Role In Osteosarcoma Research Articles

Expression and function of PIM kinases in osteosarcoma.

The provirus integrating site Moloney murine leukemia virus (PIM) family of serine/threonine protein kinases is composed of three members, PIM1, PIM2 and PIM3, which have been identified as oncoproteins in various malignancies. However, their role in osteosarcoma (OS) remains largely unknown. This study aimed to examine the expression patterns and the clinical significance of PIM kinases in human OS and their biological effects in human OS cell lines. Immunohistochemical staining was used to detect PIM kinases in archived pathologic material from 43patients with primary OS; in addition, the effects of PIM knockdown and overexpression on the proliferation, migration and invasion of OS cell lines were determined. We observed that all three PIM kinases were frequently expressed in OS, but only PIM1 positive expression was associated with poorer prognosis regarding overall survival of OS patients. In addition, knockdown of PIM kinases notably inhibited OS cell proliferation, migration and invasiveness, whereas overexpression of PIM kinases resulted in increased OS cell growth and motility. This study suggests that PIM1 could be a valuable prognostic marker in patients with OS, and the biological functions of PIM kinase family in the osteosarcoma cell lines indicate that they could serve as potential therapeutic targets for OS.

The predictive potential and oncogenic effects of HOXC8 expression on osteosarcoma.

Homeobox C8 (HOXC8) has been implicated in cell growth, migration, and metastasis of various cancers, yet its role in osteosarcoma remains to be explored. In the present study, resected osteosarcoma specimens from 50 patients were enrolled to evaluate the expression of HOXC8 protein by immunohistochemistry (IHC). In vitro and in vivo assays were used to determine the effect of HOXC8 on cell growth, migration, and tumor growth. HOXC8 expression was observed in 31 (62.0%) of the 50 primary tumors and significantly associated with poorly or un-differentiated specimens (P=0.031) and larger tumor size (P=0.049). Survival analysis demonstrated that HOXC8 is a candidate predictive factor in predicting patients' outcome and chemotherapeutic effect. HOXC8 knockdown led to inhibition of tumor cell proliferation and migration in vitro by inhibiting MMP-9 expression and tumor growth in vivo. Our results strongly suggest that HOXC8 is involved in the tumorigenesis of osteosarcoma and might serve as a novel predictor for patients' outcome.

MicroRNA-181a promotes proliferation and inhibits apoptosis by suppressing CFIm25 in osteosarcoma.

MicroRNA-181a (miR-181a) is upregulated in osteosarcoma, and its overexpression promotes the proliferation and inhibits the apoptosis of osteosarcoma cells. However, the mechanism of miR‑181a as an oncogene remains to be fully elucidated in osteosarcoma. Cleavage factor (CF)Im25 links alternative polyadenylation to glioblastoma tumor suppression, however, its role in osteosarcoma has not been reported. In the present study, it was confirmed that the expression of miR‑181a was upregulated in osteosarcoma, and that silencing miR‑181a inhibited the proliferation and promoted the apoptosis of osteosarcoma cells. miRNAs are short non‑coding RNAs, which regulate target mRNAs by binding predominantly to the 3'untranslated region (3'UTR), inducing either translational repression or degradation of the target. In the present study, target genes of miR‑181a were screened using miRanda, which is a commonly used prediction algorithm. It was found that miR‑181a targeted the 3'UTR of CFIm25 mRNA. Subsequent experiments confirmed that miR‑181a downregulated the expression of CFIm25 in osteosarcoma cells. Finally, it was demonstrated that the CFIm25 protein was also downregulated in osteosarcoma tissues, and inhibited the proliferation and promoted the apoptosis of the cells. Elucidating the roles of miR‑181a and CFIm25 in osteosarcoma not only assists in further understanding the pathogenesis and progression of this disease, but also offers novel targets for effective therapies.

Overexpression of miR-506 suppresses proliferation and promotes apoptosis of osteosarcoma cells by targeting astrocyte elevated gene-1.

