| 1. IntroductionOsteosarcoma (OS) is an tumor of bone, in which the neoplastic cells produce bone and Osteosarcoma is the most frequent primary sarcoma of the skeleton. While OS arises in any bone, the most common sites are the distal femur, proximal tibia and proximal humerus, in addition, other sites contains skull, mandible and pelvis, while it is less often in the frame. Beyond primary tumor growth in the appendicular and less commonly the axial skeleton, one of OS biological characteristics is its high propensity for pulmonary metastasis. Although the diagnosis, radiation and chemotherapy treatments of OS have made great progress, the prognosis of OS is still poor and the patients have higher mortality and morbidity, because of its high transfer rate and high recurrence rate after surgical resection. Previous studies showed that the survival rate of the non-metastatic osteosarcoma is 50%-78%, but the one of the metastatic osteosarcoma is only 10% to 10%. So, it became the research focus to detect the mechanisms of osteosarcoma metastasis, and it has a great significance to deeply exploring these mechanisms to understand this disease and look for new intervention targets and treatment measures.The invasion and metastasis is the most main characteristics of the pathophysiology of malignant tumor, and it is the primary cause of tumor with poor prognosis. Invasion is that the malignant tumor cells invade surrounding normal tissues and proliferate in the tissues. Metastasis is that tumor cells from the primary tumor intrudes into the blood vessels or the lymphatic vessels, and go to target organs with blood and lymph. Tumor cells invade out of the vascular system, strand and proliferate in target organs to form a new tumor lesions. Invasion not only is the primary cause of tumor recurrence after surgery, but also is the key to tumor metastasis.Previous studies found that tumor cells degraded the basement membrane by secreting MMPs, serine protease and cysteine proteas, thereby these cells were separated from in situ tumor tissue; the tumor cells also resisted the anoikis by increased the expression of tyrosine kinase receptors, ?2?3-integrin, Adenomatous polyposis coli (APC) gene product, focal adhesion kinase, galectin-3 and TGF-P and so on. At the site of the tumor metastasis, The tumor emboli formed by tumor cells was adhesion to endothelial cells and then passed through the basement membrane. In this process, integrin, laminin receptor and proteolytic enzymes played an important role. In addition, angiogenesis is the key step in the tumor metastasis, and its mechanisms are that there is a unbalance between promote angiogenesis factor (VEGF, FGF, IL-18) and its antagonist factor such as platelet response protein-1 and angiostatin, which leads to endothelial cell proliferation and then angiogenesis. By visible on, a variety of genes and their products involves in the invasion and metastasis of osteosarcoma, among them, integrin, TGF-p, VEGF and MMPs are the key factors. Integrin can directly or indirectly accelerate the transformation of epithelial mesenchymal from tumor cells, and the phenomenon of this transformation is closely related with tumor invasion and metastasis. Integrin can also activate VEGF signaling pathways inducing tumor angiogenesis. TGF-P could induce the cell transformation and angiogenesis throng multiple signaling pathways such as smads, MAPKs, Ras, and so on. MMPs could promote tumor invasion and metastasis through degrading extracellular matrixSome studies revealed the molecular mechanisms of metastatic osteosarcoma. Ezrin protein is considered a key factor for tumor metastasis, and its high expression indicates a poor prognosis of patients with osteosarcoma. In addition, heparin enzyme, protein phosphatase-tension analogues (PTEN) and nerve adhesion factor-2 was also involved in mediating the metastasis of osteosarcoma. Matrix metalloproteinases (MMPs) are a group of proteolytic enzymes, which can degrade the extracellular matrix and then promote the local invasion and distant metastasis of tumor cells. Many studies have found that MMP-2 and MMP-9 play an important role in osteosarcoma metastasis. In normal tissue, the expression of MMP-2 is suppressed by natural inhibiting factors such as MMPS tissue inhibitor (TIMP), Kazal motifs, reversible regulate homocysteine concentration protein (RECK), alpha 2 macroglobulin, while