The Effects Of 5-aza-dC, TSA And The RNA Interference Plasmid Of MeCP2 On Biological Behavior In Lung Epithelial Carcinoma A549 Cells

AIM:To explore the effects of methyltransferase inhibitor 5-aza-2-deoxycytidine (5-aza-dC), histone deacetylase inhibitor trichostatinA (TSA) and the RNA interference plasmid Psilencer-2.1-U6-MeCP2 of methyl-binding protein MeCP2 on biological behavior in lung epithelial carcinoma A549 cells .Methods:Human epithelial carcinoma A549 cells were cultured in DMEM culture medium containing 10% Fetal bovine serum. The recombinant plasmid of Psilencer-2.1-U6-MeCP2 (The plasmid was builded and identitied by the subject members in 2007, in the mRNA level and protein level which can significantly reduce the expression of MeCP2 protein) were transfected into A549 cells with LipofectamilleTM 2000. The trial consisted of eight groups: (1) control group: do not give any treatment of the A549 cells;(2) experimental group 1: 24h after inoculation, A549 cells were treated with 5μmol/L 5-aza-dC;(3) experimental group 2 : 24h after inoculation, A549 cells were treated with 300nmol/L TSA;(4) experimental group 3: the transfected A549 cells ;(5) experimental group 4: 24h after inoculation, A549 cells were treated with 5μmol/L 5-aza - dC and 300nmol/L TSA;(6) experimental group 5: 24h after inoculation, the transfected A549 cells were treated with 5μmol/L of 5-aza-dC;(7) experimental group 6: 24h after inoculation, the transfected A549 cells were treated with 300nmol/L TSA;(8) experimental group 7: 24h after inoculation, the transfected A549 cells were treated with 5μmol/L 5-aza - dC and 300nmol/L TSA.The effect of apoptosis in different groups for 72 h was detected by Annexin V / PI double staining. the expression of C/EBPαmRNA in different groups for 72 h was detected by RT-PCR.Results: (1) The recombinant plasmid of Psilencer-2.1-U6-MeCP2 cloud be stably transfect into A549 cells.(2) 5-aza-dC,TSA and the recombinant plasmid of Psilencer-2.1-U6-MeCP2 can induce A549 cells apoptosis, the apoptosis of A549 cell in the groups of combination with two treatments were significantly higher than single- treatment groups (all P <0.01),the apoptosis of A549 cells in the three combined treatments group was higher than the combination with two treatments groups (all P <0.05), The differences were statistically significant.(3)5-aza-dC,TSA and the recombinant plasmid of Psilencer-2.1-U6-MeCP2 can increase the expression levels of C/EBPαmRNA,the expression levels of C/EBPαmRNA in the groups of combination with two treatments were significantly higher than single- treatment groups (all P <0.01),in the three combined treatments group was higher than the combination of the two treatments groups (all P <0.05),The differences were statistically significant.(4)There were significant differences between experimental groups and control group in relative expression of C/EBPαmRNA and cell apoptosis rate(all P < 0.05).(5)It was no significantly differences between 5-aza-dC group ,the TSA group and the recombinant plasmid of Psilencer-2.1-U6-MeCP2group in induce apoptosis and enhance the expression of C/EBPαmRNA.Conclusion: Compared with 5-aza-dC, TSA or the recombinant plasmid of Psilencer-2.1-U6-MeCP2 alone, combination treatments can significantly induced apoptosis and enhance C/EBPαmRNA expression,and the effects were most significant in three combined treatment, which will provide a theoretical basis for the demethylation of multi-drug combination therapy of lung cancer.