吉林大学学报(医学版) ›› 2023, Vol. 49 ›› Issue (3): 665-674.doi: 10.13481/j.1671-587X.20230315

• 基础研究 • 上一篇    下一篇

miR-93-5p靶向下调ROCK2表达对类风湿性关节炎成纤维样滑膜细胞增殖、迁移和侵袭的影响

杨舟,林书典(),詹宇威,肖璐,符克英,黄小蝶   

  1. 海南省人民医院 海南医学院附属海南医院风湿免疫科,海南 海口 570000
  • 收稿日期:2022-07-25 出版日期:2023-05-28 发布日期:2023-06-20
  • 通讯作者: 林书典 E-mail:yuier3558@163.com
  • 作者简介:杨 舟(1983-),女,湖南省常德市人,副主任医师,医学硕士,主要从事炎性关节炎的诊断和治疗方面的研究。
  • 基金资助:
    海南省科技厅自然科学基金面上项目(820MS128);海南省卫健委省级临床医学中心建设项目(琼卫医函(2021)75号)

Effects of down-regulation of ROCK2 expression targeted by miR-94-5p on proliferation, migration and invasion of rheumatoid arthritis fibroblast-like synoviocytes

Zhou YANG,Shudian LIN(),Yuwei ZHAN,Lu XIAO,Keying FU,Xiaodie HUANG   

  1. Department of Rheumatology,People’s Hospital,Hainan Province,Affiliated Hainan Hospital,Hainan Medical College,Haikou 570000,China
  • Received:2022-07-25 Online:2023-05-28 Published:2023-06-20
  • Contact: Shudian LIN E-mail:yuier3558@163.com

摘要:

目的 探讨miR-93-5p对类风湿性关节炎成纤维样滑膜细胞(RA-FLSs)增殖、迁移和侵袭的影响,并阐明其可能的机制。 方法 选择类风湿性关节炎(RA)患者(RA组,n=37)和接受关节置换手术的关节创伤患者(对照组,n=30)作为研究对象,从RA患者滑膜组织分离出RA-FLSs,采用免疫荧光法和流式细胞术鉴定细胞。将RA-FLSs分为空白组、mimics NC组(转染miR-93-5p mimics NC)、mimics组(转染miR-93-5p mimics)、OE-NC组(转染ROCK2过表达空载质粒)、OE-Rho相关螺旋卷曲蛋白激酶(ROCK)2组(转染ROCK2过表达质粒)、mimics+OE-NC组(共转染miR-93-5p mimics和ROCK2过表达空载质粒)和mimics+OE-ROCK2组(共转染miR-93-5p mimics和ROCK2过表达质粒)。采用实时荧光定量PCR(RT-qPCR)法检测2组患者滑膜组织和各组细胞中miR-93-5p和ROCK2 mRNA表达水平,CCK-8法检测各组细胞增殖活性,EdU染色检测各组细胞中EdU阳性细胞率,Transwell小室实验检测各组RA-FLSs迁移和侵袭数,Western blotting法检测各组细胞中ROCK2、Ki-67、增殖细胞核抗原(PCNA)、基质金属蛋白酶2(MMP-2)和基质金属蛋白酶9(MMP-9)蛋白表达水平,双荧光素酶报告基因实验验证miR-93-5p与ROCK2之间的靶向关系。 结果 免疫荧光法检测,Vimentin蛋白表达呈阳性。流式细胞术检测,第3代RA-FLSs表面CD55呈阳性表达,CD14和CD68呈阴性表达,证实分离的细胞为RA-FLSs。RT-qPCR法检测,与对照组比较,RA组患者滑膜组织中miR-93-5p表达水平明显降低(P<0.01),ROCK2 mRNA表达水平明显升高(P<0.01)。与空白组和mimics NC组比较,mimics组细胞中miR-93-5p表达水平明显升高(P<0.01),ROCK2 mRNA和蛋白表达水平明显降低(P<0.01)。CCK-8法和EdU染色检测,与mimics NC组比较,mimics组细胞增殖活性和EdU阳性细胞率明显降低(P<0.01),OE-ROCK2组细胞增殖活性和EdU阳性细胞率明显升高(P<0.01);与mimics组比较,mimics+OE-ROCK2组细胞增殖活性和EdU阳性细胞率明显升高(P<0.01)。Transwell小室实验检测,与mimics NC组比较,mimics组RA-FLSs的迁移和侵袭数明显减少(P<0.01),OE-ROCK2组RA-FLSs的迁移和侵袭数明显增加(P<0.01);与mimics组比较,mimics+OE-ROCK2组RA-FLSs的迁移和侵袭数明显增加(P<0.01)。Western blotting法检测,与mimics NC组比较,mimics组细胞中ROCK2、Ki-67、PCNA、MMP-2和MMP-9蛋白表达水平明显降低(P<0.01),OE-ROCK2组细胞中ROCK2、Ki-67、PCNA、MMP-2和MMP-9蛋白表达水平明显升高(P<0.01);与mimics组比较,mimics+OE-ROCK2组细胞中ROCK2、Ki-67、PCNA、MMP-2和MMP-9蛋白表达水平明显升高(P<0.01)。miR-93-5p与ROCK2 -3'-UTR之间存在靶向结合位点。双荧光素酶报告实验验证转染miR-93-5p mimic可明显降低ROCK野生型(ROCK2-WT)组细胞中荧光素酶活性(P<0.01)。 结论 过表达miR-93-5p通过靶向下调ROCK2表达抑制RA-FLSs的增殖、迁移和侵袭。

