Mir-450a-5p Ameliorates IL-1β-Induced Chondrocyte Apoptosis, Inflammation, and Extracellular Matrix Degradation by Down-Regulating LITAF

Abstract

ObjectiveOsteoarthritis (OA) is a degenerative joint disease characterized by cartilage degradation, causing severe pain and disability. Recent studies suggest that miR-450a-5p may regulate inflammatory pathways in OA. This study aimed to elucidate the role of miR-450a-5p in OA, providing a potential therapeutic target for the clinical treatment.MethodsCartilage tissues were collected from OA patients undergoing knee replacement surgery, and CHON-001 cells were treated with interleukin (IL)-1β to induce an OA model in vitro. Real-time quantitative polymerase chain reaction was used to detect the miR-450a-5p expression, and Western blot determined the lipopolysaccharide-induced tumor necrosis factor (TNF)-α factor (LITAF) expression. The targeting relationship between LITAF and miR-450a-5p was verified by dual-luciferase reporter assay. Cell proliferation and apoptosis were assessed using the Cell Counting Kit-8 assay and flow cytometry, respectively. Levels of IL-6, IL-10, and TNF-α were measured via enzyme-linked immunosorbent assay. In addition, Western blot was employed to detect the expressions of matrix metalloproteinase-3 (MMP-3), collagen III, and aggrecan in extracellular matrix (ECM).ResultsMiR-450a-5p expression was significantly down-regulated in OA tissues and IL-1β-induced CHON-001 cells (~60%), while LITAF expression was markedly increased (~1.8-fold). There was a negative correlation between miR-450a-5p and LITAF in OA tissues (r = -0.596, P < 0.01). MiR-450a-5p directly targeted and inhibited LITAF expression. Its overexpression promoted chondrocyte proliferation, reduced apoptosis and inflammatory cytokines, and mitigated ECM degradation.ConclusionsMiR-450a-5p inhibited LITAF expression, thereby attenuating apoptosis, inflammation, and ECM degradation in chondrocytes. It may serve as a promising therapeutic target for OA.

Keywords: CHON-001; IL-1β; LITAF; chondrocytes; miR-450a-5p; osteoarthritis.

Figure 1.

MiR-450a-5p expression is significantly elevated in cartilage tissues and IL-1β-induced chondrocytes. (A) RT-qPCR was used to detect the miR-450a-5p expression level of cartilage tissues in OA patients and non-OA patients, **P < 0.01, normal group versus OA group. (B) RT-qPCR was performed to measure the miR-450a-5p expression level in IL-1β-treated CHON-001 cells, **P < 0.01. IL-1β = interleukin-1β; OA = osteoarthritis; RT-qPCR = real-time quantitative polymerase chain reaction.

Figure 2.

MiR-450a-5p inhibits IL-1β-induced apoptosis, inflammation, and extracellular matrix degradation in chondrocytes. (A) The mRNA levels of miR-450a-5p were examined by RT-qPCR, **P < 0.01, miR-450a-5p group versus NC mimics group; (B) cell proliferation at 0, 24, 48, and 72 hours was detected by CCK-8 assay. (C) Flow cytometry was performed to assess the apoptosis level of CHON-001 cells; (D) ELISA was used to determine the levels of IL-6, IL-18, and TNF-α; (E) Western blot was utilized to detect the protein expression levels of MMP-3, collagen III, and aggrecan; **P < 0.01, IL-1β group versus control group; ^##P < 0.01, IL-1β + miR-450a-5p group versus IL-1β + NC mimics group. IL-1β = interleukin-1β; ECM = extracellular matrix; RT-qPCR = real-time quantitative polymerase chain reaction; CCK-8 = cell counting kit-8; ELISA = enzyme-linked immunosorbent assay; IL = interleukin; TNF-α = tumor necrosis factor-α; MMP-3 = matrix metalloproteinase-3.

Figure 3.

LITAF serves as a direct target of miR-450a-5p. (A) The target sequences for miR-450a-5p binding to LITAF were predicted; (B) a dual-luciferase reporter assay was used to validate the binding of miR-450a-5p to LITAF, **P < 0.01; (C) RT-qPCR was utilized to detect the LITAF mRNA level in CHON-001 cells; (D) Western blot was adopted to determine the protein level of LITAF in CHON-001 cells, **P < 0.01; (E) the expression level of LITAF was measured after transfection with miR-450a-5p, **P < 0.01; (F) RT-qPCR was used to detect the LITAF mRNA level in cartilage tissues of OA patients, **P < 0.01; (G) the correlation between the expression of miR-450a-5p and LITAF was assessed by the Pearson analysis. LITAF = lipopolysaccharide-induced tumor necrosis factor-α factor; RT-qPCR = real-time quantitative polymerase chain reaction; OA = osteoarthritis.

Figure 4.

LITAF reduces the protective effect of miR-450a-5p on IL-1β-induced chondrocytes. (A) Western blot was used to detect the changes in LITAF protein expression; (B) the proliferation ability of CHON-001 cells was measured by CCK-8; (C–D) the apoptosis level of CHON-001 cells; (E) ELISA was adopted to determine the levels of IL-6, IL-18, and TNF-α; (F–G) Western blot and quantitative statistical analysis for the protein expression levels of MMP-3, collagen III, and aggrecan, **P < 0.01 versus NC mimics + vector group, ^##P < 0.01, versus miR-450a-5p + vector group. TNF-α = tumor necrosis factor-α; LITAF = lipopolysaccharide-induced TNF-α factor; IL = interleukin; CCK-8 = cell counting kit-8; ELISA = enzyme-linked immunosorbent assay; MMP-3 = matrix metalloproteinase-3.

Figure 5.

The diagram of potential mechanism by which miR-450a-5p targets and inhibits LITAF in OA.