Clinical significance of CCL2 expression in osteosarcoma tissue

Increasing evidences have demonstrated that B7-H4 is associated with tumor development and prognosis. However, the clinical significance of B7-H4 expression in human osteosarcoma (OS) remains unclear. The aim of present study was to examine the B7-H4 expression and to explore its contribution in OS. B7-H4 expression in OS tissues was examined by immunohistochemistry. Soluble B7-H4 (sB7-H4) levels in blood were examined by ELISA. The association of B7-H4 expression with clinicopathological factors or prognosis was statistically analyzed. Our findings demonstrated that B7-H4 expression in OS tissues was significantly higher than those in paired normal bone tissues (P < 0.001). sB7-H4 level in OS serum samples was significantly higher than that in healthy controls (P = 0.005). High B7-H4 expression in tissues and sB7-H4 level were both correlated with advanced tumor stage (P < 0.001, P = 0.017, resp.) and distant metastasis (P = 0.034, P = 0.021, resp.). Additionally, high B7-H4 expression or serum sB7-H4 levels were significantly related to poor overall survival (P = 0.028, P = 0.005, resp.). B7-H4 in tissues and serum samples were an independent factor for affecting the survival time of OS patients (P = 0.004, P = 0.041, resp.). Collectively, our data suggest that the evaluation of B7-H4 expression in tissues and blood is a useful tool for predicting the progression of osteosarcoma and prognosis.

Osteosarcoma is the most prevalent type of primary malignant bone tumor. Inhibitor of growth 4 (ING4) has been demonstrated to function as a tumor suppressor through multiple pathways, and is its expression is understood to be suppressed or reduced in various malignancies. The present study aimed to investigate the expression of ING4 and to determine its prognostic value in osteosarcoma tissue. Formalin-fixed, paraffin-embedded tissue microarrays were analyzed, and contained 41 osteosarcoma specimens and 11 normal bone tissue specimens with duplicate cores. ING4 expression was evaluated by immunohistochemical staining. The association between ING4 expression in the osteosarcoma and normal bone tissues was analyzed, in addition to the association between ING4 expression and Enneking classification of the osteosarcoma tissues. A significant statistical difference was observed in the ING4 immunohistochemical staining score between the osteosarcoma and normal bone tissues (P<0.001). Furthermore, a significant negative correlation was detected between the ING4 immunohistochemical staining scores and the Enneking classification results of the 41 osteosarcoma tissues (P=0.002). Low expression of ING4 was observed in the osteosarcoma specimens, and this reduced expression of ING4 was negatively correlated with Enneking classification. Thus, the results of the present study indicate that ING4 may serve as a promising prognostic marker in osteosarcoma.

The aim of the present study was to investigate the clinical significance of Twist, vascular endothelial growth factor (VEGF) and CD34 expression in osteosarcoma (OS) in order to elucidate potential therapeutic targets for the treatment of OS. Immunohistochemistry was performed to detect the protein expression of Twist, VEGF and CD34 in OS and osteochondroma (OC) tissues. The ratio of the protein expression of Twist and VEGF in OS and OC tissues as well as at different phases of OS was compared using chi-squared tests. Microvessel density (MVD), as determined by CD34 labeling, in OS and OC tissue as well as at different phases of OS was compared using the Student's t-test. In addition, associations between Twist, VEGF and MVD were assessed using the Spearman's rank correlation test. The results revealed that out of the 32 OS tissues examined, 56.25% exhibited Twist positive expression, 71.88% exhibited VEGF positive expression and the MVD was increased compared with that of the OC tissue. The positive rate of Twist and VEGF expression in phase III OS tissues was significantly increased compared with that in phase I/II OS tissues (Twist: χ2=5.732, P=0.018; VEGF: χ2=7.513, P=0.006). The MVD in phase III OS tissues (31.08±3.36 per field) was significantly higher compared with that of the phase I/II OS tissues (41.2±4.17 per field; t=7.536, P<0.001). Spearman's rank correlation analysis revealed that Twist expression was positively associated with VEGF expression (r=0.371, P=0.002) and with MVD (r=0.393, P=0.001) in OS; in addition, VEGF expression was found to have a positive correlation with MVD (r=0.469, P=0.001). In conclusion, the results of the present study demonstrated that OS tissues exhibited elevated Twist and VEGF expression as well as MVD compared with OC tissue. In addition, metastatic OS (phase III) exhibited an increased positive rate of Twist and VEGF expression as well as MVD values compared with non-metastatic OS (phase I/II). Furthermore associations were detected between Twist and VEGF expression as well as VEGF and MVD. Therefore, inhibition of Twist expression may have potential therapeutic use for the treatment of OS.

