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Mechanistic study of miR‑369‑3p in regulating the Wnt/β‑catenin signaling pathway via targeting SPTBN1 in inflammatory response and bone destruction of rheumatoid arthritis

  • Authors:
    • Aihong Wu
    • Yuan Wang
    • Feifei Liu
    • Zhoufang Cao
    • Shuhui Du
    • Mengyu Sun
  • View Affiliations / Copyright

    Affiliations: Department of Rheumatology, Anhui Provincial Hospital of Traditional Chinese Medicine, The First Clinical Medical College of Anhui University, Hefei, Anhui 230038, P.R. China, Anhui Provincial Key Laboratory for Applied Basic and Clinical Translational Research on Rheumatological Diseases in Traditional Chinese Medicine, The First Affiliated Hospital of Anhui University of Chinese Medicine, Hefei, Anhui 230031, P.R. China
    Copyright: © Wu et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 72
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    Published online on: December 18, 2025
       https://doi.org/10.3892/mmr.2025.13782
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Abstract

Aberrant expression of microRNAs (miRNAs) has been closely linked to the progression of rheumatoid arthritis (RA). The present study explored the potential role of miR‑369‑3p in regulating immune‑driven inflammation and bone degradation in RA through the spectrin β, non‑erythrocytic 1 (SPTBN1)/Wnt/β‑catenin signaling cascade. To test this, synthetic mimics and inhibitors of miR‑369‑3p were generated and transfected into RA fibroblast‑like synoviocytes (RA‑FLSs). A pathological model was established by co‑culturing RA‑FLSs with peripheral blood mononuclear cells (PBMCs). The influence of miR‑369‑3p overexpression or suppression on RA‑FLS behavior was assessed in terms of cell survival, cell cycle distribution, proliferation and migratory capacity. Bioinformatics predictions together with luciferase reporter assays confirmed the direct interaction between miR‑369‑3p and SPTBN1. Expression levels of inflammatory cytokines, bone metabolism markers and matrix metalloproteinases were measured by ELISA, while reverse transcription‑quantitative PCR and western blotting were employed to evaluate alterations in the miR‑369‑3p/SPTBN1/Wnt/β‑catenin pathway. The results showed that miR‑369‑3p expression was markedly reduced in the PBMC‑induced RA‑FLS model. Transfection with miR‑369‑3p mimics suppressed the viability and proliferation of RA‑FLS and decreased the expression of SPTBN1, Wnt ligands and β‑catenin mRNA. By comparison, inhibition of miR‑369‑3p produced opposite effects. ELISA findings demonstrated that the miR‑369‑3p/SPTBN1 pathway modulated critical inflammatory and bone‑related markers, which were consistently confirmed across replicate experiments. These results suggested that miR‑369‑3p regulates RA pathology by targeting the SPTBN1/Wnt/β‑catenin pathway, attenuating inflammatory responses and limiting bone destruction in RA.
View Figures

Figure 1

Double-luciferase reporter gene
assay. (A) Schematic diagram of the binding site of miR-369-3p and
SPTBN1. (B) Dual-luciferase reporter assay for the interaction
between miR-369-3p and SPTBN1. (n=3). ***P<0.001; ns, not
significant. miR, microRNA; SPTBN1, spectrin β, non-erythrocytic 1;
WT, wild-type; Mut, mutant.

Figure 2

Screening for the optimal ratio and
time of RA-FLSs and RA-PBMCs and determination of inflammatory
cytokines expression levels in the RA-FLSs group and the co-culture
group. (A) Optimization of the co-culture ratio and duration for
RA-FLSs and RA-PBMCs (1:3, 48 h).(n=6). **P<0.01. (B) Expression
profiles of inflammatory cytokines (IL-17: pg/ml; IL-1β: pg/ml) in
RA-FLSs monocultures and co-culture systems (n=6). **P<0.01. RA,
rheumatoid arthritis; FLSs, fibroblast-like synoviocytes; PBMCs,
peripheral blood mononuclear cells.

Figure 3

miR-369-3p editing affects cell
viability and cycle. (A) miR-369-3p expression across groups (n=6).
***P<0.001, ****P<0.0001. (B) Cell viability in each group
(n=6). ****P<0.0001. (C) Percentage of cell cycle in each group
(n=3). *P<0.05, **P<0.01, ****P<0.0001. Cell cycle
histograms of (D) control group, (E): model group, (F) model mimics
group, (G) model mimics-NC group, (H) model inhibitor group and (I)
model inhibitor-NC group. miR, microRNA; NC, negative control.

Figure 4

The influence of miR-369-3p on
cellular proliferation and migratory activity in each group. (A)
Relative fluorescence ratio of EdU-positive cells in each group
detected by EdU assay. (B) Fluorescence images of EdU staining in
each group (EdU: green; DAPI: blue; Merged: overlay; magnification,
×200). (C) Cell migration rate histogram of each group detected by
scratch-wound healing assay (magnification, ×100; n=3).
****P<0.0001. (D) Representative images of scratch-wound healing
assay in each group at 0 and 24 h (magnification, ×100; n=3).

