miR-373 regulates inflammatory cytokine-mediated chondrocyte proliferation in osteoarthritis by targeting the P2X7 receptor

Abstract

Inflammatory cytokines commonly initiate extreme changes in the synovium and cartilage microenvironment of osteoarthritis (OA) patients, which subsequently cause cellular dysfunction, especially in chondrocytes. It has been reported that induction of the purinergic P2X7 receptor (P2X7R) can regulate the expression of a variety of inflammatory factors, including interleukin (IL)-6 and -8, leading to OA pathogenesis. However, knowledge of the mechanism of upregulation of P2X7R in OA is still incomplete, and its role in chondrocyte proliferation is also not clear. It was reported previously that the expression of P2X7R was controlled by certain microRNAs, and so we tested the expression of several microRNAs and found that microRNA-373 (miR-373) was downregulated in the chondrocytes from OA patients. Regarding the mechanism of action, miR-373 inhibited chondrocyte proliferation by suppressing the expression of P2X7R, as well as inflammatory factors such as IL-6 and IL-8. Furthermore, the proliferative and pro-inflammatory effects of miR-373 on the chondrocytes could be suppressed by a P2X7R antagonist, further suggesting that miR-373 mediates chondrocyte proliferation and inflammation by targeting P2X7R. Generally, our results suggest a novel method for OA treatment by targeting the miR-373-P2X7R pathway.

Keywords: chondrocyte; miR‐373; osteoarthritis; purinergic P2X7 receptor.

Figure 1

Expression of miR‐373 is suppressed in OA patients. (A) Analysis of error distribution in scatter plot of normalized ΔC _t values of miRNAs in plasma of healthy participants (n = 12) and OA patients (n = 11). (B) Analysis of miR‐373 expression in chondrocytes from healthy participants and OA patients by RT‐PCR. (C) Proliferation of chondrocytes in healthy participants and OA patients was determined by MTT assay at indicated time points. (D) Proliferation of chondrocytes from healthy participants and OA patients was determined by Brdu assay at indicated time points. Data are presented as mean ± SD from three independent experiments. *P < 0.05, **P < 0.01 compared with the control group.

Figure 2

miR‐373 negatively regulates chondrocyte proliferation. (A) The suppression effect of miRNAi on its mRNA expression was determined by RT‐PCR. (B) Chondrocytes in OA patients were treated with inhibitor or precursor of miR‐373. The proliferation rate was determined by MTT assay at indicated time points. (C) Chondrocytes were treated as in (B) and the proliferation rate was determined by Brdu assay at indicated time points. (D) Chondrocytes were treated as in (B) and IL‐6 and IL‐8 expression was determined by RT‐PCR. (E) Chondrocytes were treated as in (B) and the expression of P2X7R was determined by RT‐PCR. (F) Chondrocytes were treated as in (B) and the expression of P2X7R was determined by western blot. Data are presented as mean ± SD from three independent experiments. *P < 0.05, **P < 0.01 compared with the control group.

Figure 3

Expression level of P2X7R is negatively correlated with miR‐373. (A) P2X7R expression in chondrocytes of OA patients was determined by RT‐PCR, and normalized with that from healthy participants. (B) Correlation between P2X7R and miR‐373 was analyzed by Spearman's rank correlation analysis. Data are presented as mean ± SD from three independent experiments. *P < 0.05 compared with the control group.

Figure 4

Knockdown of P2X7R inhibits the proliferation of chondrocytes. (A) Knockdown effect of P2X7R siRNA was verified by western blot. (B) Chondrocytes from OA patient were transfected with P2X7R siRNA. The proliferation rate was determined with an MTT assay at indicated time points. (C) Chondrocytes were treated as in (B), and the proliferation rate was determined by Brdu assay at indicated time points. (D) Chondrocytes were treated as in (B) and IL‐6 and IL‐8 expression as mRNA level was determined by RT‐PCR. (E) Chondrocytes were treated as in (B) and the secretion of IL‐6 and IL‐8 was determined by ELISA. (F) Chondrocytes from OA patient were transfected with P2X7R siRNA and treated with 100 ng·mL⁻¹ IL‐6 or 100 ng·mL ⁻¹ IL‐8. The proliferation rate was determined by MTT assay at indicated time points. Data are presented as mean ± SD from three independent experiments. *P < 0.05 compared with the control group.

Figure 5

P2X7R antagonist reverses the inhibition effect of miRNAi on OA proliferation. (A) Chondrocytes of OA patients were treated with miRNAi with or without pretreatment with P2X7R antagonist. The proliferation rate was determined by MTT assay at indicated time points. (B) Chondrocytes were treated as in (A) and the proliferation rate was determined by Brdu assay at indicated time points. (C) Chondrocytes were treated as in (A) and IL‐6 and IL‐8 expression was determined by RT‐PCR. Data are presented as mean ± SD from three independent experiments. *P < 0.05 compared with the control group.