The Function And Molecule Mechanisms Of MeCP2 In Human Osteosarcoma

Osteosarcoma is the most common primary malignant bone tumor and usually arises from primitive transformed cells of mesenchymal origin(and thus a sarcoma), such as the distal femur, proximal tibia, and proximal humerus during the second decade of life. A second smaller peak occurs between 60 to 80 years. Overall, osteosarcoma has a moderate incidence, with 10 to 26 per million new cases worldwide each year. The 5-year survival in osteosarcoma in the first half of the twentieth century was less than 20 %. Nowadays, longterm survival for patients with localized osteosarcoma has improved to approximately 60%, and the long-term survival of patients with metastatic osteosarcoma still remains at 25 to 30%. However, the specific mechanism and effective drugs for osteosarcoma have not been investigated clearly. DNA hypermethylation plays an important role in silencing the tumor suppressor genes being one of the most consistent hallmarks of the human cancers, and the phenomenon is of a comparable significance to classic genetic mutations. Especially for recent years, DNA methylation has emerged as an attractive target for the cancer therapeutics. The repressive effects of DNA methylation are mediated in large part by the methyl-CpG binding proteins(MBDs) and also associated with histone modifying and chromosome remodeling complexes.The generation of novel aberrantly hypermethylated regions during cancer development and progression makes MBD proteins interesting targets for their biological and clinical implications. The unspecific reactivation of methylated sequences is the main problem of epigenetic treatments. Parallel with demethylation of tumour suppressor genes CpG islands, global genomic demethylation, which could cause or contribute to chromosomal instability, also occurs. Instead of targeting DNA methyltransferases, the proteins that recognise the signal encoded by DNA methylation and recruit histone modifying and chromosome remodeling complexes to these methylated sequences, such as MBDs, could also be good targets in cancer therapy. This strategy could avoid problems associated with genomic instability which inhibiting DNA methylation per se could induce. In early job, We have established a model of malignant transformation of osteoblasts and found the imprinted genes TSSC3 involved in malignant transformation of osteoblasts by gene chip.we found promoter hypermethylation may be an important epigenetic mechanism of silencing TSSC3 and loss expression of TSSC3 play an important role in the anoikis-resistant osteosarcoma cell. As a potential tumor suppressor gene, TSSC3 may become a new target for the treatment of osteosarcoma. We envisage, MBD protein may mediate the silence of TSSC3 expression.Usually, the MBDs, such as methyl-CpGbinding protein 2(MeCP2), methyl-CpG-binding domain 1(MBD1), and MBD2, could specifically bind to CpG methylated DNA and are associated with the histone deacetylase(HDAC).MethylCpG-binding protein 2(MeCP2) is a DNA methylation-related gene of the methyl-CpG-binding protein family. Methyl-CpG-binding protein 2(MeCP2) is essential in human brain development and has been linked to several cancer types and neuro-developmental disorders. Here, we investigated the epigenetic function of the MeCP2 in SaOS2 and U2 OS cell lines, and explored the antitumor effects of the gene silencing for osteosarcoma. Furthermore, this study aims to screen the MeCP2 related differentially expressed genes and discover the therapeutic targets for osteosarcoma.In this study, chromatin immunoprecipitation assay was used to detect MeCP2 binding activity with TSSC3 gene. RT-PCR and western blot assay were used to analyze the MeCP2 expression in osteosarcoma cell lines after transfection with LV-MeCP2-RNAi. Transwell invasion and migration assays were used to detect the cell invasion and migration. The cell apoptosis was examined by using the flow cytometry assay. The tumor size was also assessed to determine the therapeutic effects of gene silencing. CCK8 assay was used to detect the proliferation and SaOS2 and U2 OS cells. Apoptosis of cells was detected by flow cytometry analysis that monitored Annexin V-APC/7-DD binding and 7- ADD uptake simultaneously. Denaturing formaldehyde agarose gel electrophoresis was employed to examine the quality of total RNA 18 S and 28 S units. Gene chip technique was utilized to discover the differentially expressed genes correlated with MeCP2 gene. Differential gene screening criteria were used to screen the changed genes. The gene up-regulation or downregulationmore than 1.5 times was regarded as significant differential expression genes.The results indicated that MeCP2 haved the highest combining power with TSSC3 gene. But this effect is not the primary mechanism for regulating the expression of TSSC3 gene. LVMECP2- RNAi could decrease MeCP2 level in tumor cells compared with the untreated cells,but also reduces the expression of TSSC3 gene. LV-MECP2- RNAi inhibited the U2 OS and SaOS2 cells invasion and migration compared with the control cells. LVMECP2- RNAi triggered the U2 OS and SaOS2 cell apoptosis, and inhibited the cell proliferation significantly compared with the control cells. The gene silencing of RNAi could also decreased the tumor size significantly compared with untreated cells. The CCK8 results indicated that the cell proliferation of MeCP2 silencing cells(LV-MeCP2- RNAi) was significantly decreased compared to non-silenced cells(LV-MeCP2-RNAi-CN).MeCP2 silencing could also induce significant apoptosis compared to non-silenced cells; 107 expression changed genes were screened from a total of 49,395 transcripts. Among the total 107 transcripts, 34 transcripts were up-regulated and 73 transcripts were down-regulated There were five significant differentially expressed genes, including IGFBP4, HOXC8, LMO4, MDK, and CTGF, which correlated with the MeCP2 gene. The methylation frequency of CpG in IGFBP4 gene could achieve 55%. LVMECP2- RNAi could decrease MeCP2 level in tumor cells compared with the untreated cells, Simultaneously increase the expression of IGFBP4 gene.In conclusion, MeCP2 haved the highest combining power with TSSC3 gene. But this effect is not the primary mechanism for regulating the expression of TSSC3 gene. Silencing the MeCP2 gene could block the MeCP2 expression and inhibit the tumor cell migration, invasion, and proliferation, also decreases the tumor size by inducing the apoptosis of the tumor cells.The differentially expressed IGFBP4, HOXC8, LMO4, MDK, and CTGF genes may be involved in MeCP2 gene mediated proliferation and apoptosis in osteosarcoma cells. IGFBP4 may be targeting molecules of MECP2 gene in SaOS2 cells. Reducing the expression levels of MECP2 by RNAi can recover IGFBP4 expression and tumor suppression function, thereby altering the biological effects of tumor cells.