MiR-24-BIM-Smac/DIABLO axis controls the sensitivity to doxorubicin treatment in osteosarcoma.

Emerging evidence shows that microRNAs (miRNAs) act as critical regulators in the progression and chemoresistance of multiple tumors, including osteosarcoma (OS). In this study, we found that the level of miR-24 was increased in OS patients' serum, tumor tissues and OS cell lines. Furthermore, we found that knockdown of miR-24 by its specific inhibitors significantly increased the therapeutic effect of doxorubicin (DOX) on OS cell lines (MG-63 and HOS). Moreover, miR-24 inhibitors resensitized the doxorubicin-resistant MG-63 cells (MG-63/R) and HOS cells (HOS/R) to DOX. As the gene of Bcl-2 interacting mediator of cell death (BIM) was proved to be a target of miR-24 in MG-63/R cells, we further observed that the miR-24 inhibitors promoted the DOX-induced apoptosis via mitochondrial pathway. In addition, results of immunoprecipitation showed the release of second mitochondria derived activator of caspase/ direct IAP binding protein with low pI (Smac/DIABLO) abolished the biological activity of X-linked inhibitor of apoptosis protein (XIAP) by binding with it, which subsequently induced the activation of caspase 9, 7 and 3. In summary, those results strongly suggest that the miR-24-BIM-Smac/DIABLO axis might be a novel target for the treatment of OS.

Characterization of MicroRNA Expression Profiles in Patients with Giant Cell Tumor.

OBJECTIVE: Giant cell tumors of bone (GCTs) are bone destructive neoplasms, the bone resorption being mediated by osteoclasts. Given that microRNAs are crucially involved in tumorigenesis and the modulation of cell fate and behavior, they are promising candidates for regulation of osteoclastogenesis. However, no reliable miRNAs profile for GCT is available. Our study aimed to evaluate osteoclastogenesis-related miRNA expression in GCTs of Chinese patients. METHODS: From January 2013 to December 2014, 11 patients with GCTs were treated in our department and grouped into a GCT group. A control group comprising four patients with benign tumors of the iliac bone was established. The diagnoses were initially established by imaging examinations and intraoperative frozen sections and later confirmed by standard histologic examination. The GCT group (five male and six female patients) were aged from 17 to 61 years (mean, 32.9 years; SD, 12.8 years). Six patients with GCT underwent intralesional curettage surgery and the other five wide resection. According to Campanacci grading, four patients had Grade I tumors, three Grade II, and three Grade III. The average age of the control group was 28.75 years (SD, 14.24 years); all of them were diagnosed as having benign tumors and underwent iliac grafting. The morphology of the excised tissue was evaluated by examining standardized hematoxylin and eosin (HE) stained paraffin-embedded samples. In all, three osteoclastogenesis-related RNAs and 20 microRNAs (miRNAs) were extracted from the patients. The strength of expression was assessed by quantitative reverse transcription polymerase chain reaction (PCR ) and the results assessed by a Student's t test. RESULTS: Examination of HE stained sections revealed that the higher the Campanacci grade, the more numerous and bigger the osteoclasts (P < 0.05). PCR results indicated large amounts of osteoclast-related mRNA (cathepsin K, tartrate-resistant acid phosphatase and matrix metalloproteinase9) in GCTs (P < 0.05). Expression of six miRNAs was significantly weaker in the GCT than the control group (P < 0.05). The expression of has-mir-16-5p and has-let-7a-5p was correlated with Campanacci grade in the GCT patients (P = 0.009 and 0.034, respectively). The expression of these two miRNAs may indicate severity of bone destruction. CONCLUSION: Overall, the clinical utility of six novel miRNA markers for GCTs was demonstrated. Of these, strength of expression of hsa-mir-16-5p and hsa-let-7a-5p may indicate the grade of bone resorption.

DEC2 expression is positively correlated with HIF-1 activation and the invasiveness of human osteosarcomas.