There is increasing evidence that microRNAs (miRs) are implicated in tumor development and progression; however, their specific roles in osteosarcoma are not well understood. The aim of the present study was to investigate the role of miR-506 in the pathogenesis of osteosarcoma. The expression levels of miR-506 and astrocyte elevated gene-1 (AEG-1) mRNA were detected using quantitative polymerase chain reaction, and the protein levels of AEG-1, β-catenin, c-myc and cyclin D1 were determined using western blot analysis. The effects of miR-506 and AEG-1 on cell viability, colony forming ability and apoptosis were assessed using MTT assay, colony formation assay, and flow cytometry, respectively. Lucifer reporter assays were used to demonstrate whether AEG-1 is a direct target of miR-506. The present study identified that miR-506 was downregulated in osteosarcoma tissues and cells. Overexpression of miR-506 suppressed the proliferation and induced apoptosis in osteosarcoma cells in vitro and inhibited tumor formation in vivo. Overexpression of miR-506 significantly inhibited the luciferase activity of AEG-1 with a wild-type 3′-untranslated region, providing clear evidence that AEG-1 was a direct and functional downstream target of miR-506. Similar to the overexpression of miR-506, downregulation of AEG-1 lead to an inhibitory effect on osteosarcoma in vitro. Furthermore, overexpression of miR-506 or downregulation of AEG-1 inhibited the Wnt/β-catenin signaling pathway, and inhibition of this pathway by β-catenin small interfering RNA or CGP049090, a small molecule inhibitor, suppressed cell proliferation and induced apoptosis in vitro. Overall, the present data indicated that miR-506 functions as a tumor suppressor by targeting AEG-1 in osteosarcoma via the regulation of the Wnt/β-catenin signaling pathway.

Extracellular vesicles secreted by highly metastatic clonal variants of osteosarcoma preferentially localize to the lungs and induce metastatic behaviour in poorly metastatic clones.

Osteosarcoma (OS) is the most common pediatric bone tumor and is associated with the emergence of pulmonary metastasis. Unfortunately, the mechanistic basis for metastasis remains unclear. Tumor-derived extracellular vesicles (EVs) have been shown to play critical roles in cell-to-cell communication and metastatic progression in other cancers, but their role in OS has not been explored. We show that EVs secreted by cells derived from a highly metastatic clonal variant of the KHOS cell line can be internalized by a poorly metastatic clonal variant of the same cell line and induce a migratory and invasive phenotype. This horizontal phenotypic transfer is unidirectional and provides evidence that metastatic potential may arise via interclonal co-operation. Proteomic analysis of the EVs secreted by highly metastatic OS clonal variants results in the identification of a number of proteins and G-protein coupled receptor signaling events as potential drivers of OS metastasis and novel therapeutic targets. Finally, multiphoton microscopy with fluorescence lifetime imaging in vivo, demonstrated a preferential seeding of lung tissue by EVs derived from highly metastatic OS clonal variants. Thus, we show that EVs derived from highly metastatic clonal variants of OS may drive metastatic behaviour via interclonal co-operation and preferential colonization of the lungs.

The clinical significance of changes in ezrin expression in osteosarcoma of children and young adults.

Ezrin is a protein that functions as a cross-linker between actin cytoskeleton and plasma membrane. Its clinical role in osteosarcoma is unclear. The aim of this study was to investigate, in osteosarcoma, the prognostic value of ezrin expression at biopsy and changes in expression levels after preoperative chemotherapy. Thirty-eight newly diagnosed osteosarcoma patients aged 6-23years were included. At diagnosis, 20 patients had localized disease, the others had distant metastases. Median follow-up was 75months (range 13-135). Ezrin expression was assessed immunohistochemically in biopsy tissue and primary tumour specimens resected after chemotherapy. The influence on survival of changes in ezrin expression after chemotherapy was analysed. Ezrin expression was significantly higher after preoperative chemotherapy and changes compared to biopsy tissue were significantly lower in patients with early progression than in patients with relapse or no further evidence of disease (p=0.006 and p=0.002, respectively). Similarly, ezrin expression was higher after preoperative chemotherapy and exhibited less change in expression in deceased patients compared to patients surviving more than 5years (both p=0.001). Ezrin expression at biopsy was significantly associated with both histopathological aggressiveness (p<0.001) and tumour size (p=0.037). The results of this study provide evidence that changes in overexpression of ezrin due to preoperative chemotherapy could be a useful predictive and prognostic marker in patients with osteosarcoma.

Promotion of tumour proliferation, migration and invasion by miR-92b in targeting RECK in osteosarcoma.

