptosis of rheumatoid arthritis synovial fibroblasts. Conclusion: Interfering with the expression of circ-SOD2 could inhibit the inflammatory response, proliferation, clone formation and induce apoptosis of rheumatoid arthritis synovial fibroblasts by targeted regulation of miR-424-5p.

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