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类风湿关节炎(rheumatoid arthritis,RA)是临床常见的一种系统性自身免疫性疾病,随着疾病的发展病人关节骨与软骨组织逐渐被破坏,甚至致使病人残疾,研究[1-2]发现滑膜成纤维细胞过度增殖及迁移可促进RA的发展。环状RNA(circular RNA,circRNA)是由5′端与3′端以共价键结合形成的一种环状RNA分子,其在RA或滑膜成纤维细胞中表达异常,并可能调控细胞增殖及转移等过程[3-4]。circPTPRA高表达可促进动脉粥样硬化的发生及发展[5]。但circPTPRA与RA的相关研究尚鲜见报道。生物信息学预测显示circPTPRA与miR-140-5p存在结合位点,研究[6]表明miR-140-5p在RA滑膜成纤维细胞中表达下调,其可抑制细胞增殖及炎性因子的分泌。但circPTPRA/miR-140-5p分子轴在RA发生发展过程中的作用机制尚未阐明。因此,本研究主要探讨circPTPRA通过靶向调控miR-140-5p而影响RA滑膜成纤维细胞增殖及转移。
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与Control组比较,RA-FLSs组circPTPRA的表达量升高,miR-140-5p的表达量降低(P < 0.01)(见表 1)。
分组 n circPTPRA miR-140-5p Control组 9 1.00±0.07 1.00±0.10 RA-FLSs组 9 2.84±0.16 0.41±0.03 t — 31.61 16.95 P — < 0.01 < 0.01 表 1 RA滑膜成纤维细胞RA-FLSs中circPTPRA及miR-140-5p表达情况(x±s;ni=9)
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与si-NC组比较,si-circPTPRA组细胞活力降低,迁移细胞数减少,CyclinD1、MMP2、MMP9蛋白水平降低(P < 0.05)(见图 1、表 2)。
分组 circPTPRA CyclinD1 MMP2 MMP9 吸光度值 细胞迁移数量 NC组 1.00±0.10 0.82±0.06 0.89±0.06 0.71±0.06 0.946±0.07 167.03±10.03 si-NC组 0.98±0.07 0.81±0.08 0.90±0.07 0.70±0.07 0.937±0.07 161.01±8.77 si-circPTPRA组 0.42±0.03* 0.39±0.02* 0.43±0.03* 0.33±0.03* 0.517±0.05* 84.02±5.11* F 185.24 156.38 207.10 134.71 131.90 284.07 P < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 MS组内 0.005 0.002 0.002 0.001 0.002 67.875 q检验: 与si-NC组比较*P < 0.05 表 2 低表达circPTPRA对RA滑膜成纤维细胞RA-FLSs增殖和转移的影响(x±s;ni=9)
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与miR-NC组比较,miR-140-5p组细胞活力降低,迁移细胞数减少,CyclinD1、MMP2、MMP9蛋白水平降低(P < 0.01)(见图 2、表 3)。
分组 miR-140-5p CyclinD1 MMP2 MMP9 吸光度值 细胞迁移数量 miR-NC组 1.00±0.10 0.82±0.08 0.92±0.08 0.72±0.07 0.942±0.08 164.07±7.53 miR-140-5p组 1.96±0.11 0.43±0.03 0.48±0.03 0.34±0.03 0.549±0.05 76.06±4.06 t 19.37 13.69 15.45 14.97 12.50 30.86 P < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 表 3 miR-140-5p对RA滑膜成纤维细胞RA-FLSs增殖和转移的影响(x±s;ni=9)
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circPTPRA与miR-140-5p存在结合位点(见图 3)。miR-140-5p过表达可明显降低野生型载体WT-circPTPRA的荧光素酶活性(P < 0.01)(见表 4)。circPTPRA可负向调控miR-140-5p的表达(P < 0.05)(见表 5)。
分组 荧光素酶活性 WT-circPTPRA MUT-circPTPRA miR-NC组 1.00±0.07 1.00±0.09 miR-140-5p组 0.46±0.03 0.98±0.09 t 21.27 0.47 P < 0.01 >0.05 表 4 双荧光素酶活性检测(x±s;ni=9)
分组 circPTPRA miR-140-5p pcDNA-NC组 1.00±0.08 1.00±0.10 pcDNA-circPTPRA组 2.69±0.15* 0.39±0.04* si-NC 1.02±0.09 1.04±0.09 si-circPTPRA组 0.48±0.03*# 2.37±0.18*# F 877.96 481.52 P < 0.01 < 0.01 MS组内 0.005 0.007 q检验: 与pcDNA-NC组比较*P < 0.05;与si-NC组比较#P < 0.05 表 5 qRT-PCR检测miR-140-5p的表达(x±s;ni=9)
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与anti-miR-NC+si-circPTPRA组比较,anti-miR-140-5p+si-circPTPRA组细胞活力升高,迁移细胞数增多,CyclinD1、MMP2、MMP9蛋白水平升高(P < 0.01)(见图 4、表 6)。
