miR-124a inhibited cell proliferation, migration and invasion of rheumatoid arthritis synovial fibro-blasts by tar-geting AKT2 gene
10.3760/cma.j.issn.1007-7480.2019.11.002
- VernacularTitle: 微RNA-124a靶向AKT2基因调控类风湿关节炎滑膜成纤维细胞增殖迁移和侵袭的作用机制研究
- Author:
Ya HE
1
;
Yan WANG
;
Huaqing LU
;
Lili REN
;
Yang MENG
Author Information
1. Department of Rheumatology and Immunology, the Fifth Affiliated Hospital of Zhengzhou University, Henan 450000, China
- Publication Type:Journal Article
- Keywords:
Arthritis, rheumatoid;
Fibroblasts;
Synovial membrane;
Proliferation;
Migration;
Invasion;
miR-124a
- From:
Chinese Journal of Rheumatology
2019;23(11):724-730
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To investigate the effects of miR-124a on proliferation, migration and invasion in rheumatoid arthritis synovial fibroblasts (RASFs) and the underlying mechanisms.
Methods:RASFs were isolated and cultured from synovial tissue, then qRT-PCR was used to detect the levels of AKT2 mRNA and miR-124a in RASFs. Western blot was applied to determin the expression level of AKT2 protein. RASFs were transfected with miR-124a, anti-miR-124a, si-AKT2 or pcDNA-AKT2 to up-regulate or down-regulate the expression level of miR-124a or AKT2 protein. The cells were divided into normal group of normal synovial tissue, control NC group, miR-con group, miR-124a group, si-con group, si-AKT2 group, miR-124a+pcDNA group and miR-124a+pcDNA-AKT2 group. MTT assay was carried out to measure the proliferation of RASFs. Transwell assay was carried out to detect the migration and invasion cell number of RASFs. Dual-luciferase reporter assay system was implemented to verify the relationship between miR-124a and AKT2. Independent sample t-test and one way analysis of variance (ANOVA) test (square deviation) were used for statistical analysis.
Results:① Compared with normal group, the expression of miR-124a (0.92±0.19) decreased significantly (t=5.788, P<0.01), AKT2 mRNA (3.15±0.63) increased significantly (t=-3.486, P=0.025), AKT2 protein (2.09±0.64) increased significantly (t=-2.959, P=0.042). ② Ccompared with NC group and miR-con group, miR-124a expression (4.17±0.46) increased significantly (F=131.830, P<0.01), migration cell number (34±6) decreased significantly, invasion cell number (14.5±3.1) decreased significantly (F1=35.788, F2=27.211, P<0.01). ③ Compared with mir-con group (1.02±0.18), WT-AKT2 in miR-124a group showed a significant decrease in its relative activity (0.31±0.11) (t=5.830, P<0.01). ④ Compared with NC group and si-con group, the expression of AKT2 protein (0.97±0.03) in si-AKT2 group decreased significantly (F=128.056, P<0.01), the number of migrating cells (32±4), and the number of invasive cells (18.6±2.2) (F1=-70.082, F2=36.524, P<0.01) were decreased significantly. ⑤ Compared with miR-con group, AKT2 protein expression in miR-124a group decreased significantly (0.21±0.03); compared with miR-124a+pcDNA group, AKT2 protein expression in miR-124a+pcDNA-AKT2 group was increased significantly (F=52.487, P<0.01). ⑥ Compared with miR-con group, the number of RASFs migrating cells (30±5) and invasive cells (12.5±1.8) in miR-124a group were significantly decreased; compared with miR-124a+pcDNA group, the number of RASFs migrating cells (71±4) and invasive cells (26.4±4.5) in miR-124a+pcDNA-AKT2 group were significantly increased (F1=30.957, F2=49.960, P<0.01).
Conclusion:MiR-124a can inhibite the proliferation, migration and invasion of RASFs by targeting AKT2 gene. MiR-124a is expected as a molecular target for diagnosis and treatment of rheumatoid arthritis.