MicroRNAs play important roles in the development of cancers. Although miR-92b has been reported to promote the tumorigenesis of some cancers, its role in osteosarcoma remains unknown. In the present study, we focused on the expression, function and mechanisms of miR-92b in osteosarcoma development. The miRNA miR-92b was up-regulated in osteosarcoma cell lines and tissues; miR-92b up-regulation correlated with poor prognosis in osteosarcoma. Overexpression of miR-92b promoted osteosarcoma cell proliferation, migration and invasion, which was abrogated by miR-92b inhibition. Reversion-inducing, cysteine-rich protein with kazal motifs (RECK) was identified as the direct and functional target of miR-92b in osteosarcoma. Importantly, restoring RECK expression abrogated increases in cell growth, motility and invasiveness induced by miR-92b RECK was down-regulated in osteosarcoma tissues, and its expression level negatively correlated with miR-92b Collectively, our results indicate that miR-92b acts as an oncogenic miRNA and may be a therapeutic target in osteosarcoma.

Lentivirus-Mediated Knockdown of CTHRC1 Inhibits Osteosarcoma Cell Proliferation and Migration.

Collagen triple helix repeat containing-1 (CTHRC1), a secreted protein, is transiently expressed in the arterial wall in response to injury, indicating that it may contribute to vascular remodeling by limiting collagen matrix deposition and promoting cell migration. Recent studies showed that it is aberrantly upregulated in most human solid tumors, yet its role in osteosarcoma remains unclear. In this study, the authors investigated the role of CTHRC1 in human osteosarcoma tumorigenesis. The authors used lentivirus-mediated short hairpin RNA (shRNA) against CTHRC1 to limit its endogenous expression in U2OS and SW1353 cells. Interestingly, they found that depletion of CTHRC1 significantly inhibited cell proliferation and colony formation in U2OS and SW1353 cells. Flow cytometry assay showed that knockdown of CTHRC1 increased the cell percentage of G0/G1 phase, resulting in cell cycle arrest in U2OS cells. Moreover, CTHRC1 silencing induced the cell cycle arrest by a decrease in the cell percentage in G0/G1 phase and increased in G2/M phase in SW1353 cells. In addition, crystal violet staining suggested CTHRC1 silencing inhibited migration of U2OS and SW1353 cells. These results demonstrated that CTHRC1 might play an important role in osteosarcoma progression.

Immunohistochemical Detection of Autophagy‐Related Proteins in Canine Appendicular Osteosarcoma

Dogs diagnosed with appendicular osteosarcoma commonly succumb to metastatic disease despite undergoing the standard of care treatment for curative intent: amputation of the affected limb, followed by adjuvant chemotherapy. Post‐surgical chemotherapy improves one‐ and two‐year survival rates, but prognosis is still poor. In response to adverse environmental conditions normal cells are capable of undergoing autophagy, a survival mechanism involving self‐degradation and recycling of cellular components. Neoplastic cells take advantage of this mechanism to enhance their survival during chemotherapy. Autophagy's role in osteosarcoma has been investigated in humans however similar studies in dogs are rare. Our objective was to examine the expression patterns of a select group of autophagy‐related proteins in canine osteosarcoma amputation samples and to identify potential correlations with survival. Immunohistochemistry for beclin‐1, LC3, phospho‐mTOR, phospho‐AKT1, phospho‐S6 ribosomal protein, and p62 was performed on a tissue microarray containing naïve canine appendicular osteosarcoma cores from dogs who began the standard of care treatment (≥1 dose of chemotherapy). For survival analyses, Kaplan‐Meier survival curves for different expression patterns were compared using the log‐rank test and interactions were considered significant if p &lt; 0.05. The median survival time from the date of diagnosis was 250 days and survival time ranged from 67–897 days (n=20, 95% CI: 122–381). Intense phospho‐S6 ribosomal protein labeling was identified most consistently adjacent to osteoid matrix. Phospho‐mTOR labeling was most evident in the nuclear compartment, however cells undergoing mitosis displayed strong cytoplasmic labeling. Canine osteosarcoma cells exhibited a mixture of diffuse and punctate LC3 cytoplasmic labeling, potentially distinguishing between free cytoplasmic LC3‐I and autophagosome anchored LC3‐II, respectively. Western blot analysis of the beclin‐1 antibody produced a 58 kDa band as predicted. Three distinct intensities of cytoplasmic labeling were observed for beclin‐1 in canine osteosarcoma cells. Strong cytoplasmic beclin‐1 labeling in the majority of neoplastic cells was not observed in any cases with greater than one‐year survival (0/6); this pattern was observed only in cases with less than one‐year survival (5/14). Dogs with tumours having light (n=4), moderate (n=9), and strong (n=5) cytoplasmic labeling had median survival times of 294, 301, and 122 days, respectively. Strong cytoplasmic labeling was a significantly correlated with decreased survival time (Chisq=6.9, 2 df, p &lt; 0.05). This preliminary study provides evidence that canine osteosarcoma cells undergo autophagy in vivo, with a similar LC3 labeling pattern to that described in other tumour types in various species. Additionally, it identifies beclin‐1 as a promising candidate biomarker of chemoresponsiveness in canine appendicular osteosarcoma.Support or Funding InformationFunded by Pet Trust

