By targeting TRAF6, miR-140-3p inhibits TGF-ß1-induced human osteosarcoma epithelial-to-mesenchymal transition, migration, and invasion.

OBJECTIVES: We evaluated the effects of miR-140-3p on EMT, cellular migration, and invasion in TGF-ß1 treated human OS cells. Human fresh OS tissue and normal bone tissue specimens were gathered from 42 patients (29 male and 13 female, 11 to 24 years of age with a mean age of 17.5 ± 2.3 years) diagnosed with OS by pathology. By targeting TRAF6, miR-140-3p inhibits TGF-ß1-induced human osteosarcoma epithelial-to-mesenchymal transition, migration, and invasion. RESULTS: In this study, we found microRNA (miR)-140-3p to be down-regulated and tumor necrosis factor receptor-associated factor 6 (TRAF6) to be up-regulated in patient OS samples. Lower levels of miR-140-3p and higher levels of TRAF6 were found in the advanced Enneking stage of OS. Furthermore, both mRNA and protein levels of TRAF6 were negatively associated with miR-140-3p mRNA expression in human OS tissue. TRAF6 was verified as a direct target of miR-140-3p in TGF-ß1-treated human U2OS cells. Further, a miR-140-3p mimic dramatically inhibited while a miR-140-3p inhibitor enhanced TGF-ß1-induced epithelial-to-mesenchymal transition, migration, and invasion of U2OS cells. Small interfering RNA was found to silence TRAF6 and to partly reverse the effects of the miR-140-3p inhibitor on TGF-ß1-treated U2OS cells in vitro. CONCLUSION: These results demonstrate miR-140-3p to function as a tumor inhibitor of human OS cells by decreasing TRAF6 expression. miR-140-3p and TRAF6 may be valuable and novel biomarkers for diagnosis and treatment of OS.

A new candidate substrate for cell-matrix adhesion study: the acellular human amniotic matrix.

In vivo adhesions between cells and the extracellular matrix play a crucial role in cell differentiation, proliferation, and migration as well as tissue remodeling. Natural three-dimensional (3D) matrices, such as self-assembling matrices and Matrigel, have limitations in terms of their biomechanical properties. Here, we present a simple method to produce an acellular human amniotic matrix (AHAM) with preserved biomechanical properties and a favorable adhesion potential. On the stromal side of the AHAM, human foreskin fibroblasts (HFFs) attached and extended with bipolar spindle-shaped morphology proliferated to multilayer networks, invaded into the AHAM, and migrated in a straight line. Moreover, αV integrin, paxillin, and fibronectin were observed to colocalize after 24 h of HFF culture on the stromal side of the AHAM. Our results indicate that the AHAM may be an ideal candidate as a cell-matrix adhesion substrate to study cell adhesion and invasion as well as other functions in vitro under a tensile force that mimics the in vivo environment.

Tibial lengthening over an intramedullary nail in patients with short stature or leg-length discrepancy: a comparative study.

PURPOSE: The aim of this study was to review our experiences with tibial lengthening over an intramedullary nail in comparison to the conventional Ilizarov method. METHODS: We performed a retrospective comparison of tibial lengthening using the conventional Ilizarov method (group A: 23 limbs in 13 patients) versus over a nail (group B: 51 limbs in 26 patients). The percentage increase in tibial length, lengthening index, external fixation index, consolidation index and complications were assessed. RESULTS: The mean gain in tibial length was 7.4 cm, which represents a mean increase of 26.0%. There was no difference in lengthening index or consolidation index; however, the patients in group A wore the external fixator longer than those in group B (281.5 versus 129.0 days), which represents a larger external fixation index (40.0 versus 17.4 day/cm). Group A had a higher complication rate (1.0 versus 0.47 per tibia) than group B. CONCLUSIONS: Tibial lengthening over an intramedullary nail confers advantages over the conventional Ilizarov method, including shorter time needed for external fixation and lower complication rates.

