Mechanism of miR-218-5p in autophagy, apoptosis and oxidative stress in rheumatoid arthritis synovial fibroblasts is mediated by KLF9 and JAK/STAT3 pathways

Abstract

This study was aimed to investigate the effects of miR-218-5p on the proliferation, apoptosis, autophagy, and oxidative stress of rheumatoid arthritis synovial fibroblasts (RASFs), and the related mechanisms. Quantitative reverse transcription-PCR showed that the expression of miR-218-5p in rheumatoid arthritis synovial tissue was significantly higher than that in healthy synovial tissue. Compared with healthy synovial fibroblasts, miR-218-5p expression was obviously upregulated in RASFs, while KLF9 protein expression was markedly downregulated. Mechanistically, miR-218-5p could directly bind to the 3' untranslated region of KLF9 to inhibit the expression of KLF9. Additionally, transfection of miR-218-5p small interfering RNA (siRNA) inhibited the proliferation but promoted apoptosis and autophagy of RASFs. Simultaneously, miR-218-5p silencing reduced reactive oxygen species and malondialdehyde levels and increased superoxide dismutase and glutathione peroxidase activity to improve oxidative stress in RASFs. More importantly, the introduction of KLF9 siRNA reversed the effects of miR-218-5p siRNA transfection on RASF proliferation, apoptosis, autophagy, and oxidative stress. What is more, silencing miR-218-5p inhibited the activation of JAK2/STAT3 signaling pathway by targeting KLF9. Collectively, knockdown of miR-218-5p could regulate the proliferation, apoptosis, autophagy and oxidative stress of RASFs by increasing the expression of KLF9 and inhibiting the activation of the JAK2/STAT3 signaling pathway, which may provide a potential target for the mechanism research of RA.

Keywords: apoptosis.

Figure 1

Expression of miR-218-5p in synovial tissues of patients with RA and RASFs was upregulated. (A) Expression of miR-218-5p in healthy controls (n=30) and RA synovial tissues (n=30) was analyzed by qRT-PCR. (B) Expression of miR-218-5p in in HSFs and RASFs was analyzed by qRT-PCR. *P<0.05. HSF, healthy synovial fibroblast; qRT-PCR, quantitative reverse transcription–PCR; RA, rheumatoid arthritis; RASF, rheumatoid arthritis synovial fibroblast.

Figure 2

KLF9 was a target of miR-218-5p. (A) The expression of KLF9 protein in synovial tissue from patients with RA (n=30) and healthy controls (n=30) was detected by western blot. (B) There was a negative correlation between miR-218-5p and KLF9 protein expression in RA synovial tissues (n=30). (C) The expression of KLF9 mRNA and protein in RASFs and HSFs was analyzed by qRT-PCR and western blot. (D) TargetScan showed that miR-218-5p binds to 3′ UTR of KLF9. (E) Luciferase reporter vectors containing KLF9-Wt and KLF9-Mut were constructed and then cotransfect with miR-218-5p or miR-NC into 293 T cells to detect luciferase activity. (F) The expression of miR-218-5 in RASFs after transfection with si-miR-218-5p or miR-218-5p mimic was determined by qRT-PCR. qRT-PCR (G) and western blot assay (H) were used to analyze the expression of KLF9 mRNA and protein in RASFs after transfection with si-miR-218-5p or miR-218-5p mimic. *P<0.05. HSF, healthy synovial fibroblast; qRT-PCR, quantitative reverse transcription–PCR; RA, rheumatoid arthritis; RASF, rheumatoid arthritis synovial fibroblast; UTR, untranslated region; Wt, wild type.

Figure 3

Knockdown of miR-218-5p regulated proliferation, apoptosis and autophagy of RASFs by targeting KLF9. (A) The expression of KLF9 protein in RASFs transfected with si-miR-218-5p and si-KLF9 was detected by western blot. (B) Proliferation of RASFs transfected with si-miR-218-5p and si-KLF9 was detected by MTT assay. (C) Apoptosis of RASFs transfected with si-miR-218-5p and si-KLF9 was detected by flow cytometry. (D) The activity of caspase-3 in RASFs transfected with si-miR-218-5p and si-KLF9 was detected by caspase-3 activity detection kit. (E–F) The protein expression of LC3, Beclin-1 and p62 in RASFs transfected with si-miR-218-5p and si-KLF9 was analyzed by western blot assay. *P<0.05. MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; OD₄₉₀, optical density at 490 nm; RASF, rheumatoid arthritis synovial fibroblast.

Figure 4

Knockdown of miR-218-5p reduced oxidative stress in RASFs by targeting KLF9. The effect of cotransfection of si-miR-218-5p and si-KLF9 on oxidative stress in RASFs was evaluated by evaluating ROS level (A), MDA content (B), SOD activity (C), and GPx activity (D). *P<0.05. RASF, rheumatoid arthritis synovial fibroblast; ROS, reactive oxygen species; SOD, superoxide dismutase.

Figure 5

Knockdown of miR-218-5p inhibitedJAK2/STAT3 signaling pathway by targeting KLF9. The protein expression of Jak2, p-JAK2, STAT3, p-STAT3 in RASFs transfected with si-miR-218-5p and si-KLF9 was analyzed by western blot assay. *P<0.05. RASF, rheumatoid arthritis synovial fibroblast; ROS, reactive oxygen species; SOD, superoxide dismutase.