Overexpression of SCARA5 inhibits tumor proliferation and invasion in osteosarcoma via suppression of the FAK signaling pathway.

Scavenger receptor class A, member 5 (SCARA5) is a member of the scavenger receptor family, and is involved in several types of human malignancy; however, its roles in osteosarcoma (OS) remain to be fully elucidated. Therefore, in the present study, the biological functions of SCARA5 in OS, and the potential underlying mechanisms were investigated. SCARA5 expression in OS tissues and cell lines was detected by reverse transcription‑quantitative polymerase chain reaction and western blot analysis. The effects of SCARA5 on the proliferation and migration/invasion ability of OS cells were determined by MTT and Transwell chamber assays, respectively. Expression levels of phosphorylated focal adhesion kinase (p‑FAK), FAK, p‑Src, Src, matrix metalloproteinase (MMP)2 and MMP9 were evaluated via western blot analysis. The results of the present study demonstrated that SCARA5 was expressed at low levels in OS tissues and cell lines. The overexpression of SCARA5 significantly inhibited the proliferation, colony formation and migration/invasion abilities of the OS cells. Furthermore, SCARA5 significantly decreased the expression levels of p‑FAK, MMP‑2 and MMP‑9 in the OS cells. Taken together, these data suggested that the overexpression of SCARA5 inhibits tumor proliferation and invasion in OS via suppression of the FAK signaling pathway. Thus, novel therapeutic strategies or drugs targeted at SCARA5 may offer potential for the treatment of OS.

MicroRNA-21 promotes proliferation, invasion and suppresses apoptosis in human osteosarcoma line MG63 through PTEN/Akt pathway.

Osteosarcoma, which accounts for 5% of pediatric tumor, remains the major cause of death among orthopedic malignancies. However, the factors associated with its malignant biological behavior are still poorly understood. MicroRNAs are a class of small noncoding RNAs, which have been considered to associate with malignant progression including cell differentiation, proliferation, apoptosis, invasion, and distant metastasis. In our research, we found that microRNA-21 (miR-21) was significantly overexpressed in human osteosarcoma cell line MG63 compared to human fetal osteoblastic cell line hFOB1.19 by using quantitative real-time polymerase chain reaction (qRT-PCR). Moreover, miR-21 overexpression in MG63 caused a significant raise in cell proliferation and invasion and a significant reduction in cell apoptosis. However, miR-21 underexpression in MG63 caused an opposite result. Western blotting displayed that proteins related with proliferation, apoptosis, and invasion were significantly changed in different groups, respectively. Furthermore, we demonstrated that PTEN may be a potential target of miR-21 in MG63 cells and miR-21 could activate PI3K/Akt pathway by suppressing PTEN expression. In summary, our findings suggested that miR-21 played an active role in osteosarcoma and it could predict the occurrence and development of osteosarcoma.

The synergistic anticancer effect of cisplatin combined with Oldenlandia diffusa in osteosarcoma MG-63 cell line in vitro.