分组 miR-140-5p CyclinD1 MMP2 MMP9 吸光度值 细胞迁移数量 anti-miR-NC+si-circPTPRA组 1.00±0.07 0.38±0.02 0.45±0.04 0.32±0.02 0.523±0.04 88±4.89 anti-miR-140-5p+si-circPTPRA组 0.45±0.03 0.86±0.07 0.81±0.06 0.77±0.06 0.973±0.07 179±13.22 t 21.67 19.78 14.98 21.35 16.75 19.37 P < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 表 6 anti-miR-140-5p逆转si-circPTPRA对RA-FLSs增殖和转移的影响(x±s;ni=9)
circPTPRA靶向miR-140-5p调控类风湿关节炎滑膜成纤维细胞增殖及迁移的机制研究
Mechanism of circPTPRA on regulating the proliferation and migration of rheumatoid arthritis fibroblast-like synoviocytes by targeting miR-140-5p
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摘要:
目的探讨circPTPRA对类风湿关节炎滑膜成纤维细胞增殖、迁移的影响及其对miR-140-5p的调控作用。 方法原代分离培养人正常滑膜成纤维细胞与类风湿关节炎滑膜成纤维细胞, 将类风湿关节炎滑膜成纤维细胞分为NC组(不转染)、si-NC组(转染si-NC)、si-circPTPRA组(转染si-circPTPRA)、miR-NC组(转染miR-NC)、miR-140-5p组(转染miR-140-5p mimics)、anti-miR-NC+si-circPTPRA组(共转染anti-miR-NC和si-circPTPRA)、anti-miR-140-5p+si-circPTPRA组(共转染anti-miR-140-5p和si-circPTPRA)。qRT-PCR检测circPTPRA与miR-140-5p的表达量; MTT实验检测细胞增殖; Transwell小室实验检测细胞迁移; 双荧光素酶报告基因实验检测circPTPRA与miR-140-5p的靶向关系。 结果与正常滑膜成纤维细胞比较, 类风湿关节炎滑膜成纤维细胞中circPTPRA的表达量升高, miR-140-5p的表达量降低(P < 0.01);与si-NC组比较, si-circPTPRA组细胞活力降低, 迁移细胞数减少(P < 0.05);与miR-NC组比较, miR-140-5p组细胞活力降低, 迁移细胞数减少(P < 0.01);circPTPRA可靶向调控miR-140-5p;与anti-miR-NC+si-circPTPRA组比较, anti-miR-140-5p+si-circPTPRA组细胞活力升高, 迁移细胞数增多(P < 0.01)。 结论干扰circPTPRA表达可通过靶向调控miR-140-5p的表达而抑制类风湿关节炎滑膜成纤维细胞增殖及迁移。 Abstract:ObjectiveTo explore the effect of circPTPRA on the proliferation and migration of rheumatoid arthritis fibroblast-like synoviocytes and its regulatory effect on miR-140-5p. MethodsThe primary human normal fibroblast-like synoviocyte and rheumatoid arthritis fibroblast-like synoviocyte were isolated and cultured.The synovial fibroblasts of rheumatoid arthritis were divided into NC group(without transfection), si-NC group(transfected si-NC), si-circPTPRA group (transfected si-circPTPRA), miR-NC group (transfected miR-NC) and miR-140-5p group (transfected miR-140-5p mimics), anti-miR-NC+si-circPTPRA group (co-transfected anti-miR-NC and si-circPTPRA), anti-miR-140-5p+si-circPTPRA group (co-transfected anti-miR-140-5p and si-circPTPRA).The expression levels of circPTPRA and miR-140-5p were detected by qRT-PCR.The cell proliferation was detected by MTT test, and the cell migration was tested by Transwell chamber.The targeting relationship between circPTPRA and miR-140-5p was detected through the dual luciferase reporter gene experiment. ResultsCompared with normal fibroblast-like synoviocytes, in rheumatoid arthritis fibroblast-like synoviocytes, the expression of circPTPRA was increased, while the expression of miR-140-5p was decreased (P < 0.01).Compared with the si-NC group, in the