关键词: 类风湿性关节炎成纤维样滑膜细胞, 微小RNA-93-5p, Rho相关螺旋卷曲蛋白激酶, 细胞迁移, 细胞侵袭

Abstract:

Objective To discuss the effect of miR-93-5p on the proliferation, migration, and invasion of the rheumatoid arthritis fibroblasts-like synoviocytes(RA-FLSs),and to elucidate its possible mechanism. Methods The rheumatoid arthritis(RA) patients (RA group,n=37) and joint trauma patients who underwent joint replacement surgery (control group,n=30) were selected as the subjects.The RA-FLSs were isolated from synovial tissue of the RA patients, and identified by immunofluorescence and flow cytometry. The RA-FLSs were divided into blank group, mimics NC group (transfected with miR-93-5p mimics NC), mimics group (transfected with miR-93-5p mimics), OE-NC group (transfected with ROCK2 over-expression empty plasmid), OE-Rho related spiral coil protein kinase (ROCK)2 group (transfected with ROCK2 over-expression plasmid), mimics+OE-NC group (co-transfected with miR-93-5p mimics and ROCK2 over-expression empty plasmids),and mimics+OE-ROCK2 group (co-transfected with miR-93-5p mimics and ROCK2 over-expression plasmids). Real time fluorescence quantitative PCR (RT-qPCR) method was used to detect the expression levels of miR-93-5p and ROCK2 mRNA in synovial tissue of the patients in two groups and cells in various groups;CCK-8 method was used to detect the proliferation activities of the cells in various groups; EdU staining was used to detect the EdU positive rates of the cells in various groups;Transwell champer assay was used to detect the migration and invasion numbers of RA-FLSs in various groups; Western blotting method was used to detect the expression levels of ROCK2, Ki-67, proliferating cell nuclear antigen (PCNA),matrix metalloproteinase 2 (MMP-2), and matrix metalloproteinase 9 (MMP-9) proteins in the cells in various groups; the targeting relationship between miR-93-5p and ROCK2 was verified by double Luciferase reporter gene experiment. Results The immunofluorescence assay results showed that the expression of Vimentin protein was positive.The flow cytometry detection results showed that the expression of CD55 on surface of the third generation RA-FLSs was positive, while the expressions of CD14 and CD68 were negative, confirming that the isolated cells were the RA-FLSs. The RT-qPCR results showed that compared with control group, the expression level of miR-93-5p in synovial tissue of the patients in RA group was significantly decreased(P<0.01), while the expression level of ROCK2 mRNA was significantly increased (P<0.01). Compared with blank group and mimics NC group, the expression level of miR-93-5p of the cells in mimics group was significantly increased (P<0.01), while the expression levels of ROCK2 mRNA and protein were significantly decreased (P<0.01). The CCK-8 method and EdU staining results showed that compared with mimics NC group, the proliferation activitiy and EdU positive rate of the cells in mimics group were significantly decreased (P<0.01), while the proliferation activitiy and EdU positive rate of the cells in OE-ROCK2 group were significantly increased (P<0.01); compared with mimics group, the proliferation activitiy and EdU positive rate of the cells in mimics+OE-ROCK2 group were increased (P<0.01). The Transwell champer assay results showed that compared with mimics NC group, the migration and invasion numbers of RA-FLSs in mimics group were significantly decreased (P<0.01), while the migration and invasion numbers of RA-FLSs in OE-ROCK2 group were significantly increased (P<0.01); compared with mimics group, the migration and invasion numbers of RA-FLSs in mimics+OE-ROCK2 group were increased (P<0.01). The Western blotting method results showed that compared with mimics NC group, the expression levels of ROCK2, Ki-67, PCNA, MMP-2, and MMP-9 proteins in the cells in mimics group were significantly decreased (P<0.01), while the expression levels of ROCK2, Ki-67, PCNA, MMP-2, and MMP-9 proteins in the cells in OE-ROCK2 group were significantly increased (P<0.01); compared with mimics group, the expression levels of ROCK2, Ki-67, PCNA, MMP-2, and MMP-9 proteins in the cells in mimics+OE-ROCK2 group were significantly increased (P<0.01).There was a targeted binding site between miR-93-5p and ROCK2-3'-UTR. The double luciferase report experiment results showed that transfection of miR-93-5p mimic could significantly decrease the luciferase activity of the cells in ROCK2 wild type(ROCK2-WT) group(P<0.01). Conclusion Over-expression of miR-93-5p inhibits the proliferation, migration, and invasion of the RA-FLSs targeted by down-regulation of ROCK2 expression.

Key words: Rheumatoid arthritis fibroblast-like synoviocyte, MicroR-93-5p, Rho associated coiled-coil containing protein kinases, Cell migration, Cell invasion

中图分类号: 

  • R593.22