LINC00837/miR-671-5p/SERPINE2功能轴促进类风湿关节炎成纤维细胞样滑膜细胞的恶性病理学过程

doi:10.12122/j.issn.1673-4254.2025.02.18

摘要/Abstract

摘要：

目的探讨LINC00837/miR-671-5p/SERPINE2功能轴对类风湿关节炎成纤维样滑膜细胞（RA-FLS）病理学过程的影响。方法选用正常成纤维细胞样滑膜细胞（FLS）和RA-FLS，构建LINC00837的过表达质粒和干扰质粒转染至RA-FLS中，实验分为对照组（FLS）、模型组（RA-FLS）、pcDNA3.1-NC组、pcDNA3.1-LINC00837组、siRNA-NC组、siRNA-LINC00837组。采用双荧光素酶验证LINC00837靶向miR-671-5p及miR-671-5p靶向SERPINE2关系；使用qRT-PCR实验检测各组细胞LINC00837、miR-671-5p、SERPINE2表达量；Edu法检测细胞增殖情况，划痕愈合实验检测RA-FLS迁移数，Western blotting法检测细胞增殖、迁移相关蛋白表达水平；ELISA法检测炎性细胞因子分泌情况。结果双荧光素酶报告基因结果显示，LINC00837与miR-671-5p的3'非翻译区（3'UTR）结合，而miR-671-5p与SERPINE2的3'非翻译区（3'UTR）结合，两两之间具有结合位点（P<0.01）。同一时间段，与对照组相比，模型组LINC00837和SERPINE2表达量升高，而miR-671-5p表达量降低，细胞增殖、迁移能力提高，促炎细胞因子表达升高，抑炎细胞因子表达降低（P<0.01）；与模型组相比，pcDNA-LINC00837组细胞增殖、迁移活跃，促炎细胞因子表达升高，抑炎细胞因子表达降低（P<0.01）；与模型组相比，siRNA-LINC00837组细胞增殖、迁移能力下降，促炎细胞因子表达下降，抑炎细胞因子水平升高（P<0.01）。结论 RA-FLS中存在LINC00837/miR-671-5p/SERPINE2功能轴，该功能轴促进RA-FLS增殖、迁移及炎症因子分泌等恶性病理学过程，干预LINC00837有望成为调控RA-FLS病理学过程的潜在策略。

关键词: 类风湿关节炎, 成纤维细胞样滑膜细胞, LINC00837, 增殖迁移, 炎症因子

Abstract:

Objective To investigate the regulatory effect of LINC00837/miR-671-5p/SERPINE2 functional axis on pathological processes of fibroblast-like synovial cells (FLS) in rheumatoid arthritis (RA). Methods RA-FLS were transfected with a LINC00837 overexpression plasmid (pcDNA3.1-LINC00837), a LINC00837 interference plasmid (siRNA-LINC00837), or their respective negative control plasmids (pcDNA3.1-NC and siRNA-NC). Dual luciferase was used to verify the targeting relationship between LINC00837 and miR-671-5p and between miR-671-5p and SERPINE2. RT-qPCR was used to detect the expression levels of LINC00837, miR-671-5p and SERPINE2 in normal FLS or the transfected cells, whose proliferation and migration abilities were assessed using Edu assay and scratch healing assay and by detecting the expression levels of Ki-67, PCNA, E-cadherin and N-cadherin with Western blotting. The changes in cellular secretion of the inflammatory cytokines (TNF‑α, IL-17, IL-4 and IL-10) were examined using ELISA. Results Dual luciferase reporter gene assay showed that LINC00837 was capable of binding to the 3'-UTR of miR-671-5p, and the latter bound to the 3-UTR of SERPINE2 at specific binding sites between them. Compared with normal FLS, RA-FLS showed significantly increased expressions of LINC00837 and SERPINE2, lowered miR-671-5p expression and enhanced proliferation and migration abilities with increased expressions of pro-inflammatory cytokines and reduced expressions of anti-inflammatory cytokines. Transfection of RA-FLS with pcDNA-LINC00837 further enhanced cell proliferation and migration and the changes in the inflammatory cytokines, while transfection with si-LINC00837 produced the opposite changes. Conclusion RA-FLS have a LINC00837/miR-671-5p/SERPINE2 functional axis, which regulates cell proliferation, migration and secretion of inflammatory factors, and interventions targeting LINC00837 may provide a potential strategy to regulate the pathological processes in RA-FLS.

Key words: rheumatoid arthritis, fibroblast-like synovial cells, LINC00837, proliferation, migration, inflammatory factors

曹周芳, 汪元, 王梦娜, 孙玥, 刘菲菲. LINC00837/miR-671-5p/SERPINE2功能轴促进类风湿关节炎成纤维细胞样滑膜细胞的恶性病理学过程[J]. 南方医科大学学报, 2025, 45(2): 371-378.

Zhoufang CAO, Yuan WANG, Mengna WANG, Yue SUN, Feifei LIU. LINC00837/miR-671-5p/SERPINE2 functional axis promotes pathological processes of fibroblast-like synovial cells in rheumatoid arthritis[J]. Journal of Southern Medical University, 2025, 45(2): 371-378.

图/表 5

表1 PCR检测各组基因的引物序列

Tab.1 Primer sequences for RT-qPCR of the target genes

Gene	Amplicon size (bp)	Forward primer (5'→3')	Reverse primer (5'→3')
Hu-GAPDH	138	CGGATTTGGTCGTATTGG	GGTGGAATCATATTGGAACA
Hu-U6	94	CTCGCTTCGGCAGCACA	AACGCTTCACGAATTTGCGT
hsa-miR-671-5p		GACTATATAAGCCCTGGAGGGG	AGTGCAGGGTCCGAGGTATT
hsa-miR-671-5p RT		GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCTCCAG
Hu-Linc00837	139	GCCCGCTTAACTGCTGAAAG	GGACCAACTGCTACAACCCA
Hu-SERPINE2	170	CTCTTTCCGGCTGTGACCCTC	GCCAGTTCATGGTTCCTTCCA

图1 LINC00837与miR-671-5p及miR-671-5p与SERPINE2之间存在靶向关系

Fig.1 Targeting relationship between LINC00837 and miR-671-5p and between miR-671-5p and SERPINE2. A: Binding site between LINC00837 and miR-671-5p predicted by Targetscan. B: Binding site between miR-671-5p and SERPINE2 predicted by Targetscan. C: Fluorase activity is decreased in miR-671-5p mimic+LINC00837-WT co-transfection group. D: Fluorase activity is decreased miR-671-5p mimic+SERPINE2-WT co-transfection group. **P<0.01.

图2 转染后各组细胞LINC00837、miR-671-5p、SERPINE2水平

Fig.2 Expression of LINC00837, miR-671-5p and SERPINE2 in normal FLS (control) and RA-FLS after transfection. **P<0.01, ##P<0.01.

图3 LINC00837对细胞增殖、迁移能力的影响

Fig.3 Effects of LINC00837 overexpression or knockdown on proliferation and migration abilities of RA-FLS. A, C: Cell proliferation detected by Edu assay (Original magnification: ×200); B, D: Cell migration detected by scratch healing assay (×100). **P<0.01,##P<0.01.

图5 LINC00837对细胞炎症因子TNF-α、IL-17、IL-4、IL-10表达的影响

Fig.5 Effects of LINC00837 overexpression or knockdown on expressions of inflammatory factors TNF-α, IL-17, IL-4 and IL-10 in RA-FLS. **P<0.01, ##P<0.01.

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