BackgroundOldenlandia diffusa (OD) is a well-known traditional Chinese medicine, which is used to prevent and treat many disorders, especially cancers. However, its role in osteosarcoma has not been well understood. Here, we used OD and cisplatin individually and combined in osteosarcoma MG-63 cell to explore whether OD could induce cellular apoptosis and suppress the ability of proliferation and invasion of osteosarcoma MG-63 cell.MethodsThe changes of cellular shape were analyzed by optical microscopy. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide assay was used to analyze cell survival rate in vitro. Flow cytometry was performed to detect cell cycle and cell death. Scratch migration assay was used to evaluate cell migration and invasion. Western blot was performed to determine the expression levels of pro-apoptotic and anti-apoptotic protein.ResultsIn this study, we found that the survival rate reduced significantly in the combined group compared with the individual group and control group. The apoptosis-inducing effect of combined application was much more significant than that of individual application. The invasion ability of combined application was significantly lower than that of the individual application. In the combined group, there were high expression levels of pro-apoptotic protein and low expression of anti-apoptotic protein. Cell-cycle analysis showed a change in the cell-cycle distribution and arrested cells in G2-M phase.ConclusionIn this study, we found that OD inhibited proliferation and induced apoptosis in the human osteosarcoma MG-63 cell line in a time-dependent and dose-dependent manner. In addition, OD displayed inhibitory activity on MG-63 cell proliferation and invasion and the study also showed that OD activity might be mediated by caspase activation. These data suggest that OD might represent a novel, efficient candidate agent for further experimentation in osteosarcoma treatment.

MicroRNA-154 functions as a tumor suppressor in osteosarcoma by targeting Wnt5a.

MicroRNAs (miRNAs) are small non-coding RNAs that are involved in tumor initiation and development by suppressing target gene expression. miRNA-154 has been shown to be important in tumorigenesis in many types of cancers. However, its role in osteosarcoma (OS) remains unknown. In the present study, we focused on the roles and mechanisms of miR‑154 in OS development. The results of quantitative RT‑PCR showed that miR‑154 expression was decreased in primary OS tumor samples and cell lines compared to levels in the matched adjacent normal tissues and human normal osteoblast cells (NHOst). Restoration of expression in U2OS cells inhibited cell proliferation, colony formation, migration and invasion, as well as induced cell cycle arrest at the G1 stage. Bioinformatic prediction suggested that Wnt5a is a target gene of miR‑154. It was further verified that Wnt5a is a target gene of miR‑150 in OS cells using luciferase assay, mRNA and protein expression analysis. Wnt5a was upregulated in OS cell lines and primary tumor samples, and its mRNA expression level was negatively correlated with the miR‑154 level in the OS tissues. Restored expression of Wnt5a weakened miR‑154‑mediated suppression of tumor progression. Taken together, these findings suggest that miR‑154 functions as a tumor suppressor in OS by partially suppressing Wnt5a expression.

SPAG9 controls the cell motility, invasion and angiogenesis of human osteosarcoma cells.

Sperm-associated antigen 9 (SPAG9) is an oncoprotein involved in the progression of various human malignancies; however, its role in osteosarcoma (OS) remains poorly evaluated. The present study used Matrigel™ cell migration and invasion assays, tube formation assay, Cell Counting kit-8, quantitative polymerase chain reaction, western blotting, and enzyme-linked immunosorbent assay to investigate the role of SPAG9 in OS cell motility, invasion and angiogenesis. The results of the present study demonstrated that SPAG9 expression was upregulated in OS tissues, as compared with adjacent normal tissues, and knockdown of SPAG9 in an OS cell line inhibited cell motility and invasion via inactivation of metalloproteinase (MMP)-2 and MMP-9. Furthermore, the present study demonstrated that silencing of SPAG9 in OS cells inhibited tube formation, the proliferation of human umbilical vascular endothelial cells, and suppressed vascular endothelial growth factor (VEGF) expression and secretion, contributing to a reduction in angiogenesis. The results of the present study indicated that SPAG9 may be an important regulator in OS and may be involved in metastasis. Therefore SPAG9 may be a promising target for the treatment of metastatic OS.

Long noncoding RNA MALAT1 as a potential therapeutic target in osteosarcoma.

