| Objective:Osteosarcoma is a highly malignant primary tumor occurring in the bone and/or associated tissues. As disease progresses, osteosarcoma can progressively harm the patients’ health by causing pain, lump formation, organ dysfunction, and fractures. In the late stage, the majority of patients would develop metastases, ending up with systemic failure. So far, the exact mechanism of osteosarcoma pathogenesis remains unclear. Recent studies have found that COX-2 expression is gradually increased in the development of osteosarcoma, suggesting its involvement in the disease progression. Moreover, COX-2 has also been shown be play an important role in the pathogenesis of several diseases, including gastric cancer, pancreatic cancer, and bladder cancer, which make COX-2 a promising biomarker in disease diagnosis. Furthermore, miR-143 has been demonstrated to influence the expression of COX-2, and affect the disease progression. However, the expression of COX-2 in osteosarcoma, especially concerning its relationship with miR-143, has not yet been fully understood. In this study, the expression profiles of COX-2 and miRNA-143 in tumor tissue and blood samples from osteosarcoma patients were detected. The relationship between the expression profiles and the disease severity had also been discussed. Our findings would provide new insights into the early diagnosis and clinical treatment of osteosarcoma.Methods:Totally 46 patients diagnosed with osteosarcoma were enrolled in this study, who had been admitted to our hospital from December 2011 to December 2013, including 22 stage Ⅰ patients(16 male and 6 female with average 18.6±3.7 years old), 18 stage Ⅱ patients(12 male and 6 female with average 19.8±4.8 years old), and 6 stage Ⅲ patients(1 male and 5 female with average 18.1±2.9 years old). In addition, 26 normal subjects (16 male and 10 female with average 18.8±5.0 years old) were used as controls.20 osteosarcoma samples were obtained from these patients with adjacent tissues used as negative control. Osteosarcoma samples included 9 from stage Ⅰ patients(6 male and 3 female with average 17.6±4.0 years old),8 from stage Ⅱ patients(2 male and 6 female with average 16.4±2.8 years old), and 3 from stage Ⅲ patients (2 male and 1 female with average 17.3±2.5 years old), All patients were diagnosed referring to WHO 2002 classification for bone tumors and Enneking staging system. Prior written and informed consent were obtained from every patient and the study was approved by the ethics review board of Nanjing General Hospital of Nanjing Military Command. Samples included two sources:one is from the venous blood in the morning before taking food, the other is from the surgical resected tissue. After collection, all the specimen were cryopreserved. Quantitative real-time PCR was performed to determine the relative expression of COX-2 mRNA and miR-143 in the tumor tissue or blood samples. Western blot analysis was performed to determine the relative expression of COX-2 protein in the tumor tissue or blood samples. Data were expressed as mean ±SD. SPSS18.0 software package was used for statistical analysis. Normality test and one-way ANOVA were performed for the comparison. When the homogeneity of variance is set up, LSD methods were performed for comparison between groups. If not, Dunnett’s T3 methods were performed. P<0.05 was considered statistically significant.Results:To investigate the mRNA and protein expression levels of COX-2 in osteosarcoma tumor tissue, quantitative real-time PCR and Western blot analysis were performed, respectively, in normal control subjects and patients with stage Ⅰ-Ⅲ osteosarcoma. Results from quantitative real-time PCR showed that, the mRNA expression levels of COX-2 in osteosarcoma patents were significantly higher than the normal control subjects (P< 0.05). Moreover, the mRNA expression level of COX-2 in patients with stage Ⅲ osteosarcoma was significantly higher than patients with stage Ⅱ osteosarcoma, which was significantly higher than stage Ⅰ osteosarcoma patients, indicating that the COX-2 mRNA expression level was increased along with the disease severity. On the other hand, Western blot analysis showed that the protein expression levels of COX-2 exhibited similar trends with COX-2 mRNA. These results suggest that the expression of COX-2 is increased as the disease progresses, implying