miR-140 Attenuates the Progression of Early-Stage Osteoarthritis by Retarding Chondrocyte Senescence

Abstract

Osteoarthritis (OA) is a major cause of joint pain and disability, and chondrocyte senescence is a key pathological process in OA and may be a target of new therapeutics. MicroRNA-140 (miR-140) plays a protective role in OA, but little is known about its epigenetic effect on chondrocyte senescence. In this study, we first validated the features of chondrocyte senescence characterized by increased cell cycle arrest in the G0/G1 phase and the expression of senescence-associated β-galactosidase (SA-βGal), p16^INK4a, p21, p53, and γH2AX in human knee OA. Then, we revealed in interleukin 1β (IL-1β)-induced OA chondrocytes in vitro that pretransfection with miR-140 effectively inhibited the expression of SA-βGal, p16^INK4a, p21, p53, and γH2AX. Furthermore, in vivo results from trauma-induced early-stage OA rats showed that intra-articularly injected miR-140 could rapidly reach the chondrocyte cytoplasm and induce molecular changes similar to the in vitro results, resulting in a noticeable alleviation of OA progression. Finally, bioinformatics analysis predicted the potential targets of miR-140 and a mechanistic network by which miR-140 regulates chondrocyte senescence. Collectively, miR-140 can effectively attenuate the progression of early-stage OA by retarding chondrocyte senescence, contributing new evidence of the involvement of miR-mediated epigenetic regulation of chondrocyte senescence in OA pathogenesis.

Figure 1

Chondrocyte Senescence Correlates with Human OA Pathogenesis (A) Representative micrographs of H&E and Safranin O staining of normal and OA cartilage (scale bars, 200 μm). (B) The modified Mankin score of normal and OA cartilage. (C) Representative micrographs of immunohistochemical staining of p16^INK4a, p21, p53, and γH2AX in normal and OA cartilage (scale bars, 100 μm for p16^INK4a, p21, and p53; 50 μm for γH2AX). (D–G) The percentage of (D) p16^INK4a-, (E) p21-, (F) p53-, and (G) γH2AX-positive chondrocytes in normal and OA cartilage. (H) Percentage of normal and OA cartilage-derived chondrocytes in the G0/G1, S, and G2/M phases of the cell cycle, determined by flow cytometric analysis. (I) Representative micrographs of SA-βGal staining in normal and OA cartilage-derived chondrocytes (scale bars, 200 μm). (J) Percentage of normal and OA cartilage-derived chondrocytes positive for SA-βGal. E-OA, early-stage OA; ML-OA, middle- to late-stage OA. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 2

Pretransfection with miR-140 Inhibits IL-1β-Induced Human Chondrocyte Senescence (A) PCR analysis of the relative expression level of miR-140, 24 h after IL-1β stimulation or transfection with miR-Scr or miR-140 in normal cartilage-derived chondrocytes. (B–F) Percentage of chondrocytes in the G0/G1, S, and G2/M phases of the cell cycle (B), representative micrographs of SA-βGal staining (scale bars, 200 μm) and percentage of SA-βGal-positive chondrocytes (C), gene (D) and protein (E) expression of p16^INK4a, p21, and p53, representative micrographs of γH2AX staining (scale bars, top: 100 μm; bottom: 20 μm), and the percentage of γH2AX-positive chondrocytes (F) after treatment with normal media (Cont), treatment with IL-1β, pretransfection with miR-Scr + IL-1β, or pretransfection with miR-140 + IL-1β. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 3

Intra-articular Injection (IAJ) of miR-140 Attenuates OA Progression in Rats (A) Experimental layout for the in vivo experiments in rats. (B) Representative in vivo fluorescence image 24 h after IAJ of Cy5-labeled miR-140 agomir into the right hind knee and normal saline (NS) injection into the left hind knee. (C) Representative fluorescence micrographs of sagittal frozen sections of the medial femoral condyles 24 h after IAJ of NS into the left hind knee (top) and Cy5-labeled miR-140 agomir into the right hind knee (bottom; 100× scale bars, 200 μm; 400× scale bars, 50 μm). (D) Number of rears, as measured by behavioral testing, before and after IAJ of miR-Scr or miR-140 agomir. (E–K) Gross observation (E), pathological lesion score (F), representative sagittal micrographs of H&E (G), and Safranin O (H) staining (scale bars, 100 μm) and modified Mankin score (I), cartilage thickness (J), and chondrocyte number (K) of the medial femoral condyles before and after IAJ of miR-Scr or miR-140 agomir. ACLT + DMM, anterior cruciate ligament transection (ACLT) and destabilization of the medial meniscus (DMM). *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 4

Intra-articular Injection (IAJ) of miR-140 Retards Chondrocyte Senescence in Rats (A–D) Representative micrographs of immunohistochemical staining and immunopositive percentage of p16^INK4a (A), p21 (B), p53 (C), and γH2AX (D) in sagittal sections of the medial femoral condyles after IAJ of miR-Scr or miR-140 agomir (scale bars, 100 μm for p16^INK4a, p21, and p53; 50 μm for γH2AX). *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 5

Bioinformatics Analyses of the Potential Mechanisms by which miR-140 Regulates Chondrocyte Senescence (A) The number of predicted target genes of hsa-miR-140-5p in the miRDB, miRmap, PicTar, TargetScan, and DIANA microT-CDS databases. (B) Gene Ontology (GO) enrichment analysis for the predicted target genes of hsa-miR-140-5p, showing only the top 10 items in each category; the detailed results are provided in Figure S4. (C) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis for the predicted target genes of hsa-miR-140-5p (p < 0.05). (D and E) Experimental validation of the effect of miR-140 on the proteins encoded by the predicted target genes of hsa-miR-140-5p by western blotting, including JAG1 and NUMBL in the Notch pathway (D) and IGF1R and TLR4 in the PI3K-AKT pathway (E). *p < 0.05, **p < 0.01.

Figure 6

Schematic Diagrams of Chondrocyte Senescence and miR-140-Related Mechanisms in OA Pathogenesis (A) Involvement of chondrocyte senescence in OA pathogenesis. (B) Potential mechanistic network (mainly Notch and PI3K-AKT pathways, which are well known to be associated with OA pathogenesis) by which miR-140 regulates chondrocyte senescence. The red nodes represent the bioinformatically predicted targets of miR-140, and the green nodes represent the validated targets of miR-140 and molecules that can be regulated by miR-140 in this study.