Effects Of 5-aza-2'-deoxycitydine And Trichostatin A On Expression Of MGMT And E-cadherin Gene In Human Gastric Carcinoma Cell Lines

ObjectiveInactivation of tumor suppressor genes and activation of oncogenes are inevitable molecular events in the development of human cancer. In particular, the genetic mutation or deletion of tumor suppressor genes that inhibit the formation of tumors is known as one of the main driving forces in carcinogenesis. Recent evidence has identified a second mechanism potentially responsible for inactivation of tumor suppressor genes during tumorigenesis, namely transcriptional silencing by hypermethylation. The methylation of DNA is an epigenetic modification that plays an important role in the control of gene expression in mammalian cells. The terms 'Epigenetic', which literally mean 'out of conventional genetics or mutations', are now used for an inheritable change in the pattern of gene expression without a change in DNA sequence.Gastric cancer, one of the most common tumors worldwide. Global gastric cancer mortality in all cancer mortality in the second place. An increasing number of genes that are inactivated by CpG island hypermethylation have been reported in gastric cancer, involving tumor suppressor, cell-cycle regulator, tissue-invasion-related and DNA mismatch repair genes.We want to study tumor suppressor genes MGMT and E-cad methylation in human gastric carcinoma cell lines and the relationship between tumor suppressor genes methylation and genes inactivation to find the molecular mechanism of gastric carcinoma carcinogenesis and a new way of therapy.Methods1,Human gastric carcinoma cell lines MGC-803 and MKN-45 were cultured in RPMI1640. Divide cell lines into four group and them was treated by 5-aza-2'-deoxycitydine(5-Aza-dC) and Trichostatin A(TSA). (1) Without drug control group; (2) 5-Aza-dC(5μmol/L) was used for 72 h in the treatment; (3) TSA(300nmol/L)was used for only 24 h in the treatment; (4) 5-Aza-dC was used for 48 h followed by TSA for an additional 24 h in the combined treatment.2,Methylation-specific Polymerase Chain Reaction(MSP): DNA methylation status of MGMT and E-cad gene promoter in MGC-803 and MKN-45 was assayed by MSP.3,Reverse Transcription-Polymerase-Chain Reaction(RT-PCR): The expression of MGMT and E-cad in MGC-803 and MKN-45 was detected by RT-PCR.Results1,MSP detection: MGMT gene was hypermethylated in MGC-803; E-cad gene was Hemimethylated in MKN-45. 5-Aza-dC induced the demethylation of MGMT and E-cad gene and the methylation of MGMT in MGC-803 and E-cad in MKN-45 cell lines was reversed after 5-Aza-dC treatment.2,RT-PCR detection: MGMT and E-cad mRNA was expressed in MGC-803 and MKN-45 after 5-Aza-dC treatment, but it was undetectable or express weakly before the treatment. The combination of 5-Aza-dC and TSA dramatically up-regulate expression of mRNA in gastric carcinoma cell lines.Conclusion1,The expression levels of MGMT and E-cad in MGC-803 and MKN-45 were associated with DNA methylation status. The methylation of promoter region in CpG islands is a main mechanism of MGMT and E-cad transcriptional inactivation.2,The expression of mRNA was re-expressed after 5Aza-dC treatment in gastric carcinoma cell lines with the methylation of MGMT and E-cad genes. The combination of 5-Aza-dC and TSA synergistically enhance expression of mRNA in human gastric carcinoma cells.