1. Intra-articular injection of microRNA-140 (miRNA-140) alleviates osteoarthritis (OA) progression by modulating extracellular matrix (ECM) homeostasis in rats 
H.-b. Si, Y. Zeng, S.-y. Liu, Z.-k. Zhou, Y.-n. Chen, J.-q. Cheng, Y.-r. Lu, B. Shen 
Osteoarthritis and Cartilage 
Volume 25, Issue 10, Pages (October 2017)
DOI: /j.joca
Copyright © 2017 Osteoarthritis Research Society International Terms and Conditions

2. Fig. 1
PCR analysis of miRNA-140 level in human chondrocytes and synovial fluid. (A) The relative expression levels of miRNA-140 in normal and OA cartilage-derived chondrocytes. (B) The relative levels of miRNA-140 in synovial fluid obtained from normal and OA knee joint. U6 was used as endogenous control, and the level in the normal group was set to 1. *, Compared with normal group, P < 0.05; #, compared with OA group, P < 0.05.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2017 Osteoarthritis Research Society International Terms and Conditions

3. Fig. 2
Collagen II, MMP-13 and ADAMTS-5 expression in human normal and OA cartilage derived chondrocytes. (A–C) PCR analysis of the relative expression levels of Collagen II, MMP-13 and ADAMTS-5, respectively. β-actin was used as endogenous control, and the expression level in normal group was set to 1. (D–F) Western blot analysis (top) and quantitation (bottom) of Collagen II, MMP-13 and ADAMTS-5 expression, respectively. The expression level was normalised to that of β-actin. *, Compared with normal group, P < 0.05; #, compared with moderate OA group, P < 0.05.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2017 Osteoarthritis Research Society International Terms and Conditions

4. Fig. 3
miRNA-140 regulates Collagen II, MMP-13 and ADAMTS-5 expression in human normal and OA cartilage derived chondrocytes. (A) PCR analysis of the relative expression levels of miRNA-140 after miRNA-140 transfection in normal and OA cartilage derived chondrocytes. U6 was used as endogenous control, and the expression level in the blank group was set to 1. (B–D) PCR analysis of the relative expression levels of Collagen II, MMP-13, and ADAMTS-5, respectively, after miRNA-140 transfection in normal and OA cartilage derived chondrocytes. β-actin was used as endogenous control, and the expression level in the blank group was set to 1. (E–H) Western blot analysis (E) and quantitation of Collagen II (F), MMP-13 (G), and ADAMTS-5 (H) expression after miRNA-140 transfection in normal and OA cartilage derived chondrocytes. The protein expression level was normalised to that of β-actin. *, Compared with blank group, P < 0.05.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2017 Osteoarthritis Research Society International Terms and Conditions

5. Fig. 4
miRNA-140 expression in cartilage and general findings after intra-articular injection of miRNA-140 agomir in OA rats. (A) Relative expression levels of miRNA-140 in rat cartilage after intra-articular injection of miRNA-140 agomir (n = 3 in each group). U6 was used as endogenous control, and the expression level in the normal group was set to 1. (B) The surgical wounds healed well in all the rats, and no instances of oozing, infection, necrosis, or death were observed throughout the observation period. (C) Number of rears, as measured by behavioural testing, and larger numbers of rears were indicative of less pain (n = 6 in each group). (D) Gross observation of the medial femoral condyle after intra-articular injection of miRNA-140 agomir (n = 6 in each group). (E) Comparison of cartilage pathological lesion scores between the miRNA-140 agomir and control groups (n = 6 in each group). *, Compared with control group, P < 0.05.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2017 Osteoarthritis Research Society International Terms and Conditions

