Both microRNA-455-5p and -3p repress hypoxia-inducible factor-2α expression and coordinately regulate cartilage homeostasis

Abstract

Osteoarthritis (OA), the most common aging-related joint disease, is caused by an imbalance between extracellular matrix synthesis and degradation. Here, we discover that both strands of microRNA-455 (miR-455), -5p and -3p, are up-regulated by Sox9, an essential transcription factor for cartilage differentiation and function. Both miR-455-5p and -3p are highly expressed in human chondrocytes from normal articular cartilage and in mouse primary chondrocytes. We generate miR-455 knockout mice, and find that cartilage degeneration mimicking OA and elevated expression of cartilage degeneration-related genes are observed at 6-months-old. Using a cell-based miRNA target screening system, we identify hypoxia-inducible factor-2α (HIF-2α), a catabolic factor for cartilage homeostasis, as a direct target of both miR-455-5p and -3p. In addition, overexpression of both miR-455-5p and -3p protect cartilage degeneration in a mouse OA model, demonstrating their potential therapeutic value. Furthermore, knockdown of HIF-2α in 6-month-old miR-455 knockout cartilage rescues the elevated expression of cartilage degeneration-related genes. These data demonstrate that both strands of a miRNA target the same gene to regulate articular cartilage homeostasis.

Fig. 1. Expressions of miR-455-5p and -3p in chondrocytes.

a Heatmap of miRNA microarray. The microarray data of differentially expressed miRNAs (Signal value: > 1.5-fold and < 0.6-fold change in MOI = 5, and > threefold and < 0.6-fold change in MOI = 20) whose expression changed by adenovirally expressed Sox9 are shown. LacZ-expressing chondrocytes were used as a negative control. b Relative expression of miRNAs in Sox9- or LacZ-expressing adenovirus-infected chondrocytes. The experiment was performed once with three biological replicates (n = 3). Data are represented as the mean ± SEM. ns not significant, *P < 0.05, **P < 0.01, ***P < 0.001. Two-tailed Student’s t test. c Relative expression of both strands of miRNAs in mouse primary chondrocytes. The experiment was performed independently twice with two biological replicates (n = 4). d Relative expression of miR-455s and miR-140 in OA (n = 11, biologically independent samples) or normal articular cartilage (N; n = 10, biologically independent samples). Data are represented as the mean ± SEM. Two-tailed Student’s t test. e Relative expression of pri-miR-455, Col27a1, mature miR-455s, Sox9, Col2a1 and Mmp13 in mouse primary chondrocytes with or without IL-1β stimuli (IL-1β or Ctrl). For miR-455s and Col27a1, the experiment was performed independently twice with two or three biological replicates (n = 5). For other cartilage-related genes, the experiment was performed once with three biological replicates (n = 3). Data are represented as the mean ± SEM. Two-tailed Student’s t test. Source data are provided as a Source Data file.

Fig. 2. miR-455 knockout mice show an OA-like phenotype.

a Genetic deletion in miR-455-deficient mice generated by CRISPR/Cas9. b Representative image of Safranin-O staining of wild-type (WT) and miR-455 knockout (KO) mice (2, 6-month-old). Staining was repeated at least three times with similar results. Scale bars show 200 μm. c The OARSI scores of 2 (n = 10, biologically independent samples) or 6-month-old (n = 12, biologically independent samples) wild-type (WT) and miR-455 knockout (KO) mice (2, 6-month-old). Data are represented as the mean ± SEM. Two-tailed Student’s t test. d Relative mRNA levels of cartilage markers and cartilage degeneration-related genes in knee cartilage of wild-type (WT) and miR-455 knockout (KO) mice (2, 6-month-old). Data are represented as the mean ± SEM, n = 5 biologically independent samples. ns: not significant, *P < 0.05, **P < 0.01. Two-tailed Student’s t test. Source data are provided as a Source Data file.

