LncRNA MEG3 Inhibits the Degradation of the Extracellular Matrix of Chondrocytes in Osteoarthritis via Targeting miR-93/TGFBR2 Axis

Abstract

Background: As a degenerative joint disease, osteoarthritis (OA) is characterized by articular cartilage degradation. Long noncoding RNAs (lncRNAs) act critical roles in the regulation of OA development, including affecting the proliferation, apoptosis, extracellular matrix (ECM) degradation, and inflammatory response of chondrocytes. The current study's aim was to investigate the regulatory function and the underlying molecular mechanism of lncRNA MEG3 in ECM degradation of chondrocytes in OA.

Methods: In the current study, chondrocytes were induced by interleukin-1β (IL-1β) to simulate OA condition, and further assessed cell viability, lncRNA MEG3 and miR-93 expression levels. Overexpression or knockdown of lncRNA MEG3 in chondrocytes treated with IL-1β were performed to investigate the function of MEG3 in regulating cell proliferation, apoptosis and ECM degradation using EdU assay, flow cytometry, quantitative reverse transcription polymerase chain reaction (qRT-PCR), and Western blot. The interaction between MEG3 and miR-93 was assessed using qRT-PCR. Furthermore, overexpression of miR-93 was performed as recovery experiment to explore the functional mechanism of MEG3.

Results: MEG3 was significantly downregulated in chondrocytes treated with IL-1β, whereas miR-93 was upregulated concomitantly. Overexpression of MEG3 induced the proliferation, suppressed the apoptosis, and relieved the degradation of ECM in IL-1β-induced chondrocytes. By contrast, knockdown of MEG3 suppressed the proliferation, promoted the apoptosis, and aggravated ECM degradation in IL-1β induced chondrocytes. In addition, MEG3 was found to relieve the inhibitive expression of TGFBR2 as a competitive endogenous RNA (ceRNA) of miR-93, and then activated transforming growth factor-β (TGF-β) signaling pathway, regulated chondrocytes ECM degradation in IL-1β induced chondrocytes subsequently.

Conclusion: LncRNA MEG3 targeted miR-93/TGFBR2 axis, regulated the proliferation, apoptosis and ECM degradation of chondrocytes in OA.

Keywords: extracellular matrix degradation; lncRNA MEG3; miR-93/TGFBR2; osteoarthritis.

Figure 1.

The expression of lncRNA MEG3 and miR-93 in interleukin-1β (IL-1β)-induced chondrocytes. (A) Plots of the cell viability of chondrocytes treated with IL-1β at 0, 5, 10, or 15 ng/mL concentration for 48 hours. Cell viability was assessed by MTT assay. (B) Representative images and plots of flow cytometry analysis of chondrocytes apoptosis rate with IL-1β treatment at 0, 5, 10, or 15 ng/mL concentration for 48 hours. (C and D) qRT-PCR and Western blot analysis of relative mRNA and protein expressions of MMP-13, ADAMTS-5, and COL2A1 in chondrocytes treated with IL-1β at 0, 5, 10, or 15 ng/mL concentration for 48 hours. (E) qRT-PCR analysis of relative expression of MEG3 and miR-93 in chondrocytes treated with IL-1β at 10 ng/mL concentration for 48 hours. *P < 0.05; **P < 0.01.

Figure 2.

The effect of MEG3 overexpression on the proliferation, apoptosis, and extracellular matrix (ECM) degradation of chondrocytes. (A) Representative images of green fluorescent protein (GFP) expression of chondrocytes transfected with empty vector. (B) Representative images and plots of proliferation rate of chondrocytes with interleukin-1β (IL-1β) treatment and transfection with MEG3. (C) Representative images and plots of flow cytometry analysis of the apoptosis rate of chondrocytes with IL-1β and transfection with MEG3. (D and E) qRT-PCR and Western blot analysis of relative mRNA and protein expressions of MMP-13, ADAMTS-5, and COL2A1 in chondrocytes with IL-1β treatment only, and transfection with MEG3. *P < 0.05; **P < 0.01.

Figure 3.

The effect of MEG3 knockdown on the proliferation, apoptosis, and extracellular matrix (ECM) degradation of chondrocytes. (A) Representative images and plots of proliferation rate of chondrocytes with interleukin-1β (IL-1β) treatment and transfection with si-MEG3. (B) Representative images and plots of flow cytometry analysis of the apoptosis rate of chondrocytes with IL-1β treatment and transfection with si-MEG3. (C and D) qRT-PCR and Western blot analysis of relative mRNA and protein expressions of MMP-13, ADAMTS-5, and COL2A1 in chondrocytes with IL-1β treatment and transfection with si-MEG3. *P < 0.05; **P < 0.01.

Figure 4.

The regulatory effect of MEG3 and miR-93 on transforming growth factor β receptor type II (TGFBR2) expression in interleukin-1β (IL-1β)-induced chondrocytes. (A) qRT-PCR analysis of expression of miR-93 in IL-1β-induced chondrocytes transfected with MEG3 and si-MEG3. (B) qRT-PCR and Western blot analysis of mRNA and protein expression of TGFBR2 in IL-1β-induced chondrocytes transfected with MEG3 and si-MEG3. (C) qRT-PCR analysis of relative mRNA expression of MEG3 in IL-1β-induced chondrocytes transfected with miR-93 mimic and inhibitor. (D) qRT-PCR and Western blot analysis of mRNA and protein expression of TGFBR2 in IL-1β-induced chondrocytes transfected with miR-93 mimic and inhibitor. *P < 0.05; **P < 0.01.

Figure 5.

MEG3 regulated extracellular matrix (ECM) degradation in osteoarthritis (OA) via miR-93 and transforming growth factor β receptor type II (TGFBR2). (A) Representative images and plots of proliferation rate of interleukin-1β (IL-1β)-induced chondrocytes with treatment of MEG3 in combination of miR-93. (B) Representative images and plots of flow cytometry analysis of the apoptosis rate of IL-1β-induced chondrocytes with treatment of MEG3 in combination with miR-93. (C) Western blot analysis of protein expression level of MMP-13, ADAMTS-5, TGFBR2, and COL2A1 in IL-1β-induced chondrocytes with treatment of MEG3 in combination of miR-93. (D) Representative images of immunofluorescence analysis of the expression of MMP-13 and COL2A1 in IL-1β-induced chondrocytes with treatment of MEG3 in combination of miR-93. *P < 0.05; **P < 0.01.