[miR-515-5p targeting Toll-like receptor 4 regulates myeloid differentiation primary response gene 88/nuclear factor-kappa B pathway to inhibit apoptosis and inflammatory response of osteoarthritis chondrocytes].

Objective: To explore the molecular mechanism of miR-515-5p in inhibiting chondrocyte apoptosis and alleviating inflammatory response in osteoarthritis (OA). Methods: Human cartilage cell line C28/I2 was cultured in vitro and treated with 10 ng/mL interleukin 1ß (IL-1ß) for 24 hours to construct an in vitro OA model. C28/I2 cells were transfected with miR mimics, mimics negative control (NC), over expression (oe)-NC, and oe-Toll-like receptor 4 (TLR4), respectively, and then treated with 10 ng/mL IL-1ß for 24 hours to establish OA model. Cell proliferation capacity was detected by cell counting kit 8 and 5-Ethynyl-2'-deoxyuridine, cell apoptosis and cell cycle were detected by flow cytometry, and B-cell lymphoma 2 protion (Bcl-2), Bcl-2-associated X protein (Bax), cleaved-Caspase-3, TLR4, myeloid differentiation primary response gene 88 (MyD88), p65 and phosphorylated p65 (p-p65) protein expression levels were detected by Western blot. Real-time fluorescence quantitative PCR was used to detect mRNA expression levels of miR-515-5p and TLR4, and ELISA was used to detect pro-inflammatory factor prostaglandin E2 (PGE2), tumor necrosis factor α (TNF -α), and IL-6 levels in cell supernatant. The potential binding sites between miR-515-5p and TLR4 were predicted by BiBiServ2 database, and the targeting relationship between miR-515-5p and TLR4 was verified by dual luciferase reporting assay. Results: After the treatment of C28/I2 cells with IL-1ß, the expressions of miR-515-5p and Bcl-2 protein and the proliferation ability of C28/I2 cells significantly reduced. The expression levels of Bax and cleaved-Caspase-3 protein, the levels of pro-inflammatory factors (PGE2, TNF-α, IL-6) in the supernatant of C28/I2 cells, and the apoptosis of C28/I2 cells significantly increased. In addition, the proportion of the cells at S phase and G 2 phase decreased significantly, and the proportion of cells at G 1 phase increased significantly, suggesting that the cell cycle was blocked after IL-1ß treatment. After transfection with miR mimics, the expression level of miR-515-5p in the cells significantly up-regulated, partially reversing the apoptosis of OA chondrocytes induced by IL-1ß, and alleviating the cycle arrest and inflammatory response of OA chondrocytes. After treating C28/I2 cells with IL-1ß, the mRNA and protein levels of TLR4 significantly increased. Overexpression of miR-515-5p targeted inhibition of TLR4 expression and blocked activation of MyD88/nuclear factor κB (NF-κB) pathway. Overexpression of TLR4 could partially reverse the effect of miR mimics on IL-1ß-induced apoptosis and inflammation of OA chondrocytes. Conclusion: miR-515-5p negatively regulates the expression of TLR4, inhibits the activation of MyD88/NF-κB pathway and apoptosis of OA chondrocytes, and effectively alleviates the inflammatory response of the cells.

Prevalence and prognosis of bone metastases in common solid cancers at initial diagnosis: a population-based study.

OBJECTIVE: Bone is one of the most common target sites for advanced tumours. The objective was to survey the prevalence and prognosis of bone metastases in 12 common solid malignant tumours. DESIGN: A retrospective cohort study. METHODS: A total of 1 425 332 patients with a primary cancer between 2010 and 2015 were identified using the Surveillance, Epidemiology, and End Results database. We computed the prevalence and prognosis of bone metastases in each cancer and compared their survival in different stages. The Kaplan-Meier method and Cox logistic regression were used to analyse survival and quantify the effect of bone metastases. RESULTS: This study included 89 782 patients with bone metastases at diagnosis. Lung cancer had the highest prevalence (18.05%), followed by liver cancer (6.63%), nasopharyngeal carcinoma (6.33%) and renal cancer (5.45%). Breast cancer (32.1%), prostate cancer (25.9%), thyroid cancer (46.9%) and nasopharyngeal carcinoma (24.8%) with only bone metastases had a 5-year survival rate of over 20%. Compared with patients at the stage previous to metastasis, bone metastases significantly increased the risk of mortality and decreased survival, especially for those with prostate cancer (adjusted HR: 18.24). Other concomitant extraosseous metastases worsened patient survival. Bone was the most common site of metastasis for prostate cancer, while for colorectal cancer, multiorgan metastases were predominant. CONCLUSIONS: This study provides the prevalence and prognosis of bone metastases at the initial diagnosis of common solid cancers. In addition, it demonstrates the impact of bone metastases on survival. These results can be used for early screening of metastases, clinical trial design and assessment of prognosis.

