Increasing reports suggest that deregulated microRNAs (miRNAs) might provide novel therapeutic targets for cancers. However, the expression and function of miR-300 in osteosarcoma is still unknown. In our study, we found that the expression of miR-300 was up-regulated in osteosarcoma tissues and cells compared with paired adjacent non-tumor bone tissues and osteoblastic cells using RT-qPCR. The enforced expression of miR-300 could promote cell proliferation, invasion and epithelial-mesenchymal transition (EMT). Moreover, we identified that bromodomain-containing protein 7 (BRD7), a new tumor suppressor gene, was a direct target of miR-300. Ectopic expression of BRD7 could significantly inhibit miR-300-promoted proliferation, invasion and EMT. Therefore, our results identify an important role for miR-300 in osteosarcoma through regulating BRD7 expression.

Background: MicroRNAs (miRNAs) are involved in the regulation of osteogenic differentiation and chondrification in vivo. The purpose of the present study was to explore the potential mechanism of miR-128 in osteoporosis (OP). Methods: Quantitative real-time PCR (qRT-PCR) was used to determine the expression of miR-128 in femoral neck trabecular bones of OP patients (n=40) and non-OP patients (n=40). C2C12 cells were transfected with miR-128 mimic or inhibitor to determine the effect of miR-128 on osteoblastic differentiation of C2C12 cells. Bioinformatics and luciferase reporter genes were used to determine the molecular mechanism of miR-128 in osteoblastic differentiation of C2C12 cells. Results: The qRT-PCR results showed that the expression level of miR-128 in bone samples of OP patients was significantly higher than that of non-OP patients, while miR-128 was significantly down-regulated during the osteogenic differentiation of C2C12 cells. In addition, the results showed that overexpression of miR-128 significantly inhibited the mRNA and protein expression levels of osteocalcin (OC), alkaline phosphatase (ALP) and collagen I type-α1 (COL1A1) in C2C12 cells, while miR-128 inhibitor could reverse this effect. Bioinformatics analysis and dual-luciferase reporter assay found that silencing information regulatory protein 6 (SIRT6) was a direct target of miR-128. The qRT-PCR and Western Blot results found that miR-128 significantly down-regulated the mRNA and protein expressions of SIRT6. Furthermore, silencing SIRT6 significantly inhibited the promoting effect of the miR-128 inhibitor on the expression of osteoblast markers. Conclusion: The above results confirmed that miR-128 inhibited osteoblast differentiation in OP by down-regulating SIRT6 expression, thus accelerating the development of OP.

BackgroundThe aim of this study was investigate the effects of the sesquiterpene lactone, ludartin, on cell proliferation, cell migration, apoptosis, and the cell cycle in osteosarcoma cell lines, compared with a normal osteoblast cell line.Material/MethodsOsteosarcoma cell lines, MG-63 Saos-2 U-2OS, T1-73 143B, and HOS, and normal hFOB 1.19 osteoblasts, were cultured and treated with increasing doses of ludartin, The MTT colorimetric assay was used to measure cell metabolic activity and viability. Apoptosis was studied by fluorescence-activated cell sorting (FACS) using 4′,6-diamidino-2-phenylindole (DAPI) nuclear staining and Annexin-V/propidium iodide (PI) staining. Cell cycle was studied using flow cytometry. Cell migration and invasion were studied using wound healing and Boyden chamber assays. Protein expression was measured by Western blotting.ResultsLudartin inhibited cell viability, cell migration, cell proliferation, and increased cell apoptosis, in all osteosarcoma cell lines, with an IC50 dose ranging from 15–30 μM. The greatest effects were on the Saso-2 osteosarcoma cells, with an IC50 of 15 μM. However, ludartin showed minor cytotoxic effects of the normal hFOB 1.19 osteoblasts (IC50 >100 μM). Ludartin exerted its anti-proliferative effects on Saos-2 cells via induction of apoptosis and cell cycle arrest at the G2/M checkpoint, associated with reduced expression of Cdc25c (Ser216), Cdc25c, pCdc2 (Tyr15), and Cdc2 and increased expression of p21WAF1. Ludartin inhibited cell migration and invasion of the Saos-2 cells.ConclusionsThe dose-dependent effects of ludartin on cell proliferation, migration, apoptosis, cell cycle arrest at the G2/M checkpoint involved p21WAFI in Saos-2 osteosarcoma cells.

Previous studies have demonstrated that connective tissue growth factor (CTGF) is expressed at increased levels in prostate cancer bone metastasis mouse models and patients with prostate cancer which metastasizes to the bone; however, the underlying molecular mechanism(s) remain unknown. The present study investigated the function of CTGF in osteoblast differentiation and its effect on prostate cancer bone metastasis by analyzing CTGF gene expression and transcription at different levels of invasion, metastasis of prostate cancer cells, and the influence of CTGF on proliferation and xenotransplantation. A mouse model demonstrating bone metastasis was used to investigate the function(s) of CTGF in bone metastasis and osteoblast differentiation. Results demonstrated that CTGF expression was increased in association with high bone metastasis in prostate cancer cells, and its expression was significantly decreased in whole cell lysates. CTGF expression in prostate cancer cells with high levels of bone metastasis was increased 1.9-fold compared with prostate cancer cells with low levels of bone metastasis. The expression of CTGF in mesenchymal cells was markedly increased compared with epithelial cells. Results indicated that the increased expression of CTGF does not affect the proliferation of tumor cells and possesses no influence on tumor volume. Control and CTGF plasmids were transfected into RM1 cells and led to 4 and 17% bone lesions, respectively. Increased expression of CTGF significantly enlarged the tumor area in the bone metastatic position compared with the control. Positive areas of alkaline phosphatase were significantly decreased as the concentration of CTGF increased. The results of the present study demonstrated that CTGF promotes prostate carcinoma to metastasize in the bone by dysregulating osteoblast differentiation.

Podoplanin (PDPN) is an important positive regulator of platelet aggregation and functions as a lymphatic endothelial marker. PDPN has been observed to be expressed in human tumor tissues and various cancer cell lines. In the present study, PDPN expression in patients with primary osteosarcoma was assessed at the mRNA and protein levels, and the associations between PDPN expression and pulmonary metastasis (PM) and prognosis were examined. Reverse transcription-quantitative PCR (RT-qPCR) analysis was used to detect the expression levels of PDPN in primary osteosarcoma tissues and paired normal bone tissues (n=20 pairs). In addition, immunohistochemical analysis of PDPN expression was performed in 168 paraffin-embedded osteosarcoma tissue specimens and 23 matched normal tissues. The RT-qPCR results revealed higher mRNA expression levels of PDPN in patients with PM compared with patients without PM. Further survival analyses identified Enneking stage and PM as two independent prognostic indicators. Finally, univariate analysis revealed that high PDPN protein expression was significantly associated with Enneking stage and PM in patients with osteosarcoma.