Circ-NCX1 inhibits LPS-induced chondrocyte apoptosis by regulating the miR-133a/SIRT1 axis

Abstract

Osteoarthritis (OA) is a degenerative joint disease, which is characterized by the degeneration of articular cartilage, thickening of subchondral bone, and inflammation of the synovial membrane. In this study, we aimed to investigate the effects and underlying mechanisms of circ-NCX1 in lipopolysaccharide (LPS)-induced injury in SW1353 chondrocytes, an in vitro model of OA. The levels of circ-NCX1, miR-133a, and related apoptotic proteins were determined by RT-qPCR. MTT assay was used to evaluate the cell viability. The apoptosis was determined by flow cytometry, whereas the expression of apoptosis proteins was detected by Western blot. Immunofluorescence was used to detect cleaved caspase-3 expression in cells. Luciferase reporter assay was used to verify the interaction between circ-NCX1 and miR-133a, and between miR-133a and Silent information regulator 2 homolog 1 (Sirt1). The results showed that the overexpression of circ-NCX1 significantly upregulated the chondrocyte viability and proliferation, and alleviated apoptosis in LPS-induced SW1353 cells. Immunofluorescence results showed that the overexpression of circ-NCX1 significantly reduced expression of LPS-stimulated cleaved Caspase-3. The RT-qPCR results showed that the overexpression of circ-NCX1 inhibited mRNA levels of cleaved Caspase-3 and Bax, and promoted mRNA levels of Bcl-2. Luciferase reporter assay showed that circ-NCX1 targeted miR-133a, and miR-133a directly targeted the Sirt1. In addition, overexpression of circ-NCX1 inhibited chondrocyte apoptosis and promoted Akt phosphorylation via the miR-133a/Sirt1 axis in LPS-induced chondrocytes. In conclusion, circ-NCX1 may serve as an important regulator of LPS-induced chondrocyte apoptosis through the miR-133a/Sirt1 axis, and may be involved in the development of OA.

Keywords: Sirt1; chondrocyte apoptosis; circ-NCX1; miR-133a; osteoarthritis.

FIGURE 1

Effects of circNCX1 expression on LPS‐induced chondrocytes activity. (A) The expression levels of circNCX1 in chondrocytes from different groups were detected by RT‐qPCR. (B) MTT method was used to determine the cell viability of chondrocytes induced by LPS cells 24, 48, and 72 h after transfection of circNCX1. (C) Cell cloning method was used to determine the proliferation of chondrocytes induced by LPS cells after transfection of circNCX1. Results are expressed as mean ± SD, **p < 0.01, ***p < 0.001

FIGURE 2

Effects of circNCX1 expression on apoptosis of chondrocytes induced by LPS. (A) The effect of overexpression of circNCX1 on chondrocyte apoptosis was detected by flow cytometry. (B) The expressions of apoptosis markers cleaved caspase3, Bax and Bcl‐2 in LPS‐induced chondrocytes were detected by RT‐qPCR and Western blot. (C) The expressions of apoptosis markers cleaved caspase3 based on immunofluorescence staining. (D) The expressions of apoptosis markers cleaved caspase3, Bax and Bcl‐2 in LPS‐induced chondrocytes were detected by RT‐qPCR. Results are expressed as mean ± SD, **p < 0.01, ***p < 0.001

FIGURE 3

CircNCX1 targets miR‐133a, and Sirt1 targets miR‐133a. (A) The effect of overexpression of circNCX1 on miR‐133a was detected by RT‐qPCR. (B) Sirt1 levels were detected by Western blot in chondrocytes. (C and D) CircNCX1 targets miR‐133a and Sirt1 targets miR‐133a, based on luciferase reporter assay in SW1353 cells. Results are expressed as mean ± SD, **p < 0.01, ***p < 0.001

FIGURE 4

CircNCX1 regulates chondrocyte apoptosis through the miR‐133a/Sirt1 axis. (A) Expression of miR‐133a was detected by RT‐qPCR. (B) Expression of Sirt1 was detected after overexpression of miR‐133a and circNCX1. (C) The expressions of apoptosis markers cleaved caspase3 based on immunofluorescence staining. (D) Cell viability was detected after overexpression of miR‐133a and circNCX1. (E) Cell apoptosis was detected after overexpression of miR‐133a and circNCX1. Results are expressed as mean ± SD, **p < 0.01, ***p < 0.001

FIGURE 5

CircNCX1 regulated the PI3K/AKT pathway and apoptosis in LPS‐induced chondrocytes. (A) Phosphor‐Akt, Akt, Bcl‐2, Bax and cleaved caspase3 in LPS induced chondrocytes were detected by western blot. (B) The cell apoptosis in LPS induced chondrocytes was detected by flow cytometry. Results are expressed as mean ± SD, **p < 0.01, ***p < 0.001