MicroRNA-33 suppresses CCL2 expression in chondrocytes

Abstract

CCL2-mediated macrophage infiltration in articular tissues plays a pivotal role in the development of the osteoarthritis (OA). miRNAs regulate the onset and progression of diseases via controlling the expression of a series of genes. How the CCL2 gene was regulated by miRNAs was still not fully elucidated. In the present study, we demonstrated that the binding sites of miR-33 in the 3'UTR of CCL2 gene were conserved in human, mouse and rat species. By performing gain- or loss-of-function studies, we verified that miR-33 suppressed CCL2 expression in the mRNA and protein levels. We also found that miR-33 suppressed the CCL2 levels in the supernatant of cultured primary mouse chondrocytes. With reporter gene assay, we demonstrated that miR-33 targeted at AAUGCA in the 3'UTR of CCL2 gene. In transwell migration assays, we demonstrated that the conditional medium (CM) from miR-33 deficient chondrocytes potentiated the monocyte chemotaxis in a CCL2 dependent manner. Finally, we demonstrated that the level of miR-33 was decreased, whereas the CCL2 level was increased in the articular cartilage from the OA patients compared with the control group. In summary, we identified miR-33 as a novel suppressor of CCL2 in chondrocytes. The miR-33/CCL2 axis in chondrocytes regulates monocyte chemotaxis, providing a potential mechanism of macrophage infiltration in OA.

Keywords: CCL2; chemotaxis; microRNA-124 (miR-124); microRNA-33 (miR-33); osteoarthritis; reporter gene.

Figure 1. Prediction of potential miRNAs targeting at the 3′UTR of CCL2 gene

(A and B) The potential target sites of miR-124 (A) or miR-33 (B) in the 3′UTR of CCL2 gene were conserved in human, mouse and rat species. The potential miRNAs targeting at the 3′UTR of CCL2 genes in different species were predicted according to an online software. The conserved miRNAs were picked out.

Figure 2. miR-33 is a regulator of CCL2 expression

(A) The relative mRNA levels of CCL2 in the primary mouse chondrocytes transfected with a scramble miRNA (100 nM) or an anti-miRNA of miR-33 (100 nM) (n=4, **P<0.01). (B) Immunoblotting assay of CCL2 protein in the chondrocytes described in (A). (C) CCL2 levels in the supernatant of the chondrocytes described in (A) (n=4, **P<0.01). (D) The relative miR-33 levels in the primary mouse chondrocytes transfected with a scramble miRNA (100 nM) or a miR-33 mimic (100 nM) (n=4, ***P<0.005). (E) The relative mRNA levels of CCL2 in the primary mouse chondrocytes treated with a scramble miRNA (100 nM) or a miR-33 mimic (100 nM) (n=4, **P<0.01). (F) Immunoblotting assay of CCL2 protein in the chondrocytes described in (E). (G) CCL2 levels in the supernatant of the chondrocytes described in (E) (n=4, **P<0.01).

Figure 3. miR-33 suppresses CCL2 expression via targeting at the 3′UTR

(A) The schematic diagram of potential binding sites (79–98) for miR-33 in the 3′UTR of mouse CCL2 gene. (B) Relative luciferase activity of the chondrocytes transfected with a scramble miRNA (100 nM) or an anti-miRNA of miR-33 (100 nM) plus the miRNA reporter plasmid (0.5 μg/ml) harbouring the 3′UTR of mouse CCL2 gene (n=3, **P<0.01). (C) Relative luciferase activity of the chondrocytes transfected with a scramble miRNA (100 nM) or a mimic of miR-33 (100 nM) plus the miRNA reporter plasmid (0.5 μg/ml) harbouring the 3′UTR of mouse CCL2 gene (n=3, **P<0.01). (D) The potential binding sites AAUGCA (wt) of miR-33 in the 3′UTR of CCL2 gene was mutated as ACCGAA (mut). The 3′UTRs containing the wt or mut sites were subcloned into a miRNA reporter vector. (E) Relative luciferase activity of the chondrocytes transfected with a scramble miRNA (100 nM) or an anti-miRNA of miR-33 (100 nM) plus the wt or mut miRNA reporter gene (n=3, **P<0.01). (F) Relative luciferase activity of the chondrocytes transfected with a scramble miRNA (100 nM) or miR-33 mimic (100 nM) plus the wt or mut miRNA reporter gene (n=3, **P<0.01).

Figure 4. The miR-33/CCL2 axis in chondrocytes regulates the monocyte chemotaxis

The primary chondrocytes were transfected with a scramble miRNA (100 nM), miR-33 mimic (100 nM) or anti-miR-33 (100 nM) for 36 h. Then, the supernatant was collected as CM for the chemotaxis test of monocytes in the transwell migration assays. The CCL2 antibody (anti-CCL2, 0.1 μg/ml) was used to block the effect of CCL2 in the medium. The representative images were displayed and the relative migration rate was calculated (n=6, *P<0.05 and **P<0.01).

Figure 5. Decreased miR-33 levels and elevated CCL2 levels in the cartilage of OA patients

(A) Relative miR-33 levels in the cartilages of OA patients compared with the ones of traumatic patients (control) (n=6–10, *P<0.05). (B) Relative CCL2 mRNA levels in the samples described in (A) (n=6–10, *P<0.05). (C) Relative CD-68 mRNA levels in the samples described in (A) (n=6–10, *P<0.05). (D) Relative IL-1β mRNA levels in the samples described in (A) (n=6–10, **P<0.01).