Fig. 3. Identifying the targets of miR-455s using a reporter library system.

a A schematic model of the reporter library system for screening for miR-455 targets. b Luciferase assays of various reporters in 293FT cells transfected with pcDNA-miR-455 (miR-455) or the empty vector (Ctrl). MCS, pLuc2-KAP-MCS (empty reporter). The assays were performed independently four times in duplicate (n = 8). Data are represented as the mean ± SD. ns not significant, **P < 0.01, ***P < 0.001. Two-tailed Student’s t test. c Relative mRNA levels of identified target candidates in mouse primary chondrocytes transfected with the negative control miRNA mimic (Ctrl), miR-455-5p mimic (miR-455-5p), miR-455-3p mimic (miR-455-3p) or both miR-455-5p and -3p mimics (miR-455-5p/3p). The experiment performed independently three times with two biological replicates (n = 6). Data are represented as the mean ± SEM. ns not significant, *P < 0.05, **P < 0.01, ***P < 0.001. Two-tailed Dunnett’s test. d Luciferase assays of whole and fragmented reporters in 293FT cells transfected with pcDNA-miR-455 (miR-455) or empty vector (Ctrl). Error bars show SD. The assays were performed independently three or four times in duplicate (n = 6–8). Data are represented as the mean ± SD. ns not significant, **P < 0.01, ***P < 0.001. Two-tailed Student’s t test. -: empty reporter, 3U: 3’UTR, CDS: coding region. Source data are provided as a Source Data file.

Fig. 4. EPAS1 is a direct target of both miR-455-5p and -3p.

a The four EPAS1 reporters tested in the luciferase assay. mut sequences with a point mutation, WT wild-type sequences. b Luciferase assays of various EPAS1 reporters in 293FT cells transfected with pcDNA-miR-455 (miR-455) or the empty vector (Ctrl). MCS, pLuc2-KAP-MCS (empty reporter). ns not significant. The assays were performed independently three times in duplicate (n = 6). Data are represented as the mean ± SD. ns not significant, *P < 0.05, **P < 0.01, ***P < 0.001. Two-tailed Tukey–Kramer test. c Representative image of Hif-2α, DAPI staining and bright-field (BF) of wild-type (WT) and miR-455 knockout (KO) articular cartilage (2, 6-month-old). Staining was repeated at least twice with similar results. Scale bars show 100 μm. d The percentage of Hif-2α positive cells in wild-type (WT) and miR-455 knockout (KO) articular cartilage (2, 6-month-old). Data are represented as the mean ± SD, n = 4, biologically independent samples. Two-tailed Student’s t test. e Representative image of Hif-2α and DAPI staining and bright-field view (BF) of 6-mo miR-455 knockout articular cartilage transfected with control mimic (Ctrl) or miR-455-5p and -3p mimics (miR-455). Staining was repeated twice with similar results. Scale bars show 100 μm. f The percentage of Hif-2α positive cells in 6-mo miR-455 knockout articular cartilage transfected with control mimic (Ctrl) or miR-455-5p and -3p mimics (miR-455). Data are represented as the mean ± SD, n = 4. Two-tailed Student’s t test. Source data are provided as a Source Data file.

Fig. 5. Potential therapeutic effect of miR-455s in OA.

a Schedule of treatment using transfection of miR-455s mimic in a surgically induced OA model. The mouse figure is a partial modification of the figure created by Sho Mokuda in the previous paper. b Representative image of Safranin-O staining. Staining was repeated at least twice with similar results. Scale bars show 200 μm. c OARSI scores. Data are represented as the mean ± SEM, n = 12, biologically independent samples. One-tailed Dunnett’s test. Ctrl: control, 5p: miR-455-5p, 3p: miR-455-3p, 5p/3p: miR-455-5p and -3p. d Representative image of Hif-2α or MMP13 staining. Staining was repeated at least twice with similar results. Scale bars show 100 μm. e Percentages of Hif-2α and MMP13-positive cells. Data are represented as the mean ± SD, n = 5, biologically independent samples. Two-tailed Student’s t test. Ctrl: control, 5p/3p: miR-455-5p and -3p. Source data are provided as a Source Data file.

Fig. 6. Knockdown of Epas1 in miR-455 KO knee cartilage rescues the abnormally increased expression of cartilage degeneration-related genes.

a Schedule of treatment using transfection of siRNA for Epas1 in knee cartilage of 6-month-old miR-455 knockout mice. The mouse figure is a partial modification of the figure created by Sho Mokuda in the previous paper. b Relative expression levels of Epas1, Mmp3, Mmp13, Adamts5, and Nos2 in siRNA-transfected knee cartilage of 6-month-old wild-type (WT; n = 6, biologically independent samples) or miR-455 knockout mice (KO; n = 5, biologically independent samples). Data are represented as the mean ± SEM. Two-tailed Student’s t test. siCtrl: negative control siRNA, siEpas1: siRNA for Epas1. Source data are provided as a Source Data file.