the involvement of COX-2 in the disease pathogenesis.The mRNA and protein levels of COX-2 in the blood samples from patients with osteosarcoma were next detected by real-time PCR and Western blot analysis. Our results showed that, compared with normal control subjects, the mRNA level of COX-2 was slightly elevated in blood samples from patients with stage Ⅰ osteosarcoma, however no significance was found (P> 0.05). In patients with stage Ⅱ-Ⅲ osteosarcoma, COX-2 mRNA contents in blood samples were significantly higher than the normal controls (P< 0.05), and the mRNA level was increased as the disease deteriorated. Similar results were observed with the Western blot analysis of COX-2 protein expression in blood samples from patients with osteosarcoma. These results indicate that the mRNA and protein levels of COX-2 in blood are increased in patients with osteosarcoma, even though without significant difference between early osteosarcoma patients and normal control subjects.To find out the role of miR-143 in osteosarcoma pathogenesis, its expressions in osteosarcoma tumor tissue and blood samples were detected by quantitative real-time PCR. Our results showed that the level of miR-143 in osteosarcoma tumor tissue was significantly decreased in patient with stage Ⅰ-Ⅲ osteosarcoma, compared with the normal control subjects (P< 0.05). Among those osteosarcoma patients at different stages, miR-143 level in tumor tissue was also declined along with the disease severity, i.e., miR-143 level in tumor tissue from patients with osteosarcoma at stage Ⅲ was significantly higher than stages Ⅰ-Ⅱ, and miR-143 level in stage Ⅱ osteosarcoma was significantly higher than stage I osteosarcoma (all P< 0.05). Similar results were also found with the detection of miR-143 in the blood samples from patients with stages Ⅰ-Ⅲ osteosarcoma. These results indicate that the expression of miR-143 is obviously decreased along with the severity of osteosarcoma, which is in the opposite trend to the COX-2 expression, suggesting that miR-143 and COX-2 might play different roles in the disease pathogenesis.Conclusions:COX-2 is the rate-limiting enzyme for the synthesis of prostaglandins, which is critically important for inflammation. Recent studies have indicated that COX-2 is also closely related to the progression of various types of tumor. Our results showed that the mRNA and protein expression levels of COX-2were both obviously up-regulated as the disease severity increased. These results suggest that COX-2 might be involved in the development of osteosarcoma.The blood expression level of COX-2 may reflect the degree of malignancy and metastasis of osteosarcoma cells, because tumor metastasis largely relies on blood and tissue fluid flow. Our results showed that the mRNA and protein expression levels of COX-2 in blood were gradually increased from stage Ⅰ to stage Ⅲ in osteosarcoma. However, the difference in blood COX-2 expression level between normal control and stage Ⅰ osteosarcoma did not reach statistical significance. This might be due to the fact that there are limited cells migrating in the blood circulation from the newly formed osteosarcoma tumor, some of which would even be phagocytosed by monocytes in the blood. Therefore, COX-2 expression in blood might not be a perfect predictor for tumor metastasis, especially for stage Ⅰ osteosarcoma.Our results showed that COX-2 expression levels in osteosarcoma tumor tissue were increased along with the disease severity, from normal control to osteosarcoma stages Ⅰ, Ⅱ, and Ⅲ. Meanwhile, the expression of miR-143 was sequentially decreased as the disease progressed. We suppose that the up-regulation of COX-2 might be related to the down-regulation of miR-143 in the disease progression. There might be a negative regulation on COX-2 of miR-143, which could be an important mechanism for chronic inflammation and cancer development process.The expression tendency of miR-143 in blood was similar with the performance of COX-2 in tumor tissues. However, and more importantly, we found that, unlike COX-2, the difference in blood miR-143 expression between normal controls and stage Ⅰ osteosarcoma was statistically significant, suggesting that blood miR-143 level might be a promising early predictor and diagnostic marker for osteosarcoma. |
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