[Detection of serum biomakers of osteosarcoma by proteomic profiling].

OBJECTIVE: To detect differentially expressed proteins in serum of patient with osteosarcoma. METHODS: 8 serum protein samples were recruited (4 cases of osteosarcoma and 4 cases of normal adults), cross-labeled with variant CyDye, followed by two-dimensional differential in-gel electrophoresis (2-D DIGE), image analysis, and identified by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS). RESULTS: 24 protein spot-features were significantly increased, and 34 were significantly decreased in the serum from patients with osteosarcoma relative to the controls. The mass spectrometry analysis revealed 18 unique proteins that were increased, and 25 unique proteins decreased in the serum of patients with osteosarcoma. Gelsolin was down-regulated in osteosarcoma, and Western blotting also confirmed a decreased level of gelsolin in the serum of patients with osteosarcoma. CONCLUSION: Our results indicate that gelsolin may have great potential as a biomarker of osteosarcoma and as a potential target for therapy. These preliminary data suggest that incorporation of more samples and new datasets will permit the identification of serum biomarkers for osteosarcoma.

Establishment and characteristics of two syngeneic human osteosarcoma cell lines from primary tumor and skip metastases.

AIM: To characterize and compare the different biological behaviors of 2 novel human osteosarcoma cell lines, Zos and Zos-M, established respectively from the primary tumor and the skip metastasis of an osteosarcoma patient. METHODS: In vitro studies included morphological observations, karyotype analysis, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cell proliferation assay, and cell sensitivity to chemotherapeutic drugs. Subcutaneous and intravenous inoculations into nude mice were carried out to study the tumorigenicity and the metastatic potential. RT-PCR was performed to assess the expression of the osteoblastic markers and some metastasis-related genes. RESULTS: Both cell lines remained stable for more than 100 passages in vitro without interruption. The RT-PCR examination indicated that they retained the molecular characteristics of an osteoblastic lineage. The karyotype analysis displayed aneuploidy and various structural abnormalities. Both cell lines are tumorigenic; Zos-M differs from Zos by the former's ability to develop lung metastasis after intravenous injection. The comparison of the expression patterns of some metastasis-related genes revealed that the decreased expression of cadherin-11 in Zos-M may correlate with a high potential of metastases. Moreover, both cell lines are less sensitive to the current chemotherapy protocols. CONCLUSION: The establishment of osteosarcoma cell lines, Zos and Zos-M, and related animal models provide a useful resource for studying the aggressive behavior of osteosarcoma and will be helpful for screening effective treatment strategies.

Comparative proteomic analysis of human osteosarcoma and SV40-immortalized normal osteoblastic cell lines.

AIM: Comparative proteomics provide a powerful approach in screening for alterations in protein levels and post-translational modifications that are associated with tumors. In the present study, we aimed to identify candidate biomarkers to distinguish osteosarcoma (OS) cells from normal osteoblastic cells. METHODS: We employed 3 OS cell lines (U2OS, IOR/OS9, and SaOS-2), and used the SV40-immortalized normal osteoblastic cell line (hFOB1.19) as the control. The differential protein levels in OS and osteoblastic cells were identified using 2-D gel electrophoresis followed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry analyses. Two proteins of interest, the levels of which were significantly increased in OS cells, were further characterized by Western blot analyses. RESULTS: Twenty-six proteins were identified, the expression level of which was either significantly increased or decreased in the OS cells as compared to the control cells. The expression level of the activator of 90 kDa shock protein ATPase homolog 1 (AHA1), was enhanced 12.4-, 24.1-, and 23.8-fold in SaOS-2, IOR/OS9, and U2OS cells, respectively, and the level of the stomatin-like protein 2 (SLP-2) was increased by 10.4- and 7.8-fold in IOR/OS9 and U2OS cells, respectively, as compared to normal osteoblastic cells. Those observations were confirmed by Western blot analyses. CONCLUSION: A differential proteomic analysis was successfully used to identify AHA1 and SLP-2 that were significantly overproduced in OS cells as compared to normal osteoblastic cells, suggesting that those proteins among others may be effective biomarker candidates for the identification of OS cells.

