Experimental Studies To The Effect Of 5-Aza-2'-Deoxycytidine And Sodium Butyrate On The Expression Of Runx3 Gene And The Growth Of Human Gastric Cancer Cell Lines

PartⅠPreliminary studies of the expression of Runx3 gene in human gastric cancer cell lines and the mechanism of gene silenceObjective:To examine the expression of Runx3 in vitro in human gastric cancer cell lines and to study the mechanism of the gene silence.Methods:The change of the expression of Runx3 mRNA was observed by Reverse transcription-PCR (RT-PCR) in human gastric cancer cell lines MKN-28 and MKN-45. The promoter methylation status was measured by methylation specific PCR (MSP).Results:Runx3 promoter was hypermethylated and Runx3 mRNA expression was not found in MKN-28 cell, whereas Runx3 mRNA was expressed and its promoter methylation status had no abmormality in human gastric cancer cell line MKN-45.Conclusions:The promoter hypermethylation of Runx3 is the crucial mechanism of its silence in human gastric cancer cell line MKN-28. PartⅡStudies to the effect of 5-aza-2'-deoxycytidine and sodium butyrate on growth of human gastric cancer MKN-28 cell linesObjective:To examine the effect of demethylating agent 5-aza-2'-deoxycytidine and histone deacetylase inhibitor sodium butyrate on the cell growth of human gastric cancer cell line MKN-28.Methods:The inhibition of cell growth was observed by MTT, The cell cycle and apoptosis were analyzed by flow cytometry.Results:The inhibitory effect of 5-aza-2'-deoxycytidine or sodium butyrate on the growth of MKN-28 cell were found in a dose- and-time dependent manner; Comparing with 5-aza-2'-deoxycytidine or sodium butyrate alone, the inhibitory effect of 5-aza- 2'-deoxycytidine combined with sodium butyrate was significantly increased(P<0.05). The cell cycles were not blocked by 5-aza-2'-deoxycytidine, whereas the ratio of G0/G1 phase was significantly increased by sodium butyrate and 5-aza-2'-deoxycytidine com- bined with sodium butyrate(P<0.05).The cell apoptosis rate induced by 5-aza-2'- deoxycytidine,sodium butyrate and 5-aza-2'-deoxycytidine combined with sodium butyrate were (8.3±1.3)%, (20.8±2.4)%, (33.7±3.1)% respectively.Comparing with the control group (2.0±0.5)%, the cell apoptosis rate was significantly increased(P<0.05).Conclusions : Demethylating agent 5-aza-2'-deoxycytidine and histone deacetylase inhibitor sodium butyrate could inhibit MKN-28 cell growth respectively, combined of them had synergetic effect.This inhibitory effect might be related to inducing apoptosis and arresting cell cycles. PartⅢStudies to the effect of 5-aza-2'-deoxycytidine and sodium butyrate on the expression of Runx3 gene in human gastric cancer cell linesObjective:To examine the effect of demethylating agent 5-aza-2'-deoxycytidine and histone deacetylase inhibitor sodium butyrate on the expression of Runx3 gene in human gastric cancer cell line MKN-28.Methods:The expression level of mRNA and protein of Runx3 gene were detected by RT-PCR and Westem blotting respectively. The promoter methylation status after 5-aza-2'-deoxycytidine treated was measureed by MSP.Results:The Runx3 mRNA and protein in MKN-28 cell were re-expressed by 10μmol/L 5-aza-2'-deoxycytidine for 72h, relative expression level of mRNA and protein were 0.63±0.05 and 0.38±0.04 respectively, whereas its expression cannot be induced by 2.5 mmol/L sodium butyrate alone, the Runx3 mRNA and protein were also re-expressed by 5-aza-2'-deoxycytidine combined with sodium butyrate,relative expression level of mRNA and protein were 0.86±0.02 and 0.62±0.02 respectively. Comparing with 5-aza-2'-deoxyc- ytidine, the relative expression level of mRNA and protein of Runx3 of 5-aza-2'- deoxycytidine combined with sodium butyrate was significantly increased(P<0.05). Runx3 promoter was hopermethylated after 10μmol/L 5-aza-2'-deoxycytidine treated for 72h.Conclusions:5-aza-2'-deoxycytidine could induce the re-expression of Runx3 mRNA and protein in MKN-28 cell, sodium butyrate cannot induce the re-expression of Runx3, whereas combined of them had synergetic effect. The hypermethylated status of Runx3 gene in MKN-28 cell can be reversed by demethylating agent 5-aza-2'-deoxycytidine.