Neuropilin (NRP)-1, a co-receptor for vascular endothelial growth factor (VEGF), plays an important role in angiogenesis and malignant progression of many cancers. However, the involvement of NRP-1 in osteosarcoma is not completely understood. The aim of this study was to investigate the expression pattern and clinical significance of NRP-1 in human osteosarcoma. NRP-1 mRNA and protein expression levels were detected by RT-PCR and Western blot assays, respectively, using 166 pairs of osteosarcoma and noncancerous bone tissues. Then, the association of NRP-1 expression with clinicopathological factors or survival of osteosarcoma patients was further evaluated. RT-PCR and Western blot assays revealed that NRP-1 expression in osteosarcoma tissues was significantly higher than that in corresponding noncancerous bone tissues at both mRNA and protein levels (both P<0.001). In addition, high NRP-1 expression more frequently occurred in osteosarcoma tissues with advanced clinical stage (P=0.006), positive distant metastasis (P=0.01) and poor response to chemotherapy (P=0.006). Moreover, osteosarcoma patients with high NRP-1 expression had significantly shorter overall survival and disease-free survival (both P<0.001) when compared with patients with the low expression of NRP-1. On Cox multivariate analysis, NRP-1 overexpression was an independent and significant prognostic factor to predict poor overall survival and disease-free survival (both P=0.001). This is the first study to reveal that NRP-1 overexpression may be related to the prediction of metastasis potency, response to chemotherapy and poor prognosis for osteosarcoma patients, suggesting that NRP-1 may serve as a prognostic marker for the optimization of clinical treatments.

LINC00673 promotes osteosarcoma progression through the miR-92b-3p/DUSP1 axis.

BackgroundOsteosarcoma is one of the most common malignant bone tumors with the annual global incidence of approximately four per million. Upregulated gene 4 (URG4) expression in the osteosarcoma tissue is closely associated with recurrence, metastasis, and poor prognosis of osteosarcoma. However, the biological function and underlying mechanisms of URG4 in osteosarcoma have not been elucidated. This study aimed to explore the expression and underlying mechanism of URG4 in osteosarcoma.MethodsThe expression level of URG4 in osteosarcoma and normal tissues was compared using immunohistochemistry (IHC). PCR and western blotting (WB) techniques are used to detect URG4 mRNA and protein levels. Wound healing and Transwell analysis to assess the effect of URG4 on osteosarcoma cell migration and invasion. Cell Counting Kit-8 assay and colony proliferation assay were performed to evaluate the effects of silencing URG4 on the inhibition of cell proliferation. The cell cycle distribution was detected by flow cytometry, and a xenograft mouse model was used to verify the function of URG4 in vivo.ResultsURG4 was found to be highly expressed in osteosarcoma tissues and cells, and its high expression was correlated with advanced Enneking stage, large tumor size, and tumor metastasis in osteosarcoma patients. The proliferation in osteosarcoma cell lines and cell cycle in the S phase was suppressed when siRNA was used to downregulate URG4. URG4 promoted cell proliferation and tumorigenesis in vitro and in vivo. WB verified that URG4 promotes cell proliferation in osteosarcoma via pGSK3β/β-catenin/cyclinD1 signaling.ConclusionURG4, which is high-expressed in osteosarcoma, promotes cell cycle progression via GSK3β/β-catenin/cyclin D1 signaling pathway and may be a novel biomarker and potential target for the treatment of osteosarcoma.

Osteosarcoma is a malignant tumor in bone and soft tissue tumors, characterized by high invasiveness and low survival rate. Here, we studied the effect of cAMP-responsive element binding protein 1 (CREB1) on osteosarcoma cell proliferation and invasion. mRNA levels of CREB1, miRNA-183-5p and prospero homeobox 1 (PROX1) were detected by quantitative real-time polymerase chain reaction (RT-qPCR), while CREB1 and PROX1 protein levels were evaluated utilizing Western blot in osteosarcoma cell lines and tissues. Immunohistochemistry investigated CREB1 expression in osteosarcoma tissues. Colony formation, transwell and flow cytometry experiments detected cell proliferation, invasion and apoptosis, respectively. Dual luciferase reporter assay, chromatin immunoprecipitation (ChIP) assay and RNA immunoprecipitation (RIP) assay verified target relationship between CREB1, miRNA-183-5p and PROX1. CREB1 expression was increased in osteosarcoma tissues and cells. Reduced CREB1 inhibited osteosarcoma cell proliferation and invasion, and promoted apoptosis. Moreover, CREB1 targets miRNA-183-5p and transcriptionally inhibited miRNA-183-5p expression in osteosarcoma cells. Additionally, PROX1 was a target gene of miRNA-183-5p, where expression was inhibited by miRNA-183-5p. PROX1 upregulation reversed inhibitory effect of miRNA-183-5p overexpression on osteosarcoma cell malignant phenotype. Downregulation of CREB1 inhibited osteosarcoma cell proliferation and invasion but induced apoptosis through miRNA-183-5p/PROX1 axis. CREB1 may become a new target for diagnosis and treatment of osteosarcoma.

