CircRNA-MSR Regulates LPS-Induced C28/I2 Chondrocyte Injury through miR-643/MAP2K6 Signaling Pathway

Abstract

Objective: Osteoarthritis (OA) is a degenerative joint disease characterized by deterioration of articular cartilage functions. Previous studies have confirmed the role of circular RNAs (circRNAs) in OA, but the role of mechanical stress-related circRNA (circRNA-MSR) in OA is unknown.

Design: The human chondrocytes C28/I2 were cultured and treated with lipopolysaccharide (LPS) to establish the OA model. The mRNA and protein levels were measured by qRT-PCR or Western blot. Cell viability was analyzed by MTT assay. Flow cytometry was carried out to detect cell apoptosis. The levels of TNF-α, IL-1β, and IL-6 were determined by enzyme-linked immunosorbent assay (ELISA). Pull-down assay was conducted to measure circRNA-MSR-related miRNA. Dual-luciferase reporter gene detection was performed to detect the target relationships between miR-643 and circRNA-MSR or Mitogen-activated protein kinase kinase 6 (MAP2K6). The RNA-fluorescence in situ hybridization (RNA-FISH) assay was conducted to verify the localization of circRNA-MSR and miR-643.

Results: The expressions of circRNA-MSR were upregulated in LPS stimulated C28/I2 cells. Knockdown of circRNA-MSR can inhibit LPS-induced apoptosis, inflammatory response, and extracellular matrix (ECM) degradation, and promote cell C28/I2 cells proliferation. Moreover, circRNA-MSR directly targeted miR-643. RNA-FISH exhibited that circRNA-MSR may act as a competing endogenous RNA (ceRNA) of miR-643. Over-expression of miR-643 could alleviate LPS-induced C28/I2 chondrocyte injury and promote cell proliferation. Besides, miR-643 directly bound to MAP2K6 mRNA. MiR-643 inhibition or MAP2K6 overexpression can reverse the role of circRNA-MSR knockdown on LPS-treated chondrocytes.

Conclusion: circRNA-MSR can upregulate MAP2K6 by targeting miR-643, thereby inhibiting cell proliferation and promoting apoptosis of C28/I2 cells.

Keywords: MAP2K6; circRNA-MSR; miR-643; osteoarthritis.

Figure 1.

CircRNA-MSR level was increased in C28/I2 cells with lipopolysaccharide (LPS) stimulation. (A) Cell viability of C28/I2 cells after LPS induction was measured by MTT assay. (B) Cell apoptosis of C28/I2 cells induced by LPS was determined by flow cytometry. (C) The expression of circRNA-MSR in C28/I2 cells with LPS treatment was measured by quantitative reverse transcription–polymerase chain reaction (qRT-PCR) assay. The measurement data were expressed in term of mean ± standard deviation. *P < 0.05, **P < 0.01. All the above assays were executed for three times. Multiple comparisons were using nonparametric analysis of variance.

Figure 2.

CircRNA-MSR knockdown suppressed lipopolysaccharide (LPS)-induced apoptosis of C28/I2 cells and enhanced cell viability. (A) The expression of circRNA-MSR in C28/I2 cells was measured by quantitative reverse transcription–polymerase chain reaction (qRT-PCR) assay. (B) Cell viability of C28/I2 cells was measured by MTT assay. (C) Cell apoptosis of C28/I2 cells was determined by flow cytometry. (D) The tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-6 levels were detected by enzyme-linked immunosorbent assay (ELISA). (E) The expression levels of Bax, cleaved-caspase-3, matrix metalloproteinase-13 (MMP13), Bcl-2, aggrecan, and Collagen II were measured by Western blot in C28/I2 cells. The measurement data were represented by mean ± standard deviation. *P < 0.05, **P < 0.01, ***P < 0.001. All the above assay were executed independently 3 times. Multiple comparisons were using nonparametric analysis of variance.

Figure 3.

MiR-643 is proved to be the molecular target of circRNA-MSR. (A, B) The expression of miR-643 in C28/I2 cells was measured by quantitative reverse transcription–polymerase chain reaction (qRT-PCR) assay. (C) Pull-down assay was conducted to measure circRNA-MSR-related miRNA through a specific biotin-labeled circRNA-MSR probe. (D) The interaction of circRNA-MSR and miR-643 was detected by dual-luciferase reporter detection. (E) The RNA–fluorescence in situ hybridization (RNA-FISH) assay was conducted to verify the localization of circRNA-MSR and miR-643. The measurement data were represented by mean ± standard deviation. Scale bar = 25 μm. *P < 0.05, **P < 0.01. All the above assay were executed independently 3 times. The results of the analysis shown in C and D was using the Student t test. Multiple comparisons were using nonparametric analysis of variance.

Figure 4.

Overexpression of miR-643 alleviates chondrocyte injury caused by lipopolysaccharide (LPS). (A) The expression of miR-643 in C28/I2 cells was measured by quantitative reverse transcription–polymerase chain reaction (qRT-PCR) assay. (B) Cell viability of C28/I2 cells was measured by MTT assay. (C) Cell apoptosis of C28/I2 cells was determined by flow cytometry. (D) The tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-6 levels were detected by enzyme-linked immunosorbent assay (ELISA). (E) The protein expressions of Bax, cleaved-caspase-3, matrix metalloproteinase-13 (MMP13), Bcl-2, aggrecan, and collagen II were measured by Western blot in C28/I2 cells. The measurement data were represented by mean ± standard deviation. *P < 0.05, **P < 0.01, ***P < 0.001. All the above assays were executed independently 3 times. Multiple comparisons were using nonparametric analysis of variance.

Figure 5.

MiR-643 can target MAP2K6 and inhibit its expression. (A, B) The mRNA and protein expressions of MAP2K6 in C28/I2 cells were detected by quantitative reverse transcription–polymerase chain reaction (qRT-PCR) assay and Western blot. (C, D) The mRNA and protein expressions of MAP2K6 in C28/I2 cells with miR-643 mimic transfection was measured. (E) The interaction between miR-643 and MAP2K6 was detected by dual-luciferase reporter detection. The measurement data were represented by mean ± standard deviation. *P < 0.05, **P < 0.01, ***P < 0.001. All the above assays were executed independently 3 times. Multiple comparisons were using nonparametric analysis of variance.

Figure 6.

circRNA-MSR promotes apoptosis, inflammatory factors secretion and extracellular matrix degradation of chondrocytes through miR-643/MAP2K6 pathway. (A) The mRNA expression of MAP2K6 and miR-643 in C28/I2 cells were detected by quantitative reverse transcription–polymerase chain reaction (qRT-PCR) assay. (B) Cell viability of C28/I2 cells was measured by MTT assay. (C) Cell apoptosis of C28/I2 cells was determined by flow cytometry. (D) The tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-6 levels were detected by enzyme-linked immunosorbent assay (ELISA). (E) The protein expressions of Bax, cleaved caspase-3, MMP13, Bcl-2, aggrecan, and collagen II were measured by Western blot. The measurement data were represented by mean ± standard deviation. *P < 0.05, **P < 0.01. All the above assays were executed independently 3 times. Multiple comparisons were using nonparametric analysis of variance.