Fig 1: XIST promotes extracellular matrix degradation by acting as a competing endogenous RNA of miR-1277-5p in AC/OA and AC/IL-1ß-5.0 cells. Normal XIST overexpression plasmids (wt-pcDNA-XIST) led to a notable upregulation of MMP-13, but the effect was attenuated by the overexpression of miR-1277-5p (co-transfection of wt-pcDNA-XIST and miR-1277-5p mimics). When the putative miR-1277-5p binding site in XIST was mutated (transfection of mut-pcDNA-XIST), the facilitative effect was eliminated. The phenomenon was detected by (A) immunofluorescence analysis, (B) RT-qPCR and (C) western blot analysis (lysates were analyzed by immunoblotting with MMP-13 and GAPDH antibodies; the left panel presents data from 3 independent experiments). **P<0.01 vs. wt-pcDNA-XIST; ##P<0.01 vs. wt-pcDNA-XIST + miR-1277-5p mimic group. wt-pcDNA-XIST, but not mut-pcDNA-XIST, promoted ADAMTS5 expression, and the effect was repressed when the putative miR-1277-5p binding site in XIST was mutated (transfection of mut-pcDNA-XIST). As with MMP-13, elevation of miR-1277-5p also reversed the facilitative effect of wt-pcDNA-XIST on ADAMTS5 expression, as determined by (D) immunofluorescence analysis, (E) RT-qPCR and (F) western blot analysis (lysates were analyzed by immunoblotting with ADAMTS5 and GAPDH antibodies; the left panel presents data from 5 independent experiments). **P<0.01 vs. wt-pcDNA-XIST; ##P<0.01 vs. wt-pcDNA-XIST + miR-1277-5p mimic group. All data were normalized to the control group, and all error bars represent standard deviation (n=3). XIST, X-inactive-specific transcript; miR, microRNA; AC, articular chondrocyte; OA, osteoarthritis; IL-1ß, interleukin-1ß; wt, wild type; mut, mutant; MMP-13, matrix metalloproteinase 13; ADAMTS5, ADAM metallopeptidase with thrombospondin type 1 motif 5; RT-qPCR, reverse transcription-quantitative polymerase chain reaction.
Fig 2: Autophagy enhanced the inhibitory effect of cyanidin on IL-1ß-induced NPCs apoptosis and ECM degradation. (A,B) The expression of autophagy related proteins LC3, beclin-1 and p62 were determined by western blots. (C) LC3-positive autophagosomes was observed by immunofluorescence staining (Bar = 25 µm). (D,E) Western blots were used to analyse the expression of Bax, Bcl-2, cleaved caspase-3. (F,G) The expression of collagen II, aggrecan, MMP-13 and ADAMTS-5 were analysed using western blot assays. *p < 0.05 vs. control; #p < 0.05 vs. NPCs treated with IL-1ß only; &p < 0.05 vs. NPCs treated with IL-1ß + cyanidin.
Fig 3: Schematic diagram of the mechanism concluded from the present study. XIST, X-inactive-specific transcript; ADAMTS5, ADAM metallopeptidase with thrombospondin type 1 motif 5; MMP-13, matrix metalloproteinase 13; ECM< extracellular matrix; miR, microRNA.
Fig 4: Cyanidin mitigated ECM synthesis and degradation and apoptosis in IL-1ß-induced NPCs. (A–E) The expression of aggrecan, collagen II, MMP-13 and ADAMTS-5 in NPCs were analysed by western blot assays. (F,G) Flow cytometry was used to detect the apoptosis of NPCs. (H–K) The expression of Bax, Bcl-2 and cleaved caspase-3 in NPCs were analysed by western blot assays. *p < 0.05 vs. control; #p < 0.05 vs. NPCs treated with IL-1ß only.
Fig 5: miR-1277-5p attenuates extracellular matrix degradation by directly targeting MMP-13 and ADAMTS5 in AC/OA and AC/IL-1ß-5.0 cells. (A) XIST, MMP-13 and ADAMTS5 share the same miR-1277-5p binding site within their 3'UTRs. (B) Downregulation of miR-1277-5p in OA tissue specimens and in AC/OA, AC/IL-1ß-1.0 and AC/IL-1ß-5.0 cells, as confirmed by reverse transcription-quantitative polymerase chain reaction. **P<0.01 vs. non-OA group; ##P<0.01 vs. AC group. (C) miR-1277-5p was upregulated by transfection of miR-1277-5p mimics as determined by reverse transcription-quantitative polymerase chain reaction. **P<0.01 vs. mimic con group. (D) Upregulation of miR-1277-5p (transfection of miR-1277-5p mimics) led to a notable decrease in MMP-13 and ADAMTS5 expression, as measured by immunofluorescence analysis. **P<0.01 vs. mimic control group; magnification, ×100; scale bar, 100 µm. (E) Diagram of the luciferase reporter plasmids with the wt- or mut-MMP-13 and -ADAMTS5 3'UTRs. (F) Compared with the effect of the mimic con, co-transfection of miR-1277-5p mimic and wt-MMP-13 led to a distinct decrease in fluorescence, but the suppressive effect was rescued by a mutation in the putative miR-1277-5p binding sites in the MMP-13 3'UTR (co-transfection of mut-MMP-13 and miR-1277-5p mimic), as verified by dual luciferase assays. **P<0.01 and &P>0.05 vs. mimic con group. (G) The same effects were observed during the verification of the interaction between miR-1277-5p and ADAMTS5 3'UTR. **P<0.01 and &P>0.05 vs. mimic con group. All error bars represented standard deviation (n=3). miR, microRNA; MMP-13, matrix metallopro-teinase 13; ADAMTS5, ADAM metallopeptidase with thrombospondin type 1 motif 5; AC, articular chondrocyte; OA, osteoarthritis; IL-1ß, interleukin-1ß; UTR, untranslated region; wt, wild type; mut, mutant; con, control; LUC+, luciferase.
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