Overexpression of FTO alleviates osteoarthritis by regulating the processing of miR-515-5p and the TLR4/MyD88/NF-κB axis.

OBJECTIVE: Osteoarthritis (OA) is regarded as the most prevalent chronic joint disease. Fat-mass and obesity-associated gene (FTO) is involved in OA alleviation. This study elucidated the role of FTO in OA and the associated mechanism. METHODS: We established a cell injury model by stimulating human normal chondrocytes (C28/I2) with lipopolysaccharide (LPS), and measured cell viability, apoptosis, and inflammatory cytokines using CCK-8, flow cytometry, Western blot, and ELISA. TLR4, MyD88, p/t-p65, and p/t-IκBα levels, FTO, COX-2, and iNOS mRNA levels, and m6A methylation levels were measured by Western blot, RT-qPCR, and colorimetry. RNA immunoprecipitation and co-immunoprecipitation were conducted to confirm the interaction between FTO and DGCR8. pri-miR-515-5p process was regulated in an m6A-dependent manner. After predicting the presence of several binding sites between miR-515-5p and TLR4 on Targetscan, we further confirmed their relationship by dual-luciferase assay. OA rat models were established by monosodium iodoacetate injection. The pathological changes in knee joint were observed by HE staining. RESULTS: FTO was diminished in LPS-induced C28/I2 cells. With the increase of LPS concentration, cell viability was repressed, apoptosis rate was increased, and inflammatory markers were promoted, which were annulled by FTO overexpression. FTO interacted with DGCR8 and modulated the pri-miR-515-5p processing in an m6A-dependent manner. miR-515-5p silencing partially averted the inhibitory effect of FTO on LPS-induced cell injury. Given that TLR4 was a direct target of miR-515-5p, miR-515-5p inactivated the MyD88/NF-κB pathway by targeting TLR4. FTO overexpression improved cartilage structure in OA rats, reduced apoptosis, inhibited inflammation in synovial fluid, and repressed the TLR4/MyD88/NF-κB axis. CONCLUSION: FTO alleviated OA in an m6A-dependent manner via the miR-515-5p/TLR4/MyD88/NF-κB axis.

The Effects of microRNA-515-5p on the Toll-Like Receptor 4 (TLR4)/JNK Signaling Pathway and WNT1-Inducible-Signaling Pathway Protein 1 (WISP-1) Expression in Rheumatoid Arthritis Fibroblast-Like Synovial (RAFLS) Cells Following Treatment with Receptor Activator of Nuclear Factor-kappa-B Ligand (RANKL).

BACKGROUND This study aimed to investigate the effects of microRNA-515-5p (miR-515-5p) on the expression of the WNT1-inducible-signaling pathway protein 1 (WISP-1) gene in rheumatoid arthritis fibroblast-like synovial (RAFLS) cells following treatment with the receptor activator of nuclear factor-kappa-B ligand (RANKL). MATERIAL AND METHODS RAFLS cells were cultured in vitro and were divided into six study groups: a normal control group; a miR-515-5p mimic group; a miR-515-5p inhibitor group; a RANKL (50 ng/ml) treatment group; a miR-515-5p mimic+RANKL treatment group; and a miR-515-5p inhibitor+RANKL treatment group. The luciferase assay was used to determine the effects of miR-515-5p on the WISP1 expression. Cell proliferation, cell apoptosis, the cell cycle, and protein expression were determined using the Cell Counting Kit-8 (CCK-8) assay, flow cytometry, Western blot, and real-time polymerase chain reaction (RT-PCR). RESULTS The luciferase assay showed that the effects of miR-515-on the 3'-UTR of WISP1 inhibited the gene expression. The miR-515-5p mimics promoted cell proliferation, reduced apoptosis, and promoted the cell cycle. The miR-515-5p mimics reduced, the expression of TLR4, WISP1, and JNK at the mRNA level, while the miR-515-5p inhibitor promoted the expression of TLR4, WISP1, and JNK. Both the miR-515-5p inhibitor and mimic promoted the phosphorylation of AKT in RAFLS cells treated with or without RANKL compared with the control, and the miR-515-5p inhibitor promoted the phosphorylation of JNK in the RAFLS cells. CONCLUSIONS In RAFLS cells, miR-515-5p inhibited the expression of the WISP1 gene, and treatment with RANKL inhibited the TLR4/JNK signaling pathway.

Eukaryotic translation initiation factor 3H suppression inhibits osteocarcinoma cell growth and tumorigenesis.

