BackgroundBreast cancer (BRCA) is the most common cancer in women, while the bones are one of the most common sites of metastasis. Although new diagnostic methods or radiation or chemotherapies and targeted therapies have made huge advances, the occurrence of bone metastasis is also linked with poorer survival. Enhancer RNAs (eRNAs) have been demonstrated to participate in the progression of tumorigenesis and metastasis. However, the role of eRNAs in BRCA bone metastasis remains largely unclear.MethodGene expression profiling of 1,211 primary BRCA and 17 bone metastases samples were retrieved from The Cancer Genome Atlas (TCGA) database, and the significant prognostic eRNAs were identified by Cox regression and least absolute shrinkage and selection operator (LASSO) regression. The acceptable accuracy and discrimination of the nomogram were indicated by the receiver operating characteristic (ROC) and the calibration curves. Then target genes of eRNA, immune cell percentage by CIBERSORT analysis, immune genes by single-sample gene set enrichment analysis (ssGSEA), hallmark of cancer signaling pathway by gene set variation analysis (GSVA), and reverse phase protein array (RPPA) protein chip were used to build a co-expression regulation network and identified the key eRNAs in bone metastasis of BRCA. Finally, Cell Counting Kit-8 (CCK8) assay, cell cycle assay, and transwell assay were used to study changes in cell proliferation, migration, and invasiveness. Immunoprecipitation assay and Western blotting were used to test the interaction and the regulation signaling pathways.ResultsThe 27 hub eRNAs were selected, and a survival-related linear risk assessment model with a relatively high accuracy (area under curve (AUC): 0.726) was constructed. In addition, seven immune-related eRNAs (SLIT2, CLEC3B, LBPL1, FRY, RASGEF1B, DST, and ITIH5) as prognostic signatures for bone metastasis of BRCA were further confirmed by LASSO and multivariate Cox regression and CIBERSORT analysis. Finally, in vitro assay demonstrated that overexpression of SLIT2 reduced proliferation and metastasis in BRCA cells. Using high-throughput co-expression regulation network, we identified that SLIT2 may regulating P38 MAPK/c-Fos signaling pathway to promote the effects of metastasis.ConclusionBased on the co-expression network for bone metastasis of BRCA, we screened key eRNAs to explore a prognostic model in predicting the bone metastasis by bioinformatics analysis. Besides, we identified the potential regulatory signaling pathway of SLIT2 in BRCA bone metastasis, which provides a promising therapeutic strategy for metastasis of BRCA.

Osteoarthritis (OA) is a common inflammatory joint disease. MicroRNAs (miRNAs/miRs) have been reported to be involved in the pathogenesis of OA; however, the role of miRNAs in OA remains largely unexplained. The purpose of the present study was to investigate the expression and role of miR-195-5p in OA, and to further explore the mechanism. The expression level of miR-195-5p was measured using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). TargetScan and a luciferase reporter assay were used to reveal the associations between miR-195-5p and REGγ (also known as PSME3). To investigate the role of miR-195-5p in OA, a cell model of OA was established by treating ATDC5 cells with lipopolysaccharide (LPS). Then an MTT assay was conducted to detect cell proliferation ability, and an Annexin V-fluorescein isothiocyanate/propidium iodide apoptosis detection kit was used to measure cell apoptosis. In addition, the levels of interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-α were determined using ELISA. Furthermore, gene and protein expression was measured via RT-qPCR and western blot assay, respectively. The results revealed that miR-195-5p was significantly upregulated in the articular cartilage tissues of patients with OA and in LPS stimulated ATDC5 cells. REGγ was a direct target of miR-195-5p. The repressed cell proliferation ability and enhanced cell apoptosis of ATDC5 cells induced by LPS were reversed by miR-195-5p downregulation. Furthermore, LPS stimulation significantly upregulated the levels of IL-1β, IL-6 and TNF-α, while miR-195-5p downregulation markedly reduced the expression of inflammatory factors induced by LPS. The results also revealed that a miR-195-5p inhibitor inhibited the LPS induced repression of the Wnt/β-catenin signaling pathway and activation of nuclear factor (NF)-κB signaling pathway in ATDC5 cells. Notably, the results of the present study also indicated that all of the effects of the miR-195-5p inhibitor on ATDC5 cells were reversed by REGγ silencing. In conclusion, the results indicated that the miR-195-5p inhibitor served a protective role in OA by inhibiting chondrocyte apoptosis and inflammatory responses by regulating the Wnt/β-catenin and NF-κB signaling pathways.

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Departmental sources Background: The purpose of this study was to explore the effects of the Na+/K+ ATPase inhibitor ouabain in regulating osteosarcoma (OS) cell stemness. Material/Methods: Western blot, qPCR, sphere-forming analysis, DNA methylation analysis, and Ca 2+ concentration detection were performed to evaluate the stem-like traits of cells and ouabain-induced effects and related mechanisms on OS cell stemness. Cell viability assessment was performed to evaluate the effect of ouabain on OS cell chemosensitivity. Results: Ouabain reduced the ALDH1 activity, the expression of critical stemness regulators, sphere size and number, and migration, invasion, and adhesion ability, but had little effects on cell viability. Additionally, the intracellular Ca 2+ concentration and methylation level of the critical stemness regulators were higher in OS cells than in spheres formed by OS cells. Mechanistic studies revealed that ouabain leads to DNA methylation of stemness markers through increasing intracellular Ca 2+ concentration. Notably, inhibition of Ca 2+ channel or DNA methylation rescued the inhibition of ouabain on OS cell stemness. Additionally, ouabain enhances cisplatin sensitivity of OS cells, which is involved in Ca 2+ channel and DNA methylation. Conclusions: This work provides a potential compound for treating OS patients, especially OS patients with chemoresistance.

The aim of the present study was to investigate the correlation between glucocorticoid activity regulation, prostaglandin E2 (PGE2) synthesis, and synovial inflammation inhibition activity, through microsomal prostaglandin E synthase-1 (mPGES-1) expression regulated by the glucocorticoid pre-receptor regulator, 11β-hydroxysteroid dehydrogenase-1 (11β-HSD1). In the present study, fibroblast-like synovial cells of rats were studied as a cell model. Cells were stimulated with 10 ng/ml interleukin (IL)-1β for 24 h, and were subsequently, within the next 24 h, treated with or without 10−6 mmol/l corticosterone alone or with 100 nmol/l PF915275. At the end of the second 24 h, PGE2 levels in culture supernatants were assayed. Cells were harvested for mRNA evaluation of 11β-HSD1, mPGES-1, IL-1β and tumor necrosis factor (TNF)-α, and protein detection of 11β-HSD1 and mPGES-1 using reverse transcription-qualitative polymerase chain reaction and western blot analysis, respectively. Corticosterone was demonstrated to suppress the mRNA expression levels of inflammatory factors, such as TNF-α and PGE2, induced by IL-1β in vitro. Simultaneously, expression levels of 11β-HSD1 decreased significantly at the mRNA and protein levels (P<0.05). Cortisol concentration in the medium of the group treated with corticosterone was significantly increased (P<0.05) compared with that of the control group; however, the cortisol concentration was decreased in the medium when the conversion bioactivity of 11β-HSD1 was inhibited by PF915275, while the changes in 11β-HSD1 and mPGES-1 mRNA expression levels and PGE2 content were reversed in the medium. These results indicated that a significant positive correlation (P<0.01) may exist between mRNA and protein expression levels. To conclude, 11β-HSD1 is a key regulator for the synthesis of mPGES-1 and PGE2 in the inflammatory synovial cells in vitro, suggesting a potential interference target for osteoarthritis.