Identifying prognostic factors for osteosarcoma (OS) aids in the selection of patients who require more aggressive management. XB130 is a newly characterized adaptor protein that was reported to be to be a prognostic factor of certain tumor types. However, the association between XB130 expression and the prognosis of OS remains unknown. In the present study, we investigated the association between XB130 expression and clinicopathologic features and prognosis in patients suffering osteosarcoma, and further investigated its potential role on OS cells in vitro and vivo. A retrospective immunohistochemical study of XB130 was performed on archival formalin-fixed paraffin-embedded specimens from 60 pairs of osteosarcoma and noncancerous bone tissues, and compared the expression of XB130 with clinicopathological parameters. We then investigate the effect of XB130 silencing on invasion in vitro and lung metastasis in vivo of the human osteosarcoma cell line. Immunohistochemical assays revealed that XB130 expression in osteosarcoma tissues was significantly higher than that in corresponding noncancerous bone tissues (P=0.001). In addition, high XB130 expression more frequently occurred in osteosarcoma tissues with advanced clinical stage (P=0.002) and positive distant metastasis (P=0.001). Moreover, osteosarcoma patients with high XB130 expression had significantly shorter overall survival and disease-free survival (both P<0.001) when compared with patients with the low expression of XB130. The univariate analysis and multivariate analysis shown that high XB130 expression and distant metastasis were the independent poor prognostic factor. We showed that XB130 depletion by RNA interference inhibited invasion of XB130-rich U2OS cells in vitro and lung metastasis in vivo. This is the first study to reveal that XB130 overexpression may be related to the prediction of metastasis potency and poor prognosis for osteosarcoma patients, suggesting that XB130 may serve as a prognostic marker for the optimization of clinical treatments. Furthermore, XB130 is the potential molecular target for osteosarcoma therapy.

Background/Aims: Serine/threonine kinase 35 (STK35) may be associated with Parkinson disease and human colorectal cancer, but there have been no reports on the expression levels or roles of STK35 in osteosarcoma. Methods: STK35 mRNA expression was determined in osteosarcoma and bone cyst tissues by real-time PCR. Cell proliferation and apoptosis were assessed by Cell Counting Kit-8 (CCK-8) assay and flow cytometry analysis, respectively. Results: STK35 was up-regulated in osteosarcoma tissues as indicated by analyzing publicly available expression data (GEO dataset E-MEXP-3628) and real-time PCR analysis on our own cohort. We subsequently investigated the effects of STK35 knockdown on two osteosarcoma cell lines, MG63 and U2OS. STK35 knockdown inhibited the growth of osteosarcoma cells in vitro and in xenograft tumors. Meanwhile, STK35 knockdown enhanced apoptosis. Expression of the active forms and the activity of two major executioner caspases, caspase 3 and caspase 7, were also increased in osteosarcoma cells with STK35 silenced. Additionally, Gene Set Enrichment Analysis (GSEA) identified that the JAK/STAT signaling pathway was positively correlated with STK35 expression. The mRNA expression of STK35 was repressed by STAT3 small interfering RNA (siRNA), but not by siRNA of STAT4, STAT5A or STAT6. A luciferase reporter assay further demonstrated that STAT3 transcriptionally regulated STK35 expression. A chromatin immunoprecipitation (ChIP) assay confirmed the direct recruitment of STAT3 to the STK35 promoter. The promotion effects of STAT3 knockdown on cell apoptosis were partially abolished by STK35 overexpression. Furthermore, STK35 mRNA expression was positively correlated with STAT3 mRNA expression in osteosarcoma tissues by Pearson correlation analysis. Conclusions: These results collectively reveal that STAT3 regulates the transcription of STK35 in osteosarcoma. STK35 may exert an oncogenic role in osteosarcoma.