in osteosarcoma tissues, the expression of MMP-2 is significantly increased. A study found that compared with the osteosarcoma patients who had the low expression of MMP-2, the ones who had the high expression of MMP-2 had lower survival time and survival rate, and Cox survival analysis also showed that MMP-2 was closely related to the prognosis. MMP-9 is overexpressed in osteosarcoma tissues. Some studies found that it could significantly reduce the bone sarcoma cells infiltration and metastasis through suppressing the mRNA level and protein activity of MMP-9. The overexpression of MMP9 in osteosarcoma tissues was closely related with the downregulation of RECK.The RECK gene encodes a 110 kDa glycoprotein that contains serine-proteinase-inhibitor-like domains and is associated with the cell membrane through a GPI anchorm, which was firstly found in v-Ki-Ras-transformed NIH 3T3 cells during the induction of a flat morphology. RECK could inhibit the expression and activity of MMPs through coding GPI anchored glycoprotein, so it is considered to be an endogenous inhibiting factor of MMPs. In recent years, a large number of studies found that the mRNA and protein expression of RECK was lowered significantly in many kinds of tumors such as nasopharyngeal carcinoma, glioma, bladder cancer, non small cell lung cancer, gastric cancer, breast cancer, pancreatic cancer, hepatocellular carcinoma and prostate cancer, and so on. Studies found that in colon cancer, gastric cancer, liver cancer and lung cancer tissues, hypermethylation methylation phenomenon in RECK gene indicated that the prognosis was poor. These results indicated that RECK might inhibit MMPs and then inhibit tumor cell metastasis. Recent studies also found that the low expression level of RECK is an independent predictor of poor prognosis in osteosarcoma. At the same time, a growing number of studies found that the hypermethylation in the promoter region of RECK gene was closely related with the occurrence of tumor development. Oncogene RAS could downregulate the expression of RECK by mediating the methylation of the of RECK gene, however, DNA methyltransferase inhibitor could maintain the expression of RECK and suppress the metastasis of tumor cells. In non-small cell lung cancer, the expression level of RECK was decreased through the promoter methylation of it and this decrease was significantly correlated with mutations of K-ras gene and lymph node metastasis. In oral squamous cell carcinomas, colorectal cancer, gastric cancer, pancreatic cancer, liver cancer, breast cancer and other malignant tumors, researchers found that the promoter methylation of RECK is associated with the metastasis of tumor.So, RECK played an important role in development and metastasis of tumor. However, now fewer literature studies detected the function of RECK in osteosarcoma and the mechanisms. We aimed to investigate the expression levels of RECK in normal and osteosarcoma tissue and detect the function of the promoter hypermethylation of RECK gene and the underlying mechanisms.2 Objectives(1) We detected the mRNA and protein expression levels of RECK in the tissues from normal subjects, the patients of local restrictive osteosarcoma and aggressive metastasis after primary resection of the osteosarcoma. At the same time, we investigated the change of RECK expression levels after the cells were prepared with TGF-beta, VEGF and 5-azacytidine.(2) To explore the levels of promoter methylation of RECK gene and invadosome protein markers in different tissue cells and to reveal the mechanisms of RECK's effects.3 Methods3.1 Patient samples of osteosarcomaThe samples were obtained from 15 control subjects with normal bone,15 patients with locally restricted osteosarcoma patients,15 patients with aggressive metastasis after primary resection of the osteosarcoma (all adult male patients>40 years of age). Tissues were pooled from 5 independent subjects in each group, and triplicate independent samples were finally agglomerated to examine the trends of changes of the different parameters examined.3.2 Cellular enrichmentWhen all samples arrived in the pathology department, they were snap frozen in liquid nitrogen. Tissues were cut into small pieces and then treated with 0.2% proteinase for 1h, furthermore, incubated for 6h in collagenase. The final concentration of the enzymes were determined by Primary pilots prior to