Recent studies have revealed that long noncoding RNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) plays an important role in the development of several solid tumors. However, the function of MALAT1 in the tumorigenesis of osteosarcoma remains unknown. In the present study, levels of MALAT1 in human osteosarcoma cell lines and tissues were detected by quantitative real-time polymerase chain reaction (RT-PCR). The roles of MALAT1 in osteosarcoma were investigated by using in vitro and in vivo assays. We observed that MALAT1 expression was up-regulated in human osteosarcoma cell lines and tissues. In vitro knockdown of MALAT1 by siRNA significantly inhibited cell proliferation and migration, and induced cell cycle arrest and apoptosis in osteosarcoma cells. In addition, MALAT1 knockdown markedly suppressed the formation of tubular network structures and caused breakage of stress fibers in osteosarcoma cell lines U2OS and MNNG/HOS. Furthermore, MALAT1 knockdown delayed tumor growth in an osteosarcoma xenograft model. Specifically, we found that administration of MALAT1 siRNA decreased the protein levels of RhoA and its downstream effectors Rho-associated coiled-coil containing protein kinases (ROCKs). Taken together, these findings suggest that MALAT1 plays an oncogenic role in osteosarcoma and may be a promising therapeutic target for the treatment of osteosarcoma patients. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:932-941, 2016.

MiR-874 suppresses the proliferation and metastasis of osteosarcoma by targeting E2F3.

Increasing evidence indicates that microRNAs (miRNAs) play critical roles in osteosarcoma (OS) occurrence and development. MicroRNA-874 (miR-874) has proven to be dysregulated in several human cancers. However, the biological function and underlying molecular mechanism of miR-874 in OS remain unclear. In this study, we aimed to investigate the biological role and potential mechanism of miR-874 in OS. Here, we found that miR-874 expression was significantly decreased in OS cell lines and tissues by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), and its expression was correlated with tumor-node-metastasis (TNM) stage, tumor size, and lymph node metastasis (all P < 0.01). Functional study revealed that overexpression of miR-874 in OS cells could remarkably inhibit proliferation, migration, and invasion and induce cell apoptosis. In addition, E2F transcription factor 3 (E2F3) was confirmed as a target of miR-874 in OS cells. E2F3 mRNA expression was upregulated and was inversely correlated with the level of miR-874 in OS tissues. Importantly, downregulation of E2F3 mimicked the effect of overexpression miR-874 in OS cells, and E2F3 overexpression partially attenuated the tumor-suppressive effects of miR-874 in OS cells. Taken together, these findings suggested that miR-874 might suppress the growth and metastasis of OS cells partially by targeting E2F3.

Loss of Runx2 sensitises osteosarcoma to chemotherapy-induced apoptosis.

Background:Osteosarcoma (OS) is the most common bone malignancy in the paediatric population, principally affecting adolescents and young adults. Minimal advancements in patient prognosis have been made over the past two decades because of the poor understanding of disease biology. Runx2, a critical transcription factor in bone development, is frequently amplified and overexpressed in OS. However, the molecular and biological consequences of Runx2 overexpression remain unclear.Methods:si/shRNA and overexpression technology to alter Runx2 levels in OS cells. In vitro assessment of doxorubicin (doxo)-induced apoptosis and in vivo chemosensitivity studies. Small-molecule inhibitor of c-Myc transcriptional activity was used to assess its role.Results:Loss of Runx2 sensitises cells to doxo-induced apoptosis both in vitro and in vivo. Furthermore, in conjunction with chemotherapy, decreasing Runx2 protein levels activates both the intrinsic and extrinsic apoptotic pathways. Transplanted tumour studies demonstrated that loss of endogenous Runx2 protein expression enhances caspase-3 cleavage and tumour necrosis in response to chemotherapy. Finally, upon doxo-treated Runx2 knockdown OS cells there was evidence of enhanced c-Myc expression and transcriptional activity. Inhibition of c-Myc under these conditions resulted in decreased activation of apoptosis, therefore insinuating a role for c-Myc in dox-induced activation of apoptotic pathways.Conclusions:Therefore, we have established a novel molecular mechanism by which Runx2 provides a chemoprotective role in OS, indicating that in conjunction to standard chemotherapy, targeting Runx2 may be a new therapeutic strategy for patients with OS.

Network analysis of microRNAs and genes in human osteosarcoma.