Osteosarcoma is the most common primary bone tumour in children and adolescents. Accumulating evidence has shown that microRNAs (miRNAs) participate in the development of almost all types of cancer. Here, we investigated the role of miR‐224 in the development and progression of osteosarcoma. We demonstrated that miR‐224 was down‐regulated in osteosarcoma cell lines and tissues. Lower miR‐224 levels were correlated with shorter survivalin osteosarcoma patients. Furthermore, overexpression of miR‐224 suppressed osteosarcoma cell proliferation, migration and invasion and contributed to the increased sensitivity of MG‐63 cells to cisplatin. We identified Rac1 as a direct target gene of miR‐224 in osteosarcoma. Rac1 expression was up‐regulated in the osteosarcoma cell lines and tissues, and there was an inverse correlation between Rac1 and miR‐224 expression in osteosarcoma tissues. Furthermore, rescuing Rac1 expression decreased the sensitivity of miR‐224‐overexpressing MG‐63 cells to cisplatin. We also demonstrated that ectopic expression of Rac1 promoted the proliferation, migration and invasion of miR‐224‐overexpressing MG‐63 cells. These data suggest that miR‐224 plays a tumour suppressor role in the development of osteosarcoma and is related to the sensitivity of osteosarcoma to cisplatin.

Recent studies have shown that microRNA-451 (miR-451) was significantly decreased in osteosarcoma tissues and was identified as a tumor suppressor in other types of human cancers. However, its clinical significance and molecular mechanisms in osteosarcoma are still not well understood. MiR-451 levels are evaluated by quantitative reverse transcription-polymerase chain reaction (RT-PCR) in osteosarcoma cell lines and in 68 pairs of osteosarcoma and adjacent noncancerous tissues. Then, the associations of miR-451 expression with clinicopathological features of patients were determined. The effects of miR-451 in osteosarcoma cells were examined by MTT and Matrigel invasion assay. The functional target of miR-451 were determined by bioinformatics analysis and validated by luciferase reporter analyses and Western blot assay. Our results showed that the expression of miR-451 was significantly downregulated in osteosarcoma tissues compared with corresponding noncancerous tissues (P < 0.01). Particularly, statistical analysis of primary human osteosarcoma indicated that decreased expression of miR-451 was correlated with metastasis and recurrence. Moreover, the miR-451 force-expression suppressed cell proliferation and invasion in vitro. Based on bioinformatics analysis, we found that chemokine ligand 16 (CXCL16) was identified as a direct functional target of miR-451. Consistent with the effects of miR-451, silencing CXCL16 could phenocopy the effects of miR-451 on phenotypes of osteosarcoma cells. Furthermore, CXCL16 expression was upregulated in osteosarcoma tissues and inversely associated with miR-451 in human osteosarcoma tissues. Our data reveal a downregulated expression of miR-451 in osteosarcoma tissues, which is inversely associated with CXCL16 levels. These observations demonstrated that miR-451 may play an important role in tumor growth and metastasis in osteosarcoma.

We investigated the role of nuclear factor-κB (NF-κB) and phosphatase and tensin homolog deleted in chromosome 10 (PTEN) in the pathogenesis of osteosarcoma and its relationship with prognosis. Immunohistochemical method was used to detect the expression of NF-κB and PTEN in osteosarcoma and adjacent tissues. RT-PCR was used to detect the expression of NF-κB and PTEN mRNA in osteosarcoma and adjacent tissues. Western blotting was used to detect the expression of NF-κB and PTEN in osteosarcoma and adjacent tissues and compare their differences. The expression of NF-κB and PTEN was detected in osteosarcoma and adjacent tissues. The positive rate of NF-κB was 75.3 and 32.9%, respectively; while the positive rate of PTEN was 67.1 and 90.4%, respectively. The positive expression of NF-κB and PTEN was statistically significant. There was a negative correlation between NF-κB and PTEN expression (r=−0.502, p<0.05). The positive and negative expression of NF-κB and PTEN was statistically significant for the five-year survival (p<0.05). At gene and protein level, osteosarcoma tissues had higher expression of NF-κB, and lower expression of PTEN, which was significantly different from the adjacent tissues. In osteosarcoma, NF-κB is highly expressed, but PTEN is expressed at low level, and the two are negatively correlated. This is of great significance for the early diagnosis of osteosarcoma and prognosis.

