Effects Of5-Aza-CdR On The Proliferation And RASSF1A Gene Expression Of Osteosarcoma Cells MG63

Objective:Explore the difference in RASSF1A tumor-suppressor ge-nes mRNA and protein expression and the influence of cell prolife-ration and apoptosis after different concentration of methylation shift enzyme inhibitors5-Aza-CdR (5-Aza-2-deoxycytidine,5-nitrogen miscellaneous-2deoxidizing cell nucleoside) interventting osteosarco-ma MG63.Methods:(1) Cell proliferation condition detection:Training of osteosarcoma MG63cells,2-3days passaged once. Take the growth good cell, respectively for different doses (0,5,10,50μmol/L)5-Aza-CdR treatment, for the different time (24h,48h,72h,96h), MTT method to detect each cell proliferation.(2) Expression of mRNA of RASSF1A gene in cell lines changes after different concentration5-Aza-CdR processing MG63:train osteosarcoma MG63cells, and extract total RNA cells, use RT-PCR amplification to detect the level of transcription mRNA in MG63cell line that processed by different doses (0,5,10,50μmol/L)5-Aza-CdR after72h.(3) Expression of RASSF1a protein changes in cell lines after different concentration5-Aza-CdR processing MG63:with different doses detect MG63respectively (0,5,10,50μmol/L)5-Aza-CdR processing72h, through the0.2%pancreatic enzyme treatment, collect cells after centrifugal, the western-blot test RASSFla protein expression level in MG63cell lines.Results:(1) Cell proliferation changes:osteosarcoma MG63cells inducted by0-50μmol/L5-Aza-CdR for96h, MG63cell proliferation reduce, cell growth speed decreased obviously, the difference was statistically significant (P<0.05).(2) RASSF1A gene expression changes:the RASSF1A gene expression significantly raised in osteosarcoma MG63cells after inducted by0-50μmol/L5-Aza-CdR for72h by RT-PCR and WB detection, compared to experimental the difference was statistically significant (P<0.05).(3) RASSF1A gene expression of tumor suppressor role:we found,5-Aza-CdR processing MG63cell lines, cell proliferation rate decreased obviously, at the same time, the RASSF1A gene transcription and translation level obviously increased.Conclusion:Osteosarcoma MG63cells intervented by5-the Aza-CdR, with the increasing of the concentration of intervention,the expression of suppressor genes RASSF1A mRNA and the protein rise, and at the same time the proliferation rate of MG63cells reduce.