Bufalin induces apoptosis in human osteosarcoma U-2OS and U-2OS methotrexate300-resistant cell lines.

AIM: To investigate the antiproliferative activity and apoptosis-inducing effects of bufalin on human osteosarcoma cell lines. METHODS: U-2OS and U-2OS methotrexate (MTX) 300-resistant cell lines were treated with bufalin. Cell viability was assessed using the MTT assay. Cell-cycle status, apoptosis-inducing effects, and the expression of apoptosis-related proteins were evaluated by flow cytometry, fluorescent staining, DNA fragmentation assays, and Western blotting. The effect of bufalin on dihydrofolate reductase (DHFR) expression was studied by RTPCR and Western blotting. RESULTS: Bufalin inhibited cell growth in both U-2OS and U-2OS MTX300 cells. The induction of G2/M cell-cycle arrest was also seen in the cells treated with bufalin. The induction of apoptosis by bufalin was confirmed by increased expression of the tumor suppressor protein p53 and the increased ratio of the Bax/Bcl-2 proteins. Bufalin induced apoptosis to the same extent in both cell lines without regard to DHFR levels in the cells. CONCLUSION: Bufalin inhibited the growth of and induced apoptosis in both MTX-sensitive and MTX-resistant human osteosarcoma U-2OS cells. The apoptosis-inducing effect of bufalin was not influenced by the presence of high levels of the DHFR protein.

2-D DIGE and MALDI-TOF-MS analysis of the serum proteome in human osteosarcoma.

We performed 2-D DIGE on proteins prepared from serum obtained from patients with osteosarcoma (OS) and controls, to identify differentially expressed proteins that might serve as serum biomarkers for OS prognosis. Proteins found to be differentially expressed were identified by MALDI-TOF mass spectrometric analysis, coupled with database interrogation. We compared serum samples from four individuals with OS to four age- and sex-matched healthy controls. We identified 24 protein spot-features that were significantly increased, and 34 that were significantly decreased in serum from patients with OS relative to the controls. The MS analysis revealed 18 unique proteins that were increased, and 25 unique proteins that were decreased in OS serum samples. Western blot and ELISA analysis confirmed increased levels of amyloid-related serum protein (SAA) in the OS serum samples. The increased expression levels of SAA were decreased after using MTX and cisplatin combination chemotherapy, and were further decreased after operation. Moreover, increased expression levels of sera SAA were seen in the relapsed patients. Our results suggested that the determination of serum SAA in OS patients might be utilized as a marker for relapse and in evaluation of the efficacy of therapy.

[Analysis of the factors affecting the recurrence of giant cell tumor of bone].

OBJECTIVE: To analyze the clinical factors affecting the recurrence of giant cell tumors (GCT) of bone. METHODS: The complete data of 146 cases with GCT were reviewed. Thirteen clinical factors were analyzed by chi(2) analysis. And the related Campanacci's grade system and Jaffe's grade system was analyzed by Crosstabs analysis. Multipal factors were analyzed by Logistic regression analysis. RESULTS: Nineteen of 146 cases recurred, and recurrence rate was 13.0%. Recurrence rates of curettage and enblock resection groups were 18.8% and 6.3% respectively. And recurrence rates of curettage with or without of extensive procedure were 12.9% and 38.9%. Five cases had lung metastasis, and two cases presented with malignant transformation. The metastasis rate and the rate of malignant transformation were 3.4% and 1.4% respectively. The two factors of surgery method and burst out of bone-envelope appearance were related with the recurrence. Moreover, Logistic regression revealed that the surgery method significantly affected the recurrence. And Campanacci's grade system and Jaffe's grade system were not related to each other. CONCLUSIONS: Surgery method is the main factor affected the recurrence of GCT, and Campanacci's grade system or Jaffe's grade system has no prognostic value.