Procollagen C-proteinase enhancer protein (PCOLCE) was originally identified as an enhancer to facilitate the catalysis of procollagens by BMP1. PCOLCE participates in the reconstitution of extracellular and corneal repair. The elevation of PCOLCE in blood indicates that breast cancer has metastasized into the bones. However, direct research on PCOLCE has not been reported.Methods: ECM candidates were identified by RNA-seq analysis from 4 normal and 16 osteosarcoma tissues. The in vitro migration and invasion abilities of osteosarcoma cells were determined by a Transwell assay. A spontaneous metastatic osteosarcoma model was established to assess osteosarcoma metastasis in vivo. The N-linked glycosylated amino acids were identified by PNGase F treatment combined with Western blotting. The mechanism of TWIST1 regulating PCOLCE transcription was elucidated by luciferase, qPCR and ChIP assays.Results: PCOLCE was markedly up-regulated in human osteosarcoma tissues compared to its expression in noncancerous adjacent tissues; high PCOLCE expression in tissues correlated with a poor patient prognosis, and the knockdown of PCOLCE by shRNAs impaired the migration, invasion and lung metastasis of osteosarcoma cells. The overexpression of wild-type PCOLCE, but not its N29Q mutant, promoted migration, invasion and metastasis, indicating that the glycosylation of PCOLCE at Asn29 is necessary for its functions in osteosarcoma. TWIST1, a key transcription factor in metastasis, was also overexpressed in osteosarcoma tissues and positively correlated with either PCOLCE or its potential procollagen substrates, such as COL1A1, COL1A2, COL5A1, COL8A2 and COL10A1.Conclusion: Our findings are the first to provide evidence that PCOLCE plays a critical role in promoting the lung metastasis of osteosarcoma, and this up-regulation of PCOLCE by TWIST1 may lead to a new therapeutic strategy to treat patients with metastatic osteosarcoma.

To investigate the expression of miR-218 and its clinical significance in osteosarcoma tissues and explore its effect on proliferation and apoptosis in osteosarcoma cells. miR-218 expression was detected in 76 samples of surgically resected osteosarcoma and matched normal tumor-adjacent tissues using quantitative reverse transcription polymerase chain reaction (qRT-PCR). MiR-218 was over-expressed by exogenous miR-218 plasmids in Saos-2 cells, and then BrdU cell proliferation assay and flow cytometry were used to determine cell proliferation and apoptosis. The expression of miR-218 in osteosarcoma tissues was significantly lower than those in normal tumor-adjacent tissues (t=8.735, P<0.001). MiR-218 expression in tumor tissues was significantly correlated with tumor size (χ(2)=5.380, P=0.020), clinical stage (χ(2)=6.692, P=0.010) and distant metastasis (χ(2)=4.180, P=0.041). MiR-218 was obviously over-expressed by exogenous miR-218 plasmids (t=19.42, P<0.001), and miR-218 overexpression significantly reduced cell proliferation (t=9.045, P<0.001) and induced apoptosis (t=12.38, P<0.001) in Saos-2 cells. The low-expression of miR-218 is correlated with the poor clinicopathological features in osteosarcoma. Moreover, miR-218 overexpression reduces cancer cell proliferation and induces apoptosis in Saos-2 cells, suggesting that miR-218 may play a key role in the progression of human osteosarcoma.

ObjectiveSOX9 plays an important role in bone formation and tumorigenesis. However, its involvement in osteosarcoma is still unclear. The aim of this study was to investigate the expression pattern and the clinical significance of SOX9 in human osteosarcoma.MethodsSOX9 mRNA and protein expression levels were detected by RT-PCR and Western blot assays, respectively, using 30 pairs of osteosarcoma and noncancerous bone tissues. Then, immunohistochemistry was performed to analyze the association of SOX9 expression in 166 osteosarcoma tissues with clinicopathological factors or survival of patients.ResultsSOX9 expression at mRNA and protein levels were both significantly higher in osteosarcoma tissues than those in corresponding noncancerous bone tissues (both P < 0.001). Immunohistochemical staining indicated that SOX9 localized to the nucleus and high SOX9 expression was observed in 120 of 166 (72.3%) osteosarcoma specimens. In addition, high SOX9 expression was more frequently occurred in osteosarcoma tissues with advanced clinical stage (P = 0.02), positive distant metastasis (P = 0.008) and poor response to chemotherapy (P = 0.02). Osteosarcoma patients with high SOX9 expression had shorter overall survival and disease-free survival (both P < 0.001). Furthermore, the multivariate analysis confirmed that upregulation of SOX9 was an independent and significant prognostic factor to predict poor overall survival and disease-free survival (both P = 0.006).ConclusionsOur data show for the first time that SOX9 is upregulated in aggressive osteosarcoma tissues indicating that SOX9 may participate in the osteosarcoma progression. More importantly, SOX9 status is a useful prognostic factor for predicting the prognosis of osteosarcoma, suggesting that SOX9 may contribute to the optimization of clinical treatments for osteosarcoma patients.Virtual slidesThe virtual slides for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1318085636110837.