tissue matrix degradation. The incubate was first filtered through a fine mesh. And then, the filtered solution was subsequently centrifuged to obtain a cellular pellet. after allowing the cells to settle down and decanting the excess fluid, the pellet was obtained. This enriched cell pellet was either used for the different analyses.3.3 The cells'stimulationTGF-P, VEGF and 5-azacytidine were used to stimulate these cells.3.4 RNA extraction, reverse transcription and Real-time quantitative PCR(RT-PCR)Total mRNA was extracted from the cell lysates using RNeasy Mini kit. All reactions were performed on an ice bucket. RT-PCR were performed to detect the mRNA levels of RECK in different tissue cells.3.5 Polymerase chain reaction for detection of methylationAfter isolation of genomic DNA, bisulfite modification was performed. Methylation-specific polymerase chain reactions were performed in triplicates to compare the differences in methylation of RECK promoters.3.6 Western blotTotal proteins were extracted from different tissue cells. The protein expression levels of RECK were detected by western blot.3.7 ELISAELISA was used to evaluate the protein expression of RECK in control tissues and native tumor tissues, as well as after stimulation with TGF-?, VEGF and 5-azacytidine.3.8 StatisticsData were expressed as meansąSEM. the analyses of variance (ANOVA) was used to compare the differences between multiple groups. Post hoc analyses were performed by the Tukey's honestly significant difference (HSD) test.4. Results4.1 Decrease in RECK mRNA in metastatic variants of osteosarcomaWe compared the differences in the mRNA expression levels of RECK in the control group, locally restricted osteosarcoma group, aggressive metastasis group, locally restricted osteosarcoma+VEGF group, locally restricted osteosarcoma+TGF-? group, control+5-azacytidine group, metastasis+5-azacytidine group. We found that RECK mRNA was significantly reduced in cellular concentrates from the metastatic variants compared with the control group. The reduction of RECK mRNA level in cellular concentrates from the metastatic variants was reversed by 5-azacytidine, while VEGF and TGF-? reduced the mRNA levels of RECK in locally restricted osteosarcoma.5-azacytidine increased the mRNA levels of RECK in metastatic osteosarcoma, which provided indirect evidence that the changes in expression of RECK transcripts may be related to its promoter methylation.4.2 Decrease in RECK protein synthesis in metastatic variants of osteosarcomaWe detected the protein expression levels of RECK by use of ELIAS. Similar with the results of RECK mRNA test, the protein expression of RECK was significantly reduced in cellular concentrates obtained from the metastatic variants compared with the control group. The reduction of RECK protein level in cellular concentrates from the metastatic variants was reversed by 5-azacytidine, while VEGF and TGF-? reduced the protein levels of RECK in locally restricted osteosarcoma. In addition, western blotting was performed to examine the protein expression levels in control and metastatic osteosarcoma group. Compared with the control group, the protein expression of RECK in the metastatic variants was significantly decreased.4.3 RECK promoter methylation in osteosarcomaCompared with control tissues, RECK promoter was hypermethylated in the metastatic variants. The hypermethylation of RECK promoter in the metastatic variants was reversed by 5-azacytidine. This hypermethylator phenotype could be induced by the osteosarcoma cells stimulated with TGF-? or VEGF.4.4 The increased expression of invadosome protein markers in metastatic osteosarcomaThe protein expression levels of invadosome protein markers included MMP9, focal adhesion kinase and beta-integrins (?1 and ?3) were detected. These protein expressions were significantly increased in metastatic osteosarcoma compared with the other groups.5. Conclusion1. The mRNA and protein expression of RECK was significantly decreased in osteosarcoma, especially in the metastatic variants. The hypermethylation of RECK promoter maybe the mechanism of the reduction of RECK expression.2. The reduction of RECK expression was correlated with invadosome protein markers, which suggested RECK could suppress the metastasis of osteosarcoma, while the downregulation of RECK could activate the process. |
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