si-circPTPRA group, the cell viability was decreased, the number of migrating cells was decreased (P < 0.05).Compared with the miR-NC group, in the miR-140-5p group, the cell viability and the number of migrating cells were decreased (P < 0.01).CircPTPRA could target miR-140-5p.Compared with the anti-miR-NC+si-circPTPRA group, the cell viability was increased as well as the number of migrating cells in the anti-miR-140-5p+si-circPTPRA group (P < 0.01). ConclusionsInterference with the expression of circPTPRA can inhibit the proliferation and migration of rheumatoid arthritis fibroblast-like synoviocyte by targeting the expression of miR-140-5p. -
Key words:
- rheumatoid arthritis /
- fibroblast-like synoviocytes /
- circPTPRA /
- miR-140-5p /
- cell proliferation /
- migration
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表 1 RA滑膜成纤维细胞RA-FLSs中circPTPRA及miR-140-5p表达情况(x±s;ni=9)
分组 n circPTPRA miR-140-5p Control组 9 1.00±0.07 1.00±0.10 RA-FLSs组 9 2.84±0.16 0.41±0.03 t — 31.61 16.95 P — < 0.01 < 0.01 表 2 低表达circPTPRA对RA滑膜成纤维细胞RA-FLSs增殖和转移的影响(x±s;ni=9)
分组 circPTPRA CyclinD1 MMP2 MMP9 吸光度值 细胞迁移数量 NC组 1.00±0.10 0.82±0.06 0.89±0.06 0.71±0.06 0.946±0.07 167.03±10.03 si-NC组 0.98±0.07 0.81±0.08 0.90±0.07 0.70±0.07 0.937±0.07 161.01±8.77 si-circPTPRA组 0.42±0.03* 0.39±0.02* 0.43±0.03* 0.33±0.03* 0.517±0.05* 84.02±5.11* F 185.24 156.38 207.10 134.71 131.90 284.07 P < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 MS组内 0.005 0.002 0.002 0.001 0.002 67.875 q检验: 与si-NC组比较*P < 0.05 表 3 miR-140-5p对RA滑膜成纤维细胞RA-FLSs增殖和转移的影响(x±s;ni=9)
分组 miR-140-5p CyclinD1 MMP2 MMP9 吸光度值 细胞迁移数量 miR-NC组 1.00±0.10 0.82±0.08 0.92±0.08 0.72±0.07 0.942±0.08 164.07±7.53 miR-140-5p组 1.96±0.11 0.43±0.03 0.48±0.03 0.34±0.03 0.549±0.05 76.06±4.06 t 19.37 13.69 15.45 14.97 12.50 30.86 P < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 表 4 双荧光素酶活性检测(x±s;ni=9)
分组 荧光素酶活性 WT-circPTPRA MUT-circPTPRA miR-NC组 1.00±0.07 1.00±0.09 miR-140-5p组 0.46±0.03 0.98±0.09 t 21.27 0.47 P < 0.01 >0.05 表 5 qRT-PCR检测miR-140-5p的表达(x±s;ni=9)
分组 circPTPRA miR-140-5p pcDNA-NC组 1.00±0.08 1.00±0.10 pcDNA-circPTPRA组 2.69±0.15* 0.39±0.04* si-NC 1.02±0.09 1.04±0.09 si-circPTPRA组 0.48±0.03*# 2.37±0.18*# F 877.96 481.52 P < 0.01 < 0.01 MS组内 0.005 0.007 q检验: 与pcDNA-NC组比较*P < 0.05;与si-NC组比较#P < 0.05 表 6 anti-miR-140-5p逆转si-circPTPRA对RA-FLSs增殖和转移的影响(x±s;ni=9)
分组 miR-140-5p CyclinD1 MMP2 MMP9 吸光度值 细胞迁移数量 anti-miR-NC+si-circPTPRA组 1.00±0.07 0.38±0.02 0.45±0.04 0.32±0.02 0.523±0.04 88±4.89 anti-miR-140-5p+si-circPTPRA组 0.45±0.03 0.86±0.07 0.81±0.06 0.77±0.06 0.973±0.07 179±13.22 t 21.67 19.78 14.98 21.35 16.75 19.37 P < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 -
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