MicroRNA-335-5p对类风湿性关节炎滑膜成纤维细胞的影响*

The effects of microRNA-335-5p on rheumatoid arthritis synovial fibroblasts*

摘要:

目的 探讨microRNA-335-5p（miR-335-5p）对类风湿性关节炎滑膜成纤维细胞（RASFs）增殖、 侵袭和凋亡的影响。方法 选取2018年3月—2019年3月湖州市第三人民医院收治的50例类风湿性关节炎 （RA）患者和50例接受关节置换手术的关节外伤患者的滑膜组织标本，并分离培养RASFs。采用实时荧光定 量聚合酶链反应检测滑膜组织和RASFs中miR-335-5p和DKK1的表达，荧光素酶报告基因检测评估miR335-5p对DKK1荧光素酶活性的影响。将miR-335-5p模拟物、si-DKK1和pcDNA-DKK1转染RASFs，分别 通过ELISA比色法、Transwell法和流式细胞术检测细胞增殖、侵袭和凋亡情况。结果 与正常组织和RASFs相 比，miR-335-5p在RA组织和RASFs中下调（P <0.05），而DKK1在RA组织和RASFs中上调（P <0.05）。荧光 素酶报告基因检测显示，miR-335-5p可特异性结合DKK1的3′UTR，抑制其荧光素酶活性（P <0.05）。此外， miR-335-5p 可降低 DKK1 的表达（P <0.05）。过表达 miR-335-5p 或敲除 DKK1 可抑制 RASFs 的增殖和 侵袭，诱导 RASFs 凋亡（P <0.05）。而过表达 DKK1 可逆转 miR-335-5p 对 RASFs 的影响（P <0.05）。结论 miR-335-5p可通过直接靶向DKK1的表达，抑制RASFs的增殖和侵袭，诱导其凋亡。

Abstract:

Objective To analyze the effects of microRNA-335-5p (miR-335-5p) on the proliferation, invasion and apoptosis of rheumatoid arthritis synovial fibroblasts (RASFs). Methods Synovial tissue samples were collected from 50 patients with rheumatoid arthritis (RA) and 50 patients with joint trauma who underwent joint replacement surgery from March 2018 to March 2019, and the synovial fibroblasts were isolated and cultured. The expressions of miR-335-5p and Dickkopf-1 (DKK1) in synovial tissue and cells were detected by quantitative real-time polymerase chain reaction (qRT-PCR). Luciferase reporter assay was used to evaluate the effect of miR335-5p on luciferase activity of DKK1. RASFs were transfected with miR-335-5p mimics, si-DKK1 and pcDNADKK1, and cell proliferation, invasion and apoptosis were detected by BrdU assay, transwell assay and flow cytometry, respectively. Results Compared with the control tissues and cells, miR-335-5p was significantly downregulated in RA tissues and RASFs (P < 0.05), while DKK1 was significantly up-regulated in RA tissues and RASFs (P < 0.05). Dual-luciferase reporter assay showed that miR-335-5p could specifically bind to the 3'- untranslated regions (3'-UTR) of DKK1 gene and significantly inhibit the luciferase activity (P < 0.05). In addition, miR-335-5p significantly reduced DKK1 expression (P < 0.05), and overexpression of miR-335-5p or DKK1 knockout suppressed the proliferation and invasion of RASFs and induced RASFs apoptosis (P < 0.05). Furthermore, overexpression of DKK1 reversed the effects of miR-335-5p on RASFs (P < 0.05). Conclusion MiR-335-5p can directly target the expression of DKK1 to inhibit the proliferation and invasion of RASFs and to induce RASFs apoptosis.