6. Fig. 5
Histological staining and assessment after intra-articular injection of miRNA-140 agomir in OA rats (n = 6 in each group). (A, B) HE and toluidine blue staining of sagittal sections of the medial femoral condyle, respectively (200× magnification, scale bar = 50 μm). (C) Cartilage chondrocyte counts, as determined by HE staining, in 400 × 200-μm grids extending from the cartilage surface to the deep zone. (D) Medial femoral condyle cartilage thickness (from the cartilage surface to the deep zone), as determined by toluidine staining. (E) The modified Mankin scores, based on the HE and toluidine blue staining results, and the score represented the most severe histologic changes. In each section, the quantitative analyses were counted at three regions, that is, the anterior, middle and posterior of medial condylar cartilage, respectively, and then averaged. The black box (400 × 200 μm) represents the area used for quantifying cell numbers. The two-way arrow represents the condylar cartilage thickness. *, Compared with control group, P < 0.05.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2017 Osteoarthritis Research Society International Terms and Conditions

7. Fig. 6
Immunohistochemical analyses of Collagen II, MMP-13 and ADAMTS-5 expression after intra-articular injection of miRNA-140 agomir in OA rats (n = 6 in each group). (A–C) Immunohistochemical analysis of Collagen II, MMP-13 and ADAMTS-5 protein expression, respectively, in sagittal sections of the medial femoral condyle (200× magnification, scale bar = 50 μm). (D–F) Quantification of Collagen II-, MMP-13-, and ADAMTS-5-positive cells, respectively, based on the staining results. (G–I) The MOD of Collagen II, MMP-13 and ADAMTS-5 protein expression in cartilage, respectively, based on the staining results. The black box (400 × 200 μm) represents the area used for quantification of the positive cells and OD. In each section, the quantitative analyses were counted at three regions, that is, the anterior, middle and posterior of medial condylar cartilage, respectively, and then averaged. *, Compared with control group, P < 0.05.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
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8. Fig. S1
miRNA-140 expression in human chondrocytes treated with miRNA-140 mimic at different concentrations and for different times. (A) Comparisons of miRNA-140 expression levels between groups treated with different concentrations of miRNA-140 mimic. miRNA-140 expression levels were significantly increased in the treated group compared with the blank group when the concentration of the miRNA-140 mimic was ≥50 nM (n = 3 in each group. *, Compared with blank group, P < 0.05; #, compared with 25 nM group, P < 0.05; &, compared with 50 nM group, P < 0.05). (B) Comparisons of miRNA-140 expression levels between groups at different times after transfection. miRNA-140 expression levels were significantly increased beginning at 24 h after transfection with miRNA-140 mimic at a working concentration ≥50 nM (n = 3 in each group. *, Compared with pre-transfection group, P < 0.05; #, compared with 24 h group, P < 0.05; &, compared with 48 h group, P < 0.05).
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2017 Osteoarthritis Research Society International Terms and Conditions

9. Fig. S2
MMP-13 gene and protein expression levels in chondrocytes treated with miRNA-140 mimic at different concentrations and for different times. (A) Comparisons of MMP-13 gene expression levels between groups treated with different concentrations of miRNA-140 mimic. MMP-13 expression levels were significantly decreased in the treated group compared with the blank group when the concentration of miRNA-140 mimic was ≥50 nM (n = 3 in each group. *, Compared with blank group, P < 0.05; #, compared with 25 nM group, P < 0.05). (B) Comparisons of relative MMP-13 gene expression levels between groups at different times after transfection. MMP-13 gene expression levels were significantly decreased at 48 h after transfection with miRNA-140 mimic at a working concentration ≥50 nM (n = 3 in each group. *, Compared with pre-transfection group, P < 0.05; #, compared with 24 h group, P < 0.05; &, compared with 48 h group, P < 0.05). (C) The western blotting bands for MMP-13 protein expression in groups treated with miRNA-140 at different concentrations and for different times. (D) Quantification of the western blot bands. MMP-13 protein expression levels were significantly decreased at 72 h after transfection with miRNA-140 mimic at a working concentration ≥50 nM (n = 3 in each group. *, Compared with blank group, P < 0.05; #, compared with 25 nM group, P < 0.05).
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2017 Osteoarthritis Research Society International Terms and Conditions

