| Objective：It is a multi-factor and multi-stage process for osteosarcoma occurs,andit is the result of the activation of oncogenes and tumor suppressor gene inactivation. Inaddition to genetic alterations, hypermethylation of promoter on tumor suppressor geneis the most common epigenetic mechanisms，because they do not involve changes inDNA sequences which are more easily reversed. If hypermethylation of tumorsuppressor gene promoter region can be reversed, so that the gene expression regain.theoretically tumor suppressor gene can play its function of inhibiting the growth oftumor cell.Chemotherapy plays an important role in the treatment of osteosarcoma, butosteosarcoma chemotherapy drug resistance is constantly increasing. Studies haveshown that promoter methylation is probably the basis of the potential of osteosarcomaresistance to chemotherapy drugs, by demethylation to reverse drug resistance ofosteosarcoma, thereby effectively enhancing the survival of patients with osteosarcomarate.RASSF1A as a tumor suppressor gene, G1cell cycle control and inhibition oftumor growth to S phase transition. The main mechanism of inactivation of RASSF1Agene promoter region is5’CpG island hypermethylation. RASSFI A gene deletion, lossof negative regulation of cell cycle function, cell prone to malignant transformation,promote tumorigenesis and development.The study includes:1,5Aza-dc with chemotherapy drugs used MG63osteosarcoma cells, to explore synergies between the two, and enhance the efficacy ofchemotherapy drugs;2,5Aza-dc on MG63osteosarcoma cells RASSF1A genemethylation affect the level; influence3,5Aza-dc on protein expression of RASSF1AMG63osteosarcoma cells, in order to further explore and provide meaningful data andtheoretical basis for the treatment of osteosarcoma by changing the methylation of tumor suppressor genes.Methods:Human osteosarcoma cell line MG63application demethylating agent5-Aza-2’-deoxycytidine (5Aza-dc) induction treatment.To detect5Aza-dc impact andADM alone and combination therapy on MG63cell viability by MTTmethod;Application of flow cytometry apoptosis rate changes. MSP application method,RASSF1A gene methylation status before and after treatment5Aza-dc. Using theWestern blot method, the RASSF1A gene protein expression levels before and aftertreatment were analyzed5Aza-dc.Results: MTT assay and flow cytometry,5Aza-dc concentrations were2.0,4.0and8.0μmol/L, within a certain range as the dose increased, the inhibition of cellproliferation MG63more obvious,5Aza-dc role48h, MG63cell survival rates were83.4%,62.8%and43.2%. Flow cytometry,5Aza-dc can be significantly increasedapoptosis rate MG63cells in a dose-dependent manner, the concentration of8.0μmol/L when the effect is most obvious.MSP, Western blot displayed before the application5Aza-dc treatment, MG63cells showed methylation status of RASSF1A gene,RASSF1A gene protein expression deletion by2.0,4.0and5Aza-dc role8.0μmol/Lafter48h, MSP Show MG63cells RASSF1A gene methylation reversal; Western blotdisplay RASSF1A gene protein recovery5Aza-dc collaboration with ADM cansignificantly enhance the killing effect of MG63cells. By MTT assay, ADM role8μg/mL,16μg/mL,32μg/mL,64μg/mL for48h, MG63cell survival rates were92.3%,84.8%,74.1%,60.5%, added to a final concentration of2.0μmol/L after5Aza-dc, under the same conditions cell viability dropped87.2%,75.8%,45.6%and33.7%.Conclusions：1.5Aza-dc can better MG63osteosarcoma cells to reverse abnormal DNAmethylation, methylation-induced activation due to high re-transcriptional genesilencing induced expression of the gene.2.5Aza-dc can effectively induce apoptosis in osteosarcoma MG63.3.5Aza-dc and ADM have a synergistic effect, enhancing the sensitivity of MG63osteosarcoma cells to chemotherapeutic drugs. |
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