Effect Of5-Aza-CdR On Growth And Methylation Of RUNX3Gene In Human Pancreatic Carcinoma Cell Line MiaPaca2

Objective:To investigate the effect of demethylating agent5-Aza-2’-deoxycytidine on the Proliferation of human pancreatic cancer cell line MiaPaca2and the expression and methylation of runt-related transcription factor3of human. To discuss the primary mechanism for inactivation of the RUNX3gene in pancreatic cancer cells, and further explore the new ideas in treatment of pancreatic cancer by changing the genetic methylation status.Methods:(1) Human pancreatic cancer cell line MiaPaca2cells which were cultured in vitro were treated with different concentrations of5-Aza-CdR(2.5μmol/L5μmol/L、10μmol/L、20μmol/L)for24hour～96hour. Morphological changes of MiaPaca2cells were observed by inverted microscope; The activity of the cell proliferation was analyzed by MTT.(2) Extract the genomic DNA of pancreatic cancer cell line MiaPaca2cells which were treated with10μmol/L5-Aza-CdR for72h and without5-Aza-CdR, respectively. The methylation status of RUNX3gene was detected by methylation-specific polymerase chain reaction(MSP).(3) Extract the total RNA of MiaPaca2cells which were treated with5μmol/L、10μmol/L5-Aza-CdR for72h and without5-Aza-CdR, respectively. The expression of RUNX3mRNA was detected by semi-quantitative reverse transcription-polymerase chain reaction(RT-PCR).Results:(1) The growth of the cells was inhibited after the treatment of5-Aza-CdR. The greater the drug concentration and the longer the duration, the more obvious inhibition.(2) The RUNX3gene of MiaPaca2cells which were treated without5-Aza-CdR was complete methylation status, and the RUNX3gene of the cells which were treated with10μmol/L5-Aza-CdR for72h was incomplete methylation status. The methylation status of RUNX3gene was reversed by5-Aza-CdR.(3) RUNX3mRNA of MiaPaca2cells which were treated without5-Aza-CdR had no expression, and the expression of RUNX3mRNA was restored when the MiaPaca2cells were treated with5μmol/L and10μmol/L5-Aza-CdR for72h. The level of expression and the dose of5-Aza-CdR have a certain dose-effect relationgship.Conclusions:The methylation of RUNX3gene is significantly related with the occurrence and development of pancreatic carcinoma. Aberrant methylation may be the important mechanism of the loss of RUNX3mRNA. The RUNX3gene of MiaPaca2cells is complete methylation status, and the expression of RUNX3mRNA is lost.5-Aza-CdR may effectively cause reversion of RUNX3methylation status, and reactivate the expression of the gene, thus it may inhibit cell growth and induce apoptosis of tumor cells. RUNX3gene could be a new biology mark and target of gene therapy in pancreatic carcinoma. The application of demethylating drugs which can change the methylation of the RUNX3gene may provide a new idea for the biological treatment of pancreatic cancer.