The present study investigated the effects of 5-azacytidine (5-azaC) on the expression level of the human runt-related transcription factor 3 (RUNX3) gene and the biological behavior of esophageal carcinoma Eca109 cells. The effect of the demethylation reagent 5-azaC on the viability of Eca109 cells was detected by the MTT assay, which demonstrated that 5-azaC inhibited the viability of Eca109 cells in a time- and dose-dependent manner. Although demethylation of other genes may occur following treatment with 5-azaC, we focused on the RUNX3 gene. When treated with 5-azaC at hypoxic levels, the expression of RUNX3 increased and the methylation degree of the RUNX3 gene was decreased significantly in Eca109 cells. 5-azaC at 50 µM demonstrated the highest RUNX3-induction activity, inducing RUNX3 mRNA and protein expression, and decreasing the degree of methylation of the RUNX3 gene. Methylation specific PCR indicated that 5-azaC induced RUNX3 expression through demethylation. The abilities of migration and invasion of Eca109 cells were inhibited by 5-azaC. The growth of Eca109 cells treated with 5-azaC in vivo was detected by a tumorigenesis experiment. 5-azaC inhibited the growth of Eca109 xenografts in nude mice. Taken together, our findings demonstrated that the RUNX3 gene is hypermethylated in Eca109 cells and that 5-azaC induces the expression of the RUNX3 gene by demethylation, which inhibits the proliferation, migration and invasion of Eca109 cells.

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