Tumor growth was measured using caliper daily, and tumor volume was calculated according to the formula: volume = length × width2 × 0.5. The human B-cell lymphoma-extra large (Bcl-xL) and myeloid cell leukemia-1 (Mcl-1) 3′UTR luciferase reporter constructs and these constructs with miR-133a target site deletion

were made as we described previously [15]. All constructs were confirmed by DNA sequencing. Luciferase reporter assay was performed as reported [15]. Briefly, at 36 h post transfection, luciferase activities were detected using Dual-Luciferase Reporter Assay System (Promega) following the manufacturer’s instructions. Data were normalized by ITF2357 solubility dmso dividing Firefly luciferase activity with that of Renilla luciferase. Cells and grinded human tissues were lysed using M-PER Protein Extraction Reagent (Pierce) supplemented with protease inhibitor cocktail (Calbiochem). Protein concentrations of the extracts were measured with BCA assay (Pierce) and equalized with the extraction reagent. Equal amounts of the extracts were loaded and subjected to SDS-PAGE, transferred onto nitrocellulose membranes, and then blotted as reported [15]. Antibodies specific to Bcl-xL, Mcl-1, β-actin, and horseradish peroxidase-coupled secondary antibodies were purchased from Cell Signaling

Technology. Densitometric analysis was done with Labworks Image Acquisition and Analysis Software (UVP, Upland, CA). The background was subtracted, and the signals of the detected bands were normalized to the amount of loading control β-actin band. Data are shown as mean ± s.d. Statistical comparisons between groups were analyzed

selleck using Student’s t-test and a two-tailed p < 0.05 was considered to indicate statistical significance. The correlation between miR-133a expression and clinical osteosarcoma stages was analyzed using Spearman's rank correlation coefficient assay in SPSS 17.0. Analysis of overall survival in osteosarcoma patients was performed using log-rank test in SPSS 17.0. The correlation between miR-133a expression and Bcl-xL or Mcl-1 protein levels was analyzed using Pearson's correlation coefficient assay in SPSS 17.0. In order to investigate the roles of miR-133a in human osteosarcoma development, we compared miR-133a expression in human normal osteoblastic cell line hFOB 1.19 with that Dimethyl sulfoxide in human osteosarcoma cell lines MG63 and U2OS by real-time qRT-PCR. And miR-133a expression was significantly decreased in osteosarcoma MG63 and U2OS cells (Fig. 1A). Furthermore, in the 92 pairs of human primary osteosarcoma tumor and adjacent normal tissue samples, miR-133a expression was significantly suppressed in tumor tissues as compared to that in adjacent normal tissues (Fig. 1B). These results suggest that miR-133a is downregulated in osteosarcoma cells, which might be involved in human osteosarcoma development. We next investigated whether the downregulation of miR-133a was correlated with osteosarcoma progression.