Eukaryotic translation initiation factor 3H subunit (EIF3H) is a member of the EIF3 family and exhibits a central role in translation initiation in higher eukaryotes. Although EIF3H expression is upregulated in numerous tumour types, its potential role in human osteosarcoma (OS) has not yet been investigated. In the present study, it was demonstrated that EIF3H mRNA expression was upregulated in the human OS cell lines Saos-2 and U2OS. A recombinant lentivirus harbouring short hairpin RNA targeting EIF3H was constructed and successfully infected human OS Saos-2 and U2OS cells, resulting in 95% downregulated EIF3H expression compared with the respective control groups. Knockdown of EIF3H significantly inhibited the proliferation and colony formation of OS cells in vitro, and tumour growth in nude mice in vivo. Flow cytometry analysis revealed cell cycle arrest and promotion of apoptosis in OS cells with EIF3H knocked down. In conclusion, the results strongly suggested that EIF3H is a critical factor mediating the growth of OS cells and may represent a novel therapeutic target.

miR-30b regulates chondrogenic differentiation of mouse embryo-derived stem cells by targeting SOX9.

The present study aimed to investigate the mechanisms underlying microRNA (miRNA)-mediated regulation of chondrogenic differentiation. Mouse embryo-derived stem cells C3H10T1/2 were cultured and chondrogenic differentiation was induced using transforming growth factor-ß3 (TGF-ß3). In addition, miRNA expression profiles were detected via miRNA array analysis, and quantitative polymerase chain reaction was performed to verify the differentially expressed miRNAs. Furthermore, bioinformatics software was used to predict the putative targets and the prediction was validated by dual-luciferase reporter assays and western blot analysis. In addition, cell proliferation and glycosaminoglycans were measured by a direct cell count method and alcian blue staining, respectively. Compared with the control group, 86 miRNAs were identified as differentially expressed in TGF-ß3-induced cells and the expression levels of 28 miRNAs were increased while the remaining 58 miRNAs exhibited a decline in expression. Amongst the differentially expressed miRNAs, miR-30b expression was observed to have significantly decreased during chondrogenic differentiation. SOX9 is a target gene of miR-30b, and miR-30b inhibits SOX9 expression during chondrogenic differentiation. Furthermore, the alcian blue staining results demonstrated that miR-30b inhibited early chondrogenic differentiation. However, the data of the present study indicated that miR-30b had no influence on C3H10T1/2 cell line proliferation. In conclusion, miR-30b is a key negative regulator of TGF-ß3-induced C3H10T1/2 cell chondrogenic differentiation, which functions by directly targeting SOX9.

miRNA-140 inhibits C3H10T1/2 mesenchymal stem cell proliferation by targeting CXCL12 during transforming growth factor-ß3-induced chondrogenic differentiation.

The aim of the present study was to investigate the role of microRNA (miRNA or miR)-140 in C3H10T1/2 mesenchymal stem cells (MSCs). Cluster analysis was used to evaluate the miRNA expression profile. The expression level of miRNA­140 was validated by reverse transcription­quantitative polymerase chain reaction (RT­qPCR). TargetScan and microRNA.org databases were used to predict target miRNAs and cartilage­associated target genes. Binding sites between miR­140 and the target gene were predicted by bioinformatics software. A dual­luciferase reporter assay was performed to determine whether miR­140 could target C­X­C motif chemokine ligand 12 (CXCL12). Following the promotion/inhibition of miR­140, 1, 7 and 14 days following transforming growth factor­ß3 (TGF­ß3)­induction, western blotting was utilized to evaluate CXCL12 protein levels. MTT assays and alcian blue staining were applied to assess C3H10T1/2 MSC viability and chondrogenic differentiation, respectively. In the TGF­ß3­induced group, RT­qPCR verified that the mRNA level of Mus musculus (mmu)­miR­140 was significantly elevated when compared with the control group. miR­140 was predicted to recognize and interact with CXCL12­3'UTR and the dual luciferase reporter assay further validated that miR­140 targeted the predicted region of CXCL12. CXCL12 was markedly decreased following miR­140 overexpression and visibly increased following miR­140 inhibition. In addition, the level of CXCL12 expression declined as the duration of induction increased. Following the promotion/inhibition of miR­140, at 1 and 7 days following TGF­ß3­induction, C3H10T1/2 MSCs inhibited or promoted cell viability, respectively, when compared with the control groups. In addition, in pellets achieved by chondrogenic differentiation following the induction of C3H10T1/2 MSCs for 7 days, alcian blue staining revealed no significant difference in characteristic extracellular matrix glycosaminoglycans between the miR­140 up and downregulated groups, and their respective control groups. The present study concludes that miRNA­140 inhibition promoted C3H10T1/2 MSC viability however, not C3H10T1/2 MSC differentiation by targeting and reducing CXCL12 protein levels during the process of TGF­ß3­induced chondrogenic differentiation. In conclusion, the present study provided a potential target for the treatment of cartilage defection.