Abstract
Objective:To investigate the effects of circular RNA NOLC1(circNOLC1)and miR-708-5p on rheumatoid arthritis(RA)synovial fibroblasts and their mechanisms of action.Methods:①Method for obtaining RA synovial fibroblasts.Synovial tissues from patients with late-stage RA were collected to isolate and culture RA synovial fibroblasts.②Method for analyzing the effect of inhibiting circNOLC1 expression on RA synovial fibroblasts.RA synovial fibroblasts were divided into a negative control group and a circNOLC1 inhibition group,transfected with non-targeting small interfering RNA(siRNA)and circNOLC1-targeting siRNA,respectively;after 48-hour culture,the circ-NOLC1 expression level,cell viability,cell proliferation ability(number of cell clones),cell migration ability(scratch healing rate),cell in-vasion ability(number of invading cells),and expression levels of E-cadherin and N-cadherin were detected in each group.③Method for analyzing the effect of overexpressing miR-708-5p on RA synovial fibroblasts.RA synovial fibroblasts were divided into a negative control group and a miR-708-5p overexpression group,transfected with miR-708-5p mimic negative control and miR-708-5p mimic,respectively;af-ter 48-hour culture,the miR-708-5p expression level,cell viability,cell proliferation ability(number of cell clones),cell migration ability(scratch healing rate),cell invasion ability(number of invading cells),and expression levels of E-cadherin and N-cadherin were detected in each group.④Method for analyzing the effect of inhibiting circNOLC1 and miR-708-5p expression on RA synovial fibroblasts.RA synovi-al fibroblasts were divided into a negative control group and a combined circNOLC1 and miR-708-5p inhibition group;the negative control group was transfected with circNOLC1-targeting siRNA and miR-708-5p inhibitor negative control,while the combined inhibition group was transfected with circNOLC1-targeting siRNA and miR-708-5p inhibitor;after 48-hour culture,various indicators of cells in each group were detected(same as in③).⑤Method for verifying the binding of miR-708-5p to the predicted target site of circNOLC1.RA synovial fibro-blasts were divided into a negative control group and a miR-708-5p overexpression group,with each group further divided into a wild-type subgroup and a mutant subgroup.The wild-type subgroup of the negative control group was transfected with miR-708-5p mimic negative con-trol and wild-type circNOLC1 psiCHECK-2 recombinant vector,and the mutant subgroup was transfected with miR-708-5p mimic negative control and mutant circNOLC1 psiCHECK-2 recombinant vector;the wild-type subgroup of the miR-708-5p overexpression group was trans-fected with miR-708-5p mimic and wild-type circNOLC1 psiCHECK-2 recombinant vector,and the mutant subgroup was transfected with miR-708-5p mimic and mutant circNOLC1 psiCHECK-2 recombinant vector.After 48-hour culture,the relative luciferase activity of RA synovial fibroblasts in each group was detected.⑥Method for verifying the co-enrichment of circNOLCl and miR-708-5p in the Ago2 com-plex.RA synovial fibroblasts were divided into an anti-immunoglobulin G(IgG)antibody group and an anti-Ago2 antibody group;after cell lysis,magnetic beads coated with anti-IgG antibody and magnetic beads coated with anti-Ago2 antibody were added to the cell lysates of the anti-IgG antibody group and the anti-Ago2 antibody group,respectively,for incubation,and the beads were collected to detect circNOLC1 and miR-708-5p levels.⑦Method for verifying the targeted physical binding of circNOLC1 to miR-708-5p.RA synovial fibroblasts were di-vided into a negative control group and a biotin-labeled miR-708-5p overexpression group,transfected with biotin-labeled miR-708-5p mimic negative control and biotin-labeled miR-708-5p mimic,respectively;after 48 hours,cells were lysed using lysis buffer,and streptavidin-coa-ted magnetic beads were added for overnight incubation;the magnetic beads from each group were collected to detect circNOLC1 levels.Results:①Analysis results of the effect of inhibiting circNOLC1 expression on RA synovial fibroblasts.The relative expression level of circ-NOLC1,cell viability,scratch