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