Role of lncRNA XIST/miR-146a Axis in Matrix Degradation and Apoptosis of Osteoarthritic Chondrocytes Through Regulation of MMP-13 and BCL2

Abstract

Growing evidence demonstrates the critical roles of long non-coding RNAs (lncRNAs) in osteoarthritis (OA) pathogenesis. The lncRNA XIST is one of the most commonly studied; however, its function remains unclear. This study aimed to research the molecular mechanism of XIST in human OA chondrocytes. Cells were transfected with small interfering RNA against XIST or with a microRNA (miR)-146a inhibitor in the presence of interleukin (IL)-1β. Viability was detected using MTT; apoptosis using cytometry; and XIST, miR-146a, B-cell lymphoma (BCL)2, and metalloproteinase (MMP)-13 expression using real-time PCR. The analysis of p50 and p65 nuclear factor (NF)-κB was conducted using PCR and immunofluorescence. Our findings showed that XIST was highly expressed in OA chondrocytes when compared to T/C-28a2 lines. Furthermore, XIST silencing significantly promoted survival and limited apoptosis, with a concomitant over expression of BCL2, reduction in MMP-13 mRNA, and NF-κB activation after IL-1β stimulus. Conversely, miR-146a was significantly down-regulated in OA cells, while its levels were increased following XIST silencing; moreover, miR-146a inhibition induced opposite results to those caused by XIST. Finally, the down-regulation of XIST was correlated to the over-expression of miR-146a, with a consequent modulation of BCL2, MMP-13, and NF-κB. This study suggests an influence of the XIST/miR-146a axis on the viability, apoptosis, and matrix degradation occurring in OA.

Keywords: apoptosis; chondrocytes; long non-coding RNA; metalloproteinases; microRNA; osteoarthritis.

Figure 1

XIST silencing regulates viability, apoptosis, and ECM degradation. Human osteoarthritic (OA) chondrocytes were analyzed in the basal condition after 24 h of transfection with small interfering RNA (siRNA) against XIST (50 nM) or negative controls (NC) (5 nM) in the presence or not of interleukin (IL)-1β (10 ng/mL). (a) Gene expression of XIST in T/C-28a2 cell line and in OA chondrocytes was assessed using quantitative real-time PCR. (b,c,f,g) Gene expression of XIST, microRNA (miR)-146a, B-cell lymphoma (BCL2), and metalloproteinase (MMP)-13 was assessed using quantitative real-time PCR. (d) Cell viability was evaluated using MTT test. (e) Apoptosis detection by flow cytometry (Annexin Alexa fluor 488 assay). Data were reported as mean ± standard deviation of triplicate values. * p < 0.05, ** p < 0.01 versus NC; ° p < 0.05, °° p < 0.01 versus IL-1β.

Figure 2

Inhibition of miR-146a reverts the effects of XIST in IL-1β-treated chondrocytes. Human osteoarthritic (OA) chondrocytes were analyzed in the basal condition, after transfection (24 h) with microRNA (miR)-146a-specific inhibitor (50 nM) or negative controls (NC) (5 nM), and in the presence or not of interleukin (IL)-1β (10 ng/mL). (a) Gene expression of miR-146a in T/C-28a2 cell line and in OA chondrocytes was assessed using quantitative real-time PCR. (b,c,f,g) Gene expression of microRNA (miR)-146a, XIST, B-cell lymphoma (BCL2), and metalloproteinase (MMP)-13 was analyzed using quantitative real-time PCR. (d) Cell viability was evaluated using MTT assay. (e) Detection of apoptosis via flow cytometry (Annexin Alexa fluor 488 assay). Data were reported as mean ± standard deviation of triplicate values. * p < 0.05, ** p < 0.01, *** p < 0.001 versus NC; ° p < 0.05 versus IL-1β.

Figure 3

XIST/miR-146a axis on the NF-κB signaling pathway. Human osteoarthritic (OA) chondrocytes were analyzed after 24 h of transfection with small interfering RNA (siRNA) against XIST (50 nM) and/or microRNA (miR)-146a-specific inhibitor (50 nM) or negative controls (NC) (5 nM) in the presence or not of interleukin (IL)-1β (10 ng/mL). (a,b) Gene expression of p50 and p65 subunits of nuclear factor (NF)-κB was analyzed using quantitative real-time PCR. Data were reported as mean ± standard deviation of triplicate values. * p < 0.05 versus NC; ° p < 0.05, °° p < 0.01 versus IL-1β; ## p < 0.01 versus si-XIST.

Figure 4

XIST/miR-146a axis of the NF-κB signaling pathway. Human osteoarthritic (OA) chondrocytes were analyzed after 24 h of transfection with small interfering RNA (siRNA) against XIST (50 nM), and/or microRNA (miR)-146a-specific inhibitor (50 nM), or negative controls (NC) (5 nM), and in the presence of interleukin (IL)-1β (10 ng/mL) (3 h). (a–j) Indirect immunofluorescence microscopy of cells incubated with monoclonal anti-p50 (a–e) and anti-p65 (f–j) subunits primary antibodies. Secondary antibody was used: a goat anti-mouse IgG-Texas Red conjugated antibody (red fluorescence). (a,f) CTRL: limited fluorescence is observed in the cytoplasm; (b,g) IL-1β: an intense signal is evident in the cytoplasm; (c,h) si-XIST + IL-1β; the label is limited. (d,i) miR-146a inhibitor + IL-1β: the signal is diffused in the cytoplasm and not reduced compared to IL-1β. (e,j) si-XIST + miR-146a inhibitor + IL-1β: the label is strongly reduced. Nuclei (blue) were stained with DAPI. Bars: 50 µm of A-L.

Figure 5

Direct targeting of XIST and miR-146a. (a–i) The analysis of the linear correlation of XIST, microRNA (miR)-146a, metalloproteinase (MMP)-13, B-cell lymphoma (BCL2), and p50 and p65 subunits of nuclear factor (NF)-κB in human osteoarthritic (OA) chondrocytes was carried out using Spearman’s correlation coefficient.