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This study aimed to explore the effects and relative mechanism of JMJD3 on knee osteoarthritis (OA). In this study, we first analyzed the expression of JMJD3 in OA cartilage using western blot and immunohistochemistry. In an in vitro study, the effects of GSK-J4, JMJD3 inhibitor, on ATDC-5 chondrocytes were evaluated by CCK-8 assay. Real-time PCR and western blot were used to examine the inhibitory effect of GSK-J4 on the inflammation and ECM degradation of chondrocytes. NF-κB p65 phosphorylation and nuclear translocation were measured by western blot and immunofluorescence. In the animal study, twenty mice were randomized into four experimental groups: sham group, DMM-induced OA + DMSO group, OA + low-dose GSK-J4 group, and OA + high-dose GSK-J4 group. After the treatment, hematoxylin–eosin and safranin O/fast green staining were used to evaluate cartilage degradation of knee joint, with OARSI scores for quantitative assessment of cartilage damage. Our results revealed that JMJD3 was overexpressed in OA cartilage and GSK-J4 could suppress the IL-1β-induced production of pro-inflammatory cytokines and catabolic enzymes, including IL-6, IL-8, MMP-9 and ADAMTS-5. Consistent with these findings, GSK-J4 could inhibit IL-1β-induced degradation of collagen II and aggrecan. Mechanistically, GSK-J4 dramatically suppressed IL-1β-stimulated NF-κB signal pathway activation. In vivo, GSK-J4 prevented cartilage damage in mouse DMM-induced OA model. This study elucidates the important role of JMJD3 in cartilage degeneration in OA, and our results indicate that JDJM3 may become a novel therapeutic target in OA therapy.

Additional file 1: Figure S1. a. Western blot demonstrating CRISPR/CAS9-mediated CD151 knockout in indicated cells. b. Heatmap of the sphingolipid metabolism genes regulated by CD151 depletion in ZOSM cells from RNA-seq. c. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of transcriptomic profiles of osteosarcoma patients based on the GEO database (GSE42352, n = 127). The red indicates the KEGG pathway related to sphingolipid metabolism. d. Heatmap of osteosarcoma patients showed changes of sphingolipid metabolism genes that express a high level of CD151 compared to tumors with low CD151 expression. Figure S2. a. Liquid chromatography coupled to mass spectrometry (LC/MS) was used to measure the concentrations of intermediates in ZOSM cells. Heatmap showing significantly differently expression metabolites altered by CD151 silencing. Shades of red and blue represent higher and lower levels of metabolites, respectively. b. The top 10 enriched pathways from integrated pathway analysis of significantly changed metabolites. The red indicates the KEGG pathway related to sphingolipid metabolism. c. Lipids were extracted from WT or CD151 KO ZOSM cells and analyzed by LC/MS. Bubble plots represent the mean log2-transformed fold-change difference between cell lines. d. The intensity of BODIPY FL-labeled ceramide or sphingomyelin was analyzed by confocal fluorescence imaging in ZOSM cells. Scale bars: 50 μm. e. ZOSM cells with CD151 depletion were incubated with Alexa 555-conjugated CTB (red) to label GM1-containing lipid rafts. Cells were analyzed by confocal microscopy or flow cytometry. Scale bars: 50 μm. Figure S3. a. Expression of genes involved in sphingolipid metabolism was measured by PCR array in ZOSM cells. Data are shown as log2-transformed fold change in CD151 silencing cells relative to control. b. Western blot analysis of SPTCL1 expression in cells with CD151 overexpression in indicated cells. c. The cellular levels of ceramide in vector and CD151 overexpression ZOSM cells with or without myriocin treatment were analyzed by BODIPY FL-labeled ceramide confocal imaging. Scale bars: 50 μm. d. The cellular levels of lipid rafts in vector and CD151 overexpression ZOSM cells with or without myriocin treatment were analyzed by Alexa 555-conjugated CTB confocal imaging. Scale bars: 50 μm. e. Mice weight quantification of established tumors with CD151 overexpression treated with vehicle or myriocin (0.5 mg/kg). Data are presented as mean ± SD, n = 5. Figure S4. a. Gene set enrichment analysis (GSEA) based on the RNA-seq using Hallmarks gene sets between WT and CD151 KO ZOSM cells. b. The c-myc expression was detected in the indicated cells with CD151 overexpression by western blot. c. Time-course analysis of c-myc protein levels in CD151 depletion ZOSM cells. c-myc band density relative to β-actin was quantified, and the ratio of c-myc protein/actin protein was artificially set as 1.0 for samples untreated with CHX to obtain half-time (T1/2) of c-myc. d. c-myc ubiquitination was analyzed in CD151 depletion cells. ZOSM cells were transfected with the indicated plasmids followed by treatment with MG132 for 6 h. Cell extracts were immunoprecipitated with an anti-HA antibody, and ubiquitination c-myc was detected by western blot. Figure S5. a-b. SPTLC1 mRNA and protein levels in CD151 depletion cells stably infected with vector or c-myc-expressing lentiviruses were analyzed by RT-qPCR (A) and Western blot (B). Data are shown as mean ± SD of triplicate experiments. c-d. Schematic representation of the promoter region in the human SPTLC1 gene (C). ChIP from ZOSM cells was performed with control IgG or c-myc antibody as indicated. The presence of the SPTLC1 binding sites was detected by qPCR using primers. Quantification of enrichments is represented as fold-enrichment over lgG control. e. ZOSM cells were transfected with an empty vector or c-myc plus the wild-type SPTLC1 promoter (WT) or mutated promoter (MUT) for measuring luciferase activity. Data are the mean ± SD, and the data are representative of three independent experiments. Figure S6. a. The representative images of Ki67 staining from SA3831 and SA4009 PDX tumor-bearing mice treated with vehicle or myriocin. Scale bars: 100 μm. Table S1.