BACKGROUND: Osteosarcoma is the most common malignancy of bone. HIF-1 (hypoxia-inducible factor 1) activation is critical for the metabolic reprogramming and progression of solid tumors, and DEC2 (differentiated embryonic chondrocyte gene 2) has been recently reported to suppress HIF-1 in human breast and endometrial cancers. However, the roles of HIF-1 and DEC2 in human osteosarcomas remain unclear. METHODS: We evaluated the correlation of DEC2 and HIF-1 expression to the prognosis, and studied the roles of DEC2 and HIF-1 activation in the invasiveness of osteosarcoma. Multiple approaches including immunohistochemical staining of clinical osteosarcoma tissues, siRNA-based knockdown and other molecular biology techniques were used. Particularly, by using a repetitive trans-well culture-based in vitro evolution system, we selected a more invasive subpopulation (U2OS-M) of osteosarcoma cells from U2OS and used it as a model to study the roles of DEC2 and HIF-1 in the invasiveness of osteosarcoma. RESULTS: We found that the expression of DEC2 was positively correlated with HIF-1α levels, and HIF-1α expression positively correlated with poor prognosis in osteosarcomas. DEC2 knockdown in osteosarcoma cell lines (U2OS, MNNG and 143B) attenuated HIF-1α accumulation and impaired the up-regulation of HIF-1 target genes in response to hypoxia. Compared with the low invasive parental U2OS, U2OS-M showed higher levels of DEC2 expression which were confirmed at both mRNA and protein levels. Importantly, we found that the increased DEC2 expression resulted in a more rapid accumulation of HIF-1α in U2OS-M cells in response to hypoxia. Finally, we found that HIF-1 activation is sufficient to upregulate DEC2 expression in osteosarcoma cells. CONCLUSION: Taken together, whereas DEC2 was found to promote HIF-1α degradation in other types of tumors, our data indicate that DEC2 facilitates HIF-1α stabilization and promotes HIF-1 activation in osteosarcoma. This implies that DEC2 may contribute to the progression and metastasis of human osteosarcoma by sensitizing tumor cells to hypoxia. On the other hand, HIF-1 activation may contribute to the expression of DEC2 in osteosarcoma. This is the first demonstration of a novel DEC2-HIF-1 vicious cycle in osteosarcoma and a tumor-type specific role for DEC2.

Baicalein suppresses the viability of MG-63 osteosarcoma cells through inhibiting c-MYC expression via Wnt signaling pathway.

The major reason responsible for the poor prognosis of osteosarcoma is the malignant proliferation of osteosarcoma cells. The activated Wnt/ß-catenin signaling induces c-MYC gene transcription and results in osteocytes' carcinomatous change, which contributes to osteosarcoma development, so c-MYC gene is one of the therapeutic targets. The role of multiple botanical extracts in the expression of ß-catenin's target gene c-MYC in osteosarcoma MG-63 cells was tested by cellomics high content screening. Baicalein was identified as the most effective one which can inhibit the proliferation and promote the apoptosis of MG-63 cells in a dose-dependent manner by cell counting kit-8 test and fluorescence-activated cell sorting, respectively. This process was associated with the decreased levels of ß-catenin and its target gene c-MYC, identified by q-PCR and Western blotting, respectively. When MG-63 cells were treated with both baicalein and JNK inhibitor SP600125, the apoptosis and expression of c-MYC were not significantly decreased. After the construct pcDNA3.1-BANCR (BRAF-regulated lncRNA 1) was transfected into MG-63 cells, RT-PCR, Western blotting and CCK-8 assay showed that BANCR was positively correlated with baicalein. These results indicated that baicalein inhibited osteosarcoma cell proliferation and promoted apoptosis by targeting c-MYC gene through Wnt signaling, in which JNK and BANCR were also involved as well as ß-catenin, suggesting a new potential mechanism for us to better understand the inhibiting effect of baicalein on osteosarcoma.

Investigation on the DNA repaired gene polymorphisms and response to chemotherapy and overall survival of osteosarcoma.

The aim of the present study was to evaluate the influence of polymorphisms in NER and HRR pathways on the response to cisplatin-based treatment and clinical outcome in osteosarcoma patients. 214 osteosarcoma patients treated with cisplatin-based chemotherapy were collected between January 2008 and January 2011. Genotypes of ERCC1 rs11615, ERCC2 rs1799793 and rs13181, NBN rs709816, RAD51 rs1801320, and XRCC3 rs861539 were conducted by Polymerase Chain Reaction Restriction Fragment Length Polymorphism (PCR-RFLP) assay. By conditional logistic regression analysis, patients carrying CC genotype of ERCC1 rs11615 showed a significant more good responder than TT genotype, and the OR (95% CI) was 2.51 (1.02-6.85). In the Cox proportional hazards model, after adjusting for potential confounding factors, we found that individuals carrying CC genotype of ERCC1 rs11615 was associated with decreased risk of death from osteosarcoma, and the HR (95% CI) was 0.43 (0.15-0.93). In conclusion, our results suggest that ERCC1 rs11615 polymorphism in the DNA repair pathways play an important role in the response to chemotherapy and overall survival of osteosarcoma.

