The Effect And Significance Of The Tumor Suppressor Gene WWOX Expression On Human Osteosarcoma MG-63

Osteosarcoma is the most common primary malignant bone tumors, espcially for young men, can be earlily pulmonary metastasis, prognosis is bad with the mortality and morbidity highly, which cause serious damage to human health. The occurrence and development of researching osteosarcoma make more and more genes involved in the process, but the pathogenesis of osteosarcoma and participation in the exact gene has not yet been elucidated. Past studies focused on genetic and chromosomal mutation of osteosarcoma,found that the P53, P16and MDM gene point mutation, amplification, lack development has a close relation with the incidence of osteosarcoma. As the molecular pathology of osteosarcoma research is gradually developing, gene epigenetics change especially the occurrence and development of methylation of osteosarcoma role gradually get attention. DNA methylation is a common way of modify gene expression, exists in nearly all higher level animal, can shut down the activity of certain genes. DNA methylation is currently in the medical community attention hot spot, CpG island methylation is a common form of methylation, in the human genome, there are about40000CpG sequences, is located in the promoter region of genes or the first exon region, CpG sequences in the genome frequency is low, but in a specific area, high density and play an important role in CpG sequences, under normal circumstances, the site is in the methylation of CpG sequences, can only CpG sequences of cytosine methylation and gene lost function. Methylation changes are important factors in the process of cancer development, contains CpG island methylation levels of abnormal rise, thus can lead to genomic instability, including chromosome mutation, elevated expression of oncogene and tumor suppressor gene expression decreased. Research in tumor methylation is concerned for the inactivation of tumor suppressor genes, tumor suppressor genes is of normal cell proliferation play a role of negative regulation of gene, its coded protein can reduce and restrain cell division, generally only when the two alleles are not work in tumor and lose function as a tumor suppressor genes of the third way, tumor suppressor genes methylation typically occur in the CpG island, generally do not occur in the genes coding region. Promoter CpG loci can appear in5’end methylated tumor suppressor gene inactivation, in malignant tumors are common, but in benign tumors are relatively rare. Contains WW domain structure of REDOX enzyme gene (WWOX) is located in the common fragile sites FRA16D tumor-suppressor genes, often because of CpG island methylation deactivation causes cancer, numerous studies have found that WWOX gene inactivation in a variety of incidence is higher in malignant tumors, study confirms that WWOX deactivation causes of malignant tumor cell apoptosis and the mechanism of dysfunction. Has confirmed that WWOX play a role of tumor suppressor genes in a variety of malignant tumor, in the building of WWOX gene defects in mice, about31%in baby mice phase, the osteosarcoma, so presumably WWOX inactivation and in the pathogenesis of osteosarcoma, but concrete action and mechanism still needs further research.Objective:To investigate the effect and significance of the tumor suppressor gene WW domain containing oxidoreductase(WWOX) expression on human osteosarcoma cell line(MG-63). Methods:Build the recombinant eukaryotic expression plasmid WWOX,and Transfect which into human osteosarcoma cell line MG-63, Flow cytometry Annexin V/PI were used to essay apoptosis rate of MG-63.Reverse transcription-polymerase chain reaction (RT-PCR) methods were used to detect amplification of WWOX and bcl-2. Western blotting methods were used to examine the expression of WWOX and bcl-2.Results:Osteosarcoma cells with stable transfection of WWOX were build. Flow cytometry Annexin V/PI shows the transfected with WWOX group osteosarcoma cells than transfection with blank plasmid group and the control group, apoptosis significantly increased [(8.21±0.03)%vs (1.13±0.03)%vs (1.12±0.02)%, P <0.05],The expression of WWOX mRNA (1.612±0.072)and protein(1.680±0.072) was increased significantly in transfected with WWOX group compared with the other groups(P<0.05),The expression of bcl-2mRNA (0.194±0.088)and protein(0.236±0.053) was decreased significantly in transfected with WWOX group compared with the other groups(P<0.05).Conclusion:WWOX can inhibit the proliferation of osteosarcoma cells and promote apoptosis of osteosarcoma cell,The mechanism of which may be associated with decreased expression of bcl-2.