Figures and data
MiRNA expression in KOA patients. (A, B) Volcano plot and heatmap showing differential miRNAs between KOA and healthy groups (healthy, n = 10; KOA, n = 15, ⎢log2 Fold Change⎪≥ 0.585, P < 0.05). (C) GO enrichment analyses of upregulated genes. (GO:0031325 positive regulation of cellular metabolic process; GO:0007268 chemical synaptic transmission; GO:0009893 positive regulation of metabolic process; GO:0070935 3’-UTR-mediated mRNA stabilization; GO:0045595 regulation of cell differentiation; GO:0043005 neuron projection;GO:0042995 cell projection; GO:0044297 cell body; GO:0005622 intracellular; GO:0005737 cytoplasm; GO:0031624 ubiquitin conjugating enzyme binding; GO:0008494 translation activator activity; GO:0044390 ubiquitin-like protein conjugating enzyme binding; GO:0016740 transferase activity; GO:0019787 ubiquitin-like protein transferase activity) (D) KEGG enrichment analyses of upregulated genes. (E) GO enrichment analyses of downregulated genes. (GO:0038063 collagen-activated tyrosine kinase receptor signaling pathway; GO:0038065 collagen-activated signaling pathway; GO:0021984 adenohypophysis development; GO:0007042 lysosomal lumen acidification; GO:0071267 L-methionine salvage; GO:0005623 cell; GO:0005622 intracellular; GO:0030057 desmosome; GO:0032590 dendrite membrane; GO:0034271 phosphatidylinositol 3-kinase complex, class III, type I; GO:0003700 DNA binding transcription factor activity; GO:0004304 estrone sulfotransferase activity; GO:0000981 RNA polymerase II transcription factor activity, sequence-specific DNA binding; GO:0008146 sulfotransferase activity; GO:0001228 transcriptional activator activity, RNA polymerase II transcription regulatory region sequence-specific DNA binding) (F) 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). M-0.5, 10 µL of 0.5 mg/mL;M-1, 10 µL of 1 mg/mL. 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 LJ 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 detection 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 infection (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 pathological 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) we use targetscan to predict 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 potential targeting Fzd6 and Gcnt2.
Primer List