Role of ß-isomerized C-terminal telopeptides (ß-CTx) and total procollagen type 1 amino-terminal propeptide (tP1NP) as osteosarcoma biomarkers.

INTRODUCTION: Few serum markers are valid and useful for the diagnosis or therapeutic effect monitoring of osteosarcoma. This study aimed to investigate the role of ß-isomerized C-terminal telopeptides (ß-CTx) and total procollagen type 1 amino-terminal propeptide (tP1NP) as serological biomarkers for osteosarcoma patients. MATERIALS AND METHODS: A total of 48 patients with osteosarcoma and 55 healthy volunteers were investigated. Serum ß-CTx and tP1NP levels were measured by electrochemiluminescence immunoassay. Data were analyzed by t test with Walth's correction and receiver operating characteristic (ROC) curve analysis. RESULTS: The baseline levels of ß-CTx and tP1NP were found to be significantly higher in patients with osteosarcoma than the healthy volunteers. The mean areas under the ROC curves were 0.919 (range, 0.864-0.973) for ß-CTx and 0.866 (range, 0.792-0.939) for tP1NP. The levels of ß-CTx and tP1NP were lower in patients with stable disease after operation than those before operation. CONCLUSION: These findings support our hypothesis that ß-CTx and tP1NP are promising serum biomarkers for diagnosing or monitoring osteosarcoma.

IL6-174G/C polymorphism and fracture risk.

Several studies have examined the associations of polymorphism in interleukin-6 (IL6) with fracture risk. However, the results were conflicting. Thus, a meta-analysis was conducted to determine the relationship between IL6-174C/G polymorphism and risk of fracture. Databases including PubMed, EMBASE, Wanfang were searched. Summary odds ratios (ORs) and corresponding 95% confidence intervals (CIs) were estimated using random-effects models. Six studies were included in this meta-analysis. IL6-174C/G polymorphism was associated with a significantly increased risk of fracture (OR=1.32; 95% CI, 1.10-1.58; I(2) =11%; Figure 1). In the subgroup analysis according to gender, women (RR=1.26; 95% CI, 1.09-1.46; I(2) =0%) was significantly associated with risk of fracture. In the age subgroup analysis, old population (RR=1.27; 95% CI, 1.11-1.48; I(2) =0%) showed increased fracture risk. However, young population did show increased risk of fracture (RR=1.95; 95% CI, 0.70-5.47; I(2) =51%). Postmenopausal women also showed an increased fracture risks (RR=1.24; 95% CI, 1.08-1.44; I(2) =0%). This meta-analysis suggested that IL6-174C/G polymorphism contributed the development of fracture.

Association between TGF-ß1 +869C/T polymorphism and fracture risk: a meta-analysis.

The association between TGF-ß1 +869C/T polymorphism and risk of fractures remained controversial. Therefore, we performed this meta-analysis to investigate this association. We searched PubMed, EMBASE, and Wangfang databases for studies before Aug 2014. Odds ratios (ORs) with 95% confidence intervals (CIs) were used to calculate the strength of association. A total of ten studies were included in this meta-analysis. TGF-ß1 +869C/T polymorphism was associated with a significantly increased risk of fracture (OR=1.41; 95% CI, 1.20-1.65; I(2) =0%). In the subgroup analysis according to gender, women was significantly associated with risk of fracture (OR=1.44; 95% CI, 1.20-1.73; I(2) =4%). In the subgroup analysis by race, Asians (OR=1.43; 95% CI, 1.06-1.92; I(2) =0%) and Caucasians (OR=1.44; 95% CI, 1.13-1.85; I(2) =15%) showed increased fracture risk. Our meta-analysis suggested that the TGF-ß1 +869C/T polymorphism may be a risk factor for developing fracture.