Additional file 1: Fig. S1. The quantification of apoptotic cells and the density of the western blot bands in Fig. 1. Fig. S2. TOD inhibited the expression of inflammatory and catabolic mediators in IL-1�� induced chondrocytes. Fig. S3. TOD suppressed Ctr expression during osteoclastogenesis in vitro.

Bo Xu,* Xinmeng Jin,* Tieyi Yang, Yan Zhang, Shuyi Liu, Liang Wu, Hui Ying, Zhi Wang Department of Orthopaedics, Gongli Hospital of Pudong New Area, Shanghai 200135, People’s Republic of China*These authors contributed equally to this workCorrespondence: Zhi WangDepartment of Orthopaedics, Gongli Hospital of Pudong New Area, NO. 219 Miaopu Road, Pudong New Area, Shanghai 200135, People’s Republic of ChinaTel +86 188 1782 1626Email bbswangzhi@163.comBackground: The immunosuppressive facet and tumorigenic role of TNF-α have been revealed in osteosarcoma (OS). Long noncoding RNA THRIL is identified to regulate TNF-α expression and participates in immune response. Thus, investigations on the clinical expression pattern of THRIL/TNF-α signal in OS would provide a potential target premise for OS patients.Methods: We collected OS (n=83), nontumor tissues (n=37) and serum samples (n=83 for OS and n=40 for healthy control) to determine the expressions and clinical significance of THRIL/TNF-α signal. Knockdown of THRIL in OS cell lines MG63 and Saos2 in vitro and in vivo was performed to confirm its function in the development of OS.Results: Elevated expression of THRIL was associated with increased TNF-α levels in OS tissues and serum samples. Combination of THRIL and TNF-α in tissues showed a more efficient diagnostic value for OS patients than either of them. Moreover, high-expressed THRIL was associated with larger tumor size, advanced Enneking stage and lung metastasis, whereas high TNF-α expression was found in patients with high histologic grade and patients who simultaneously harbor high THRIL and TNF-α showed the worst overall survival and metastasis-free survival. TNF-α signals increased OS cell vitalities in vitro but knockdown of THRIL inhibited TNF-α expressions, leading to impaired cell vitality, increased apoptosis and also downregulated epithelial to mesenchymal transition (EMT) phenotype and the ability of invasion, but these processes were restored by the treatment of TNF-α. The oncogenic role of THRIL/TNF-α signal was also confirmed in the xenograft model of MG63 cells.Conclusion: Overexpressed THRIL and TNF-α promoted OS progression with efficient diagnostic and prognostic value. THRIL/TNF-α signal supported cell growth and EMT phenotype of OS cells in vitro and in vivo.Keywords: lncRNA, THRIL, TNF-α, osteosarcoma, growth