Rearch Of Effects Of 5-aza-2'-deoxycitydine Combined With Triehostatin A On Methylation Status Of MGMT Gene In Human Lung Cancer Cells A549

Objective and significance:The occurrence and development of lung cancer is a complex multistage process involving multi-gene changes.There is a close relationship between the occurrence and development of lung cancer and the gene changes which contains inactivation of tumor suppressor genes,oncogene activation and defects of DNA repair gene.O⁶-methylguanine-DNA-methyltransferase (MGMT) gene is the most representative one of DNA repair genes.It is an efficient direct DNA repair enzyme.It is mainly to repair DNA damage caused by alkylating agent,and to protect the chromosome from the mutagenic effect,carcinogenesis and cytotoxicity of alkylating agent.It is significant for maintaining the stability of the genome.And there is a close relationship between the expression of MGMT gene and the occurrence and development of lung cancer.This study was designed to detect the growth inhibition of histonedeacetylase inhibitor(HDACI) Triehostatin A(TSA) act on lung cancer A549 cells;abserve the influence of DNA methyltransferase inhibitor(DNMTI) 5-aza-2'-deoxyeytidine(5-Aza-CdR) and TSA on the methylation status and expression of MGMT gene in A549 cell,discuss the relationship between DNA methylation status and transcription of tumor suppressor gene.And then further explore the new ideas in treatment of lung cancer by changing the genetic methylation status.Methods:1,Cultivate lung cancer cell line A549 in vitro,and then detect the inhibition rate of A549 cell after treated with TSA at different time and concentrations.2,Human lung cancer cell line A549 were treated by 5-aza-CdR and TSA separately and jiontly.Collect the cells,then extract the genomic DNA by classic method wich contains proteinase K digestion followed by phenol chloroform extraction and ethanol precipitation.The genomic RNA was extracted by Trizol.After modified by sodium bisulfite,the DNA was used to investigate the methylation status of MGMT gene in A549 cells treated separately and jiontly by 5-aza-CdR and TSA and control group by methylation-specific polymerase chain reaction(MSP). And the RNA was used to detect the expression of MGMT gene in A549 cells treated separately and jiontly by 5-aza-CdR and TSA and control group by reverse transcription-polymeras chain reaction(RT-PCR).Results:1,TSA can inhibit the growth of the lung cancer cell A549 in varying degrees.And in a certain range,the inhibition rate increased gradually with the extension of the processing time and the concentration increasing.The IC₅₀ at 24h,48h and 72h are 1622.13 nmol/L,490.91 nmol/L and 257.24 nmol/L。2,MGMT gene was in completely methylation and low-expression state in A549 cells in control group.3,After treated by 5-aza-CdR alone,MGMT gene demethylated and restorated of expression;but after treated by TSA alone,MGMT gene did not demethylate and just restorated of expression slightly.After treated by 5-aza-CdR followed by TSA,MGMT gene demethylated and the expression level significantly increased,and higher than the 5-Aza-dC and TSA group.Conclusion:1,TSA can inhibit the growth of lung cancer A549 cells2,The main reason leading to a low level gene expression, inactivation of transcription of MGMT gene in lung cancer cell A549 may be the abnormal methylation in the promoter region.3,5-aza-CdR can reverse the methylation status and reactivate the expression of tumor suppressor gene.There is a synergistic effect when treat A549 cell with 5-Asa-CdR and TSA.5-Asa-CdR and TSA can significantly enhanced the expression of tumor suppressor gene.That may provide a new way of biological therapy for lung cancer.