Effect On PTEN Methylation In Osteosarcoma Occu-rring And Influence On Cytology Behavior Of Cell Line MG-63Demethylated By5-Aza-CdR

BACKGROUND Osteosarcoma is one of primary neoplasms of the anterior mediastinum with highly variable histopathological characteristics. This heterogeneity has long hindered the development of a standardized protocol for diagnostic and prognostic evaluation of Osteosarcoma. The modified Enneking staging system, is currently the most commonly employed scoring method, but its value is limited by the fact that it assigns scores only for specific macroscopic surgical, but not histological, characteristics, making it difficult to reliably assess tumor severity in patients. Previous studies showed that multiple genetic alterations involved in many kinds of carcinomas, but the molecular mechanisms of Osteosarcoma is poorly understood. Recent data suggests that carcinomas appear to be a process that is caused by genetic alterations and by epigenetic mechanism. It mainly involves DNA methylation and histone modification. DNA methylation is one of the major mechanisms of epigenetics. Aberrant methylation of promoter regions resulting in inactivation of human tumor suppressor gene expression has been proposed to be an important mechanism in cancer. Cancer cell DNA is frequently characterized by localized gene-specific hypermethylation in TSG, the expression and status of DNA methylation of gene PTEN have already been reported in many kinds of tumor, such as lung cancer, colon cancer and oral carcinoma ea tl. Although multiple genetic and epigenetic changes have been detected in many kinds of cancer, the precise molecular mechanisms in the development and/or progression of Osteosarcoma still remain unknown. PTEN is the first TSG which has both Lipid phosphatase and Dual specificphosphatase action. The DNA mythlation can be altered by demethylation. The drug5-Aza-CdR, the DNA methyltransferase inhibitor, is the most mainly demethylation drug which has been applied for clinical treatment widely and be authorized by FDA,USA. To clarify the demethylation mechanism of the5-Aza-CdR is relatively important to the treatment of Osteosarcoma.There are3parts content in our research works. In part1, the DNA metyhlation status of PTEN in tissues of the Osteosarcoma was detected by MSP method, and the methylation results and the clinical data were analysed together. In part2, the cell behavior and the DNA methylation status of the MG-63cell were detected by the cytology test and the MSP method. In part3, To establish nude mouse animal models and intervene demethylatedly it through5-Aza-CdR injection, and the expression of PTEN and the DNA methylation of PTEN of the transplantation tumor were detected by WB and MSP methods. Part I PTEN Abnormal DNA Methylation and Its Clinical Significance in OsteosarcomaObjective:To investigate the expression and the promoter hypermethylation of PTEN and potential clinical significance in patients with Osteosarcoma.Methods:Methylation-specific PCR (MSP) method was performed for evaluating the expression and promoter region methylation patterns of PTEN in Osteosarcoma. The correlation between TSGs mRNA, TSGs methylation and clinicopathological data were analyzed.46patients with Osteosarcoma were surgically treated in the HN province tumor hospital, the2nd xiangya hospital and the xiangya hospital, and from April,2001to April,2011. The samples of Osteosarcoma were be detected by HE dye, immunohistochemistry and MSP, and the methylation status of the samples and the clinical data of the Osteosarcoma patients were analysed together for discussing the relationship between the above two.Results:There has been significant defference between the tumor tissue group and the non-tumor tissue group about the results of the expression of PTEN and the DNA methylation status of PTEN (P<0.05). PTEN hyermethylation is correlative with the tumor grades and stages of Osteosarcoma. Enneking surgery stages of tumor (P=0.002)、Price surgery classification (P=0.003)、position of tumor occurring (P= 0.030).Conclusions:1、The down-regulation of PTEN in osteosarcoma and the status of DNA methylation of promote region is hymethylation.2、PTEN hymethylation is relatively correlative with the tumor grades and stages of Osteosarcoma. Part II Influence of Human Osteosarcoma Cell MG-63was Demethylated by5-Aza-CdR on Cytology behavior and PTEN MethylationObjective:To screen completed methylation cell in the3Osteosarcoma cell lines, and to explicit the effect of demethylation of5-Aza-CdR in vitro, and to confirm the mechanism of action of the PI3K/AKt pathway can be negatively regulated by PTEN in Osteosarcoma cell line.Methods:The3Osteosarcoma cell lines MG-63,SAOS-2and U2-OS were detected by MSP, and the PTEN completely methylation cell line was screened, and this cell line was intervened by5-Aza-CdR in terms of difference drug density and active time. The protein of the PI3K and the AKt were detected by WB method in this cell line.Results:The MG-63cell line display the completely methylation in3cell lines, and the best drug density and effect time are separately10μmol/L and24h after5-Aza-CdR intervention. There has been significant difference between the intervening group and the control group (P<0.05), meanwhile, same significant difference between2groups on protein expression of the PI3K and AKt (P<0.05).Conclusions:1、The human Osteosarcoma MG-63cell line display the completely mythlation on PTEN.2、The MG-63cell line demonstrates the dependency of drug density and effect time after5-Aza-CdR intervention.3、5-Aza-CdR can exert an influence on cell behavior of MG-63.4、The protein expression of the PI3K and the AKt were down-regulated by5-Aza-CdR in Osteosarcoma cell line MG-63, and the mechanism of action of PTEN negatively regulates the PI3K/AKt pathway in Osteosarcoma was confirmed. Part Ⅲ Influence of Expression and DNA Mythlation of PTEN in Osteosarcoma Nude Mice Model Loaded Transplantation Tumor Intervened Demethylation by5-Aza-CdRObjective:To establish nude mice model loaded Osteosarcoma cell line MG-63transplantation tumor and to clear the demethylation effect mechanism of5-Aza-CdR in vivo.Methods:18nude mices which4～6weeks were used subcutaneous vaccination by1×10/200μl cell line MG-63suspension for establishing transplantation tumor loaded nude mice model. After modeling success, the animal models were divided into the group experimental(A) and control(B) separately9animals. The nude mice models of group A were intervened by5-Aza-CdR in terms of drug density10μmol/L and dose1μl/g. After4weeks, the animals were executed and the transplantation tumor were dissected. The volume of the transplantation tumor were compared between group A and B. The expression of PTEN of the transplantation tumor were detected separately by IHC method and Western-Blotting method, meanwhile, the status of DNA methylation in PTEN of it by MSP technique.Results:The human Osteosarcoma cell line MG-63transplantation tumor loaded nude mice models can be established successfully. There has been significant difference between the group A and B on the volume of transplantation tumor (P<0.05), and similar difference between the groups2on IHC results (P<0.05). The MSP results demonstrates that the PTEN hypomethylation status can be detected in transplantation tumor of group A and there has significant difference between the group A and B (P<0.05).Western-Blotting results demonstrates that the protein expression of the group A were up-regulation, and the same difference between the group A and B (P<0.05)Conclusions:1、The human Osteosarcoma cell line MG-63nude mice model loaded transplantation tumor can be established successfully through using subcutaneous vaccination method.2、The grow of the transplantation tumor of nude mice model can be inhibited by5-Aza-CdR, and the expression of PTEN of the tissue can be up-regulated and the status of PTEN hypermethylation can be inhibited by5-Aza-CdR in vivo.