Figures and data
MiRNA expression in KOA patients.
(A, B) Volcano plot and heatmap showing dif-ferential miRNAs between KOA and healthy groups (healthy, n = 10; KOA, n = 15, | log2 Fold Change | 0.585, P < 0.05). (C) GO and KEGG enrichment analyses of upregulated genes. (D) GO and KEGG enrichment analyses of downregulated genes.
Verification with GEO data and miRNA screening.
(A, B) Venn plot showing GEO dataset and our result. (C) RT-qPCR in clinical samples to verify the expression (HC, n = 5, KOA, n = 5). (D) RT-qPCR results of mouse joint samples (n = 6). Data are shown as means ± SD. *P<0.05, **P<0.01.
Chondrocyte proliferation and marker expression changes after miR-199b-5p mimic or inhibitor treatment.
(A) Second generation primary mouse chondrocytes. (B) Toluene blue staining. (C) Type Ⅱ collagen immunoassay. (D) CCK-8 assay for cell viability (n=6). (E, F) RT-qPCR detection of MMP-3 and ADAMTS5 mRNA expression (n=3). (G-I) RT-qPCR detec-tion of COL-2A1, AGGRECAN, and SOX9 mRNA expression (n=3). (J) CCK-8 cell viability assay after different doses of LPS induction (n=5). (K) CCK-8 cell viability assay after virus in-fection (n=6). (L, M) RT-qPCR detection of MMP-3 and ADAMTS5 mRNA expression (n=3). (N-P) RT-qPCR detection of COL2A1, AGGRECAN, and SOX9 mRNA expression (n=3). Data are shown as means ± SD. *P<0.05, **P<0.01.
Injection of adenovirus expressing miR-199b-5p mimic results in inflammation and pain threshold sensitivity in mice.
(A) Animal experiment schematic. (B) Behavioral detection of animal thermal pain threshold. (C, D) Detection of serum levels of IFN-γ and TNF-α in controls by ELISA. (E, F) Safranin-fast green staining and semiquantitative scoring of articular cartilage. (G) 3D reconstruction and 2D images of joints from μCT scans. Data are shown as mean ± SD. *P<0.05, **P<0.01, n=6.
Injection of adenovirus expressing miR-199b-5p inhibitor partly recovered pathologi-cal changes in KOA mice.
(A) Animal experiment schematic. (B) Behavioral detection of animal thermal pain threshold. (C, D) Detection of serum levels of IFN-γ and TNF-α in controls by ELISA. (E, F) Safranin-fast green staining and semiquantitative scoring of articular cartilage. (G) 3D reconstruction and 2D images of joints from μCT scans. Data are shown as mean ± SD. *P<0.05, **P<0.01, n=6.
Validation of the miR-199b-5p target gene.
(A) Prediction of target genes of miR- 199b-5p using search sites. (B) Target gene GO analysis. (C, D) Detection of the expression of target genes under different conditions (n=3). (E, F) Predicting the binding site of miR-199b-5p and target genes. (G, H) Validation by luciferase reporter gene assay (n=3). Data are show as mean ± SD.*P<0.05, **P<0.01, n=6. (I-L) The expression of FZD6 and GCNT2 in the synovial membrane and chondrocytes of GEO Profiles KOA.
miR-199b-5p exerts its effects on in vitro cells and in vivo mice by targeting Fzd6 and Gcnt2.
Primer List