To date, numerous studies have suggested that microRNAs (miRNAs) and genes play key roles in osteosarcoma (OS); however, the majority of these studies have been conducted with a specific focus on either the genes or the miRNAs, which has made the regulatory mechanisms of OS difficult to decipher. The aim of the present study was to systematically investigate the elements [genes, miRNAs and transcription factors (TFs)] associated with the morbidity of OS and to explore the associations among these elements, instead of focusing on one or several elements. The scattered data were collected from existing studies of OS, and three regulatory networks (abnormally expressed, related and global) were constructed to explore OS at a macroscopic level. The abnormally expressed network showed the numerous incorrect data linkages that are present when OS emerges, making it useful as a map of the faults in OS. In theory, the correction of these errors could lead to the prevention and even cure of the disease. Unlike studies in which cancer networks have been formed based purely on gene data, the present study focused on genes and miRNAs, as well as the associations among them, to form the regulatory networks of OS. The constructed regulatory networks were shown to contain numerous self-adaptation associations, which may aid in the analysis of the pathogenesis of OS. By comparing and analyzing the similarities and differences, a number of important pathways were highlighted. A notable finding was the predicted TFs obtained by the P-Match method, which could be used to further study the pathogenesis of OS. In the present study, the mechanism of OS has been systematically analyzed and a theoretical foundation for the mechanism has been provided, which may assist the development of gene therapy targeting OS.

Enhancer of zeste homolog 2 silencing inhibits tumor growth and lung metastasis in osteosarcoma

The enhancer of zeste homolog 2 (EZH2) methyltransferase is the catalytic subunit of polycomb repressive complex 2 (PRC2), which acts as a transcription repressor via the trimethylation of lysine 27 of histone 3 (H3K27me3). EZH2 has been recognised as an oncogene in several types of tumors; however, its role in osteosarcoma has not been fully elucidated. Herein, we show that EZH2 silencing inhibits tumor growth and lung metastasis in osteosarcoma by facilitating re-expression of the imprinting gene tumor-suppressing STF cDNA 3 (TSSC3). Our previous study showed that TSSC3 acts as a tumor suppressor in osteosarcoma. In this study, we found that EZH2 was abnormally elevated in osteosarcoma, and its overexpression was associated with poor prognosis in osteosarcoma. Silencing of EZH2 resulted in tumor growth inhibition, apoptosis and chemosensitivity enhancement. Moreover, suppression of EZH2 markedly inhibited tumor growth and lung metastasis in vivo. Furthermore, EZH2 knockdown facilitated the re-expression of TSSC3 by reducing H3K27me3 in the promoter region. Cotransfection with siEZH2 and siTSSC3 could partially reverse the ability of siEZH2 alone. We have demonstrated that EZH2 plays a crucial role in tumor growth and distant metastasis in osteosarcoma; its oncogenic role is related to its regulation of the expression of TSSC3.

MicroRNA-93 promotes cell proliferation via targeting of PTEN in Osteosarcoma cells.

BackgroundAberrant microRNA (miRNA) expression plays an essential role in osteosarcoma (OS) pathogenesis. Recent studies have shown that dysregulation of miRNA expression is associated with increased tumorigenesis and poor prognosis in several types of cancers, including OS. The aim of this study was to investigate the relevant microRNAs involved in the development of OS.MethodsTo explore possible oncogenic factors in OS, we used a microarray-based approach to profile changes in the expression of miRNAs and their target mRNAs in five OS cell lines and human mesenchymal stem cells (hMSCs). An miRNA, miR-93, was significantly up-regulated, whereas phosphatase and tensin homologue (PTEN) expression was significantly down-regulated in all tested OS cells, when compared with hMSCs.ResultsWhen anti-miR-93 was transfected into OS cell lines, PTEN expression was greatly increased, suggesting that PTEN might be a target of miR-93 in ES cells. The expression of phosphorylated Akt protein, which is known to be inversely correlated with that of PTEN, was significantly down-regulated in anti-miR-93-transfected cells. Furthermore, transfection of anti-miR-93 inhibited the proliferation and cell cycle progression of ES cells. In addition, the down-regulation of miR-93 in these cells significantly suppressed tumor growth in vivo.ConclusionEctopic expression of miR-93 decreased PTEN protein levels. Furthermore, miR-93 increased proliferation and decreased apoptosis in OS cells, whereas its silencing in these cells inhibited such carcinogenic processes. Taking these observations together, miR-93 can be seen to play a critical role in carcinogenesis through suppression of PTEN, and may serve as a therapeutic target for the treatment of OS.