The Effects of microRNA-515-5p on the Toll-Like Receptor 4 (TLR4)/JNK Signaling Pathway and WNT1-Inducible-Signaling Pathway Protein 1 (WISP-1) Expression in Rheumatoid Arthritis Fibroblast-Like Synovial (RAFLS) Cells Following Treatment with Receptor Activator of Nuclear Factor-kappa-B Ligand (RANKL)

Abstract

BACKGROUND This study aimed to investigate the effects of microRNA-515-5p (miR-515-5p) on the expression of the WNT1-inducible-signaling pathway protein 1 (WISP-1) gene in rheumatoid arthritis fibroblast-like synovial (RAFLS) cells following treatment with the receptor activator of nuclear factor-kappa-B ligand (RANKL). MATERIAL AND METHODS RAFLS cells were cultured in vitro and were divided into six study groups: a normal control group; a miR-515-5p mimic group; a miR-515-5p inhibitor group; a RANKL (50 ng/ml) treatment group; a miR-515-5p mimic+RANKL treatment group; and a miR-515-5p inhibitor+RANKL treatment group. The luciferase assay was used to determine the effects of miR-515-5p on the WISP1 expression. Cell proliferation, cell apoptosis, the cell cycle, and protein expression were determined using the Cell Counting Kit-8 (CCK-8) assay, flow cytometry, Western blot, and real-time polymerase chain reaction (RT-PCR). RESULTS The luciferase assay showed that the effects of miR-515-on the 3'-UTR of WISP1 inhibited the gene expression. The miR-515-5p mimics promoted cell proliferation, reduced apoptosis, and promoted the cell cycle. The miR-515-5p mimics reduced, the expression of TLR4, WISP1, and JNK at the mRNA level, while the miR-515-5p inhibitor promoted the expression of TLR4, WISP1, and JNK. Both the miR-515-5p inhibitor and mimic promoted the phosphorylation of AKT in RAFLS cells treated with or without RANKL compared with the control, and the miR-515-5p inhibitor promoted the phosphorylation of JNK in the RAFLS cells. CONCLUSIONS In RAFLS cells, miR-515-5p inhibited the expression of the WISP1 gene, and treatment with RANKL inhibited the TLR4/JNK signaling pathway.

Figure 1

microRNA-515-5p (miR-515-5p) acted on 3′-UTR of the WISP1 gene to inhibit gene expression. (A) The binding sites of WISP1 with miR-515-5p are labeled in red. (B) miR-515-5p mimics were promoted miR-515-5p expression, and the miR-515-5p inhibitor reduced miR-515-5p expression. (C) The direct action of miR-515-5p with 3′-UTR of WISP1. * P<0.05 vs. the control, ^# P<0.05 vs. blank control (one-way analysis of variance).

Figure 2

microRNA-515-5p (miR-515-5p) mimics promoted cell proliferation. * P<0.05 (one-way analysis of variance).

Figure 3

microRNA-515-5p (miR-515-5p) mimics reduced apoptosis, while the miR-515-5p inhibitor promoted apoptosis. * P<0.05; ^# P<0.05 (one-way analysis of variance).

Figure 4

microRNA-515-5p (miR-515-5p) mimics promoted, while the miR-515-5p inhibitor reduced the cell cycle. * P<0.05 vs. the control; ^# P<0.05 vs. the miR-515-5p mimics (one-way analysis of variance).

Figure 5

The microRNA-515-5p (miR-515-5p) mimics reduced the expression of TLR4, WISP1, and JNK, while the miR-515-5p inhibitor promoted the expression of TLR4, WISP1, and JNK at the mRNA level. (A) TLR4. (B) WISP1. (C) JNK. The relative expression levels of the target genes were obtained according to the objective internal reference. * P<0.05; ^# P<0.05 (one-way analysis of variance).

Figure 6

The effects of microRNA-515-5p (miR-515-5p) mimics and the miR-515-5p inhibitor on the expression of AKT, JNK, TLR4, and WISP1. (A) Representative blots. (B) Quantification data of AKT and p-AKT. (C) Quantification data of JNK and p-JNK. (D) Quantification data of TLR4 and WISP1. * P<0.05; ^# P<0.05 (one-way analysis of variance).