10. Fig. S3
Transfection of miRNA-140 mimic or inhibitor had no significant effects on cell proliferation of normal and OA cartilage derived chondrocytes. Cell proliferation was measured with MTT assay. When the chondrocytes had grown to 50–60% confluence in ninety six-well plates, they were transfected with 50 nM miRNA-140 mimic, 100 nM miRNA-140 inhibitor, or 50 nM non-specific negative oligonucleotide (miRNA control). Blank cells served as controls. After transfection, the cells were treated with 20 μl of MTT (5 mg/ml) every 24 h, after which they were incubated for an additional 4 h. The medium was subsequently removed, and 100 μl of DMSO was added to each well. At 24, 48 and 72 h after transfection, the cell number was evaluated at an absorbance at 490 nm using a microplate reader. The reference wavelength was 630 nm. (A–C) The absorbance in normal (A), middle-stage (B) and late-stage OA (C) cartilage derived chondrocytes, respectively, and no significant differences in cell proliferation were noted among the groups, indicating that transfection of miRNA-140 mimic or inhibitor has no significant effects on cell proliferation of normal and OA cartilage derived chondrocytes.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2017 Osteoarthritis Research Society International Terms and Conditions

11. Fig. S4
miRNA-140 regulates Collagen II, MMP-13 and ADAMTS-5 expression in human normal and OA cartilage derived chondrocytes. (A) PCR analysis of the relative expression levels of miRNA-140 after miRNA-140 transfection in normal and OA cartilage derived chondrocytes. U6 was used as endogenous control, and the expression level in the normal blank group was set to one. (B–D) PCR analysis of the relative expression levels of Collagen II, MMP-13, and ADAMTS-5, respectively, after miRNA-140 transfection in normal and OA cartilage derived chondrocytes. β-actin was used as endogenous control, and the expression level in the normal blank group was set to one. (E–H) Western blot analysis (E) and quantitation of Collagen II (F), MMP-13 (G), and ADAMTS-5 (H) expression after miRNA-140 transfection in normal and OA cartilage derived chondrocytes. The protein expression level was normalised to that of β-actin. *, Compared with blank group, P < 0.05.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2017 Osteoarthritis Research Society International Terms and Conditions

12. Fig. S5
Representative articular cartilage T2 maps of the knee joint at 4, 8, and 12 weeks after modelling surgery. Before the animals were sacrificed, OA progression was assessed in vivo by magnetic resonance imaging (MRI) 1 day before surgery and at 4, 8 and 12 weeks after surgery. The rats were anaesthetised by intraperitoneal injections of 10% chloral hydrate sodium (4 ml/kg; West China Hospital, Sichuan University, Chengdu, China), and MRI was performed with a 4.7 T imaging system (Siemens Sonata Medical System) equipped with a custom rat coil for transmission and reception of the signal using a multisection single-echo T2-weighted TSE sequence. The study was supervised by an experienced musculoskeletal radiologist. The protocol included turbo spin echo (TSE) sequences and T2-weighting (TE/TR = 40/3500 ms, echo train length = 8, slice thickness = 0.5 mm, acquisition time = 0:32 h), and images were acquired at a resolution of 156 × 312 μm2. The MR imaging findings-increased synovial fluid, synovial hyperplasia and subchondral bone cyst formation-also suggested that the knee joint degenerated at a slower rate in the miRNA-140 agomir group than in the control group.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2017 Osteoarthritis Research Society International Terms and Conditions

13. Fig. S6
The diagram of the specially modified miRNA-140 agomir. The antisense chain of the synthetic double-stranded miRNA-140 was fully modified by the addition of a methoxyl group, the 3′ end was modified by the addition of one cholesterol and four thio-skeletons, and the 5′ end was modified by the addition of two thio-skeletons.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2017 Osteoarthritis Research Society International Terms and Conditions