Figure 5

The effect of miR-369-3p on the
expression levels of SPTBN1, Wnt and β-catenin genes. (A)
Expression levels of the SPTBN1 gene in each group of cells (n=6).
***P<0.001, ****P<0.0001. (B) The expression levels of the
Wnt gene in cells from each group (n=6). **P<0.01,
****P<0.0001. (C) The expression levels of the β-catenin gene in
cells from each group (n=6). **P<0.01, ****P<0.0001. miR,
microRNA; NC, negative control.

Figure 6

miR-369-3p modulates the expression
levels of MMPs and inflammatory cytokines. ELISA analysis of (A)
MMP-1 (ng/ml) and (B) MMP-3 (ng/ml) expression levels in each group
(n=6). ****P<0.0001. Expression levels of inflammatory cytokines
(C) IL-17, (D) IL-1β, (E) IL-4 and (F) IL-10; pg/ml in each group
(n=6). ****P<0.0001. **P<0.01. miR, microRNA; MMP, matrix
metalloproteinases; IL, interleukin.

Figure 7

miR-369-3p regulates RA bone
destruction and the expression of the SPTBN1/Wnt/β-catenin pathway.
ELISA employed for the determination of (A) OPG (pg/ml), (B) DKK1
(ng/ml), (C) RANK (pg/ml) and (D) RANKL (pg/ml) expression in all
cell groups. (n=6). ****P<0.0001. ***P<0.001. (E-H) Western
blot employed for the determination of SPTBN1, Wnt and β-catenin
protein expression in all cell groups (n=3). ****P<0.0001.
***P<0.001. miR, microRNA; RA, rheumatoid arthritis; SPTBN1,
spectrin β, non-erythrocytic 1; OPG, osteoprotegerin; DKK1,
Dickkopf-related protein 1; RANK, receptor activator of nuclear
factor κ B; RANKL, RANK ligand.

Figure 8

Schematic diagram of the mechanism
through which miR-369-3p regulates inflammation and bone
destruction via SPTBN1/Wnt/β-catenin pathway. miR, microRNA;
SPTBN1, spectrin β, non-erythrocytic 1; MMP, matrix
metalloproteinases; IL, interleukin; DKK1, Dickkopf-related protein
1; RANK, receptor activator of nuclear factor κ B; RANKL, RANK
ligand; LRP, low-Density Lipoprotein Receptor-Related Protein; TCF,
T-Cell Factor; LEF, Lymphoid Enhancer-Binding Factor.
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Copy and paste a formatted citation
Spandidos Publications style
Wu A, Wang Y, Liu F, Cao Z, Du S and Sun M: Mechanistic study of miR‑369‑3p in regulating the Wnt/&beta;‑catenin signaling pathway via targeting SPTBN1 in inflammatory response and bone destruction of rheumatoid arthritis. Mol Med Rep 33: 72, 2026.
APA
Wu, A., Wang, Y., Liu, F., Cao, Z., Du, S., & Sun, M. (2026). Mechanistic study of miR‑369‑3p in regulating the Wnt/&beta;‑catenin signaling pathway via targeting SPTBN1 in inflammatory response and bone destruction of rheumatoid arthritis. Molecular Medicine Reports, 33, 72. https://doi.org/10.3892/mmr.2025.13782
MLA
Wu, A., Wang, Y., Liu, F., Cao, Z., Du, S., Sun, M."Mechanistic study of miR‑369‑3p in regulating the Wnt/&beta;‑catenin signaling pathway via targeting SPTBN1 in inflammatory response and bone destruction of rheumatoid arthritis". Molecular Medicine Reports 33.2 (2026): 72.
Chicago
Wu, A., Wang, Y., Liu, F., Cao, Z., Du, S., Sun, M."Mechanistic study of miR‑369‑3p in regulating the Wnt/&beta;‑catenin signaling pathway via targeting SPTBN1 in inflammatory response and bone destruction of rheumatoid arthritis". Molecular Medicine Reports 33, no. 2 (2026): 72. https://doi.org/10.3892/mmr.2025.13782
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Spandidos Publications style
Wu A, Wang Y, Liu F, Cao Z, Du S and Sun M: Mechanistic study of miR‑369‑3p in regulating the Wnt/&beta;‑catenin signaling pathway via targeting SPTBN1 in inflammatory response and bone destruction of rheumatoid arthritis. Mol Med Rep 33: 72, 2026.
APA
Wu, A., Wang, Y., Liu, F., Cao, Z., Du, S., & Sun, M. (2026). Mechanistic study of miR‑369‑3p in regulating the Wnt/&beta;‑catenin signaling pathway via targeting SPTBN1 in inflammatory response and bone destruction of rheumatoid arthritis. Molecular Medicine Reports, 33, 72. https://doi.org/10.3892/mmr.2025.13782
MLA
Wu, A., Wang, Y., Liu, F., Cao, Z., Du, S., Sun, M."Mechanistic study of miR‑369‑3p in regulating the Wnt/&beta;‑catenin signaling pathway via targeting SPTBN1 in inflammatory response and bone destruction of rheumatoid arthritis". Molecular Medicine Reports 33.2 (2026): 72.
Chicago
Wu, A., Wang, Y., Liu, F., Cao, Z., Du, S., Sun, M."Mechanistic study of miR‑369‑3p in regulating the Wnt/&beta;‑catenin signaling pathway via targeting SPTBN1 in inflammatory response and bone destruction of rheumatoid arthritis". Molecular Medicine Reports 33, no. 2 (2026): 72. https://doi.org/10.3892/mmr.2025.13782
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