healing rate,and relative expression level of N-cadherin in RA synovial fibroblasts in the circNOLC1 inhibi-tion group were all lower than those in the negative control group(1.00±0.00 vs 0.35±0.03,t=37.528,P=0.000;0.85±0.06 vs 0.44±0.04,t=9.848,P=0.000;65.48±5.87 vs 22.86±2.09％,t=11.847,P=0.000;0.64±0.04 vs 0.22±0.02,t=16.267,P=0.000),the number of cell clones and invading cells were both less than those in the negative control group(107.62±10.88 vs 42.69±4.39 cells,t=9.647,P=0.000;89.83±7.45 vs 38.08±3.45 cells,t=10.918,P=0.000),and the relative expression level of E-cadherin was higher than that in the negative control group(0.32±0.03 vs 0.75±0.05,t=12.773,P=0.000).②Analysis results of the effect of overexpressing miR-708-5p on RA synovial fibroblasts.The relative expression level of miR-708-5p in RA synovial fibroblasts in the miR-708-5p overexpression group was higher than that in the negative control group(1.00±0.00 vs 2.91±0.21,t=15.753,P=0.000),while cell viability,scratch healing rate,and relative expression level of N-cadherin were all lower than those in the negative control group(0.87±0.07 vs 0.51±0.04,t=7.734,P=0.000;69.89±6.17 vs 32.01±3.33％,t=9.358,P=0.000;0.65±0.04 vs 0.28±0.02,t=8.036,P=0.000),the number of cell clones and invading cells were both less than those in the negative control group(104.41±11.11 vs 51.37±5.12 cells,t=7.510,P=0.000;87.99±6.76 vs 49.58±4.78 cells,t=11.288,P=0.000),and the relative expres-sion level of E-cadherin was higher than that in the negative control group(0.31±0.03 vs 0.69±0.05,t=14.330,P=0.000).③Analy-sis results of the effect of inhibiting circNOLC1 and miR-708-5p expression on RA synovial fibroblasts.The relative expression level of miR-708-5p in RA synovial fibroblasts in the combined circNOLC1 and miR-708-5p inhibition group was lower than that in the negative control group(1.00±0.00 vs 0.29±0.03,t=40.992,P=0.000),cell viability,scratch healing rate,and relative expression level of N-cadherin were all higher than those in the negative control group(0.43±0.04 vs 0.78±0.06,t=8.407,P=0.000;21.07±2.29 vs 56.73±5.54％,t=10.303,P=0.000;0.21±0.02 vs 0.56±0.05,t=9.881,P=0.000),the number of cell clones and invading cells were both more than those in the negative control group(43.78±4.07 vs 89.06±7.67 cells,t=9.032,P=0.000;37.31±3.81 vs 75.81±5.57 cells,t=9.468,P=0.000),and the relative expression level of E-cadherin was lower than that in the negative control group(0.76±0.05 vs 0.41±0.04,t=11.257,P=0.000).④Verification results of the binding of miR-708-5p to the predicted target site of circNOLC1.The relative luciferase activity of RA synovial fibroblasts in the wild-type subgroup of the miR-708-5p overexpression group was lower than that in the wild-type subgroup of the negative control group(0.98±0.05 vs 0.43±0.04,t=25.769,P=0.000);the relative luciferase activity in the mutant subgroup of the miR-708-5p overexpression group was higher than that in the wild-type subgroup of the miR-708-5p overex-pression group(0.43±0.04 vs 0.99±0.07,t=12.031,P=0.000),and the difference compared with the mutant subgroup of the negative control group was not statistically significant(1.00±0.07 vs 0.99±0.07,t=0.303,P=0.766).⑤Verification results of the co-enrich-ment of circNOLC1 and miR-708-5p in the Ago2 complex.The levels of circNOLC1 and miR-708-5p in RA synovial fibroblasts in the anti-Ago2 antibody group were both higher than those in the anti-IgG antibody group(1.00±0.02 vs 16.78±2.15,t=22.018,P=0.000;1.00±0.03 vs 11.56±2.63,t=12.045,P=0.000).⑥Verification results of the targeted physical binding of circNOLC1 to miR-708-5p.The level of circNOLC1 in RA synovial fibroblasts in the biotin-labeled miR-708-5p overexpression group was higher than that in the negative control group(8.97±1.12 vs 1.00±0.04,t=21.335,P=0.000).Conclusion:The circNOLC1 can affect the viability,prolifer-ation,migration and invasion abilities of RA synovial fibroblasts by targeting and adsorbing miR-708-5p.关键词
关节炎,类风湿/滑膜/成纤维细胞/环状RNA NOLC1/miR-708-5p/细胞活性/细胞增殖/细胞迁移/细胞侵袭Key words
arthritis,rheumatoid/synovial membrane/fibroblasts/circular RNA NOLC1/miR-708-5p/cell viability/cell proliferation/cell migration/cell invasion
