Effects of 5-azacytidine on RUNX3 gene expression and the biological behavior of esophageal carcinoma cells
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Shuai Wang1, Hong Liu2, Zhou Wang1
1Department of Thoracic Surgery, Provincial Hospital Affiliated to Shandong University, Jinan, Shandong 250021, P.R. China.
Abstract:
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.
Insights
The demethylating agent 5-azacytidine (5-azaC) reactivates the RUNX3 gene in esophageal cancer cells by reducing methylation. This epigenetic reprogramming inhibits cancer cell proliferation, migration, and invasion, both in vitro and in vivo.
Area of Science:
- Molecular Biology
- Epigenetics
- Cancer Research
Background:
- Esophageal carcinoma Eca109 cells exhibit hypermethylation of the RUNX3 gene.
- RUNX3 gene expression is often silenced in various cancers, contributing to tumor progression.
Purpose of the Study:
- To investigate the effect of 5-azacytidine (5-azaC) on RUNX3 gene expression and the biological behavior of esophageal carcinoma Eca109 cells.
- To determine if 5-azaC-induced demethylation of RUNX3 can inhibit esophageal cancer cell growth and metastasis.
Main Methods:
- MTT assay to assess cell viability.
- Methylation-specific PCR to detect changes in RUNX3 gene methylation.
- Western blot and RT-PCR to measure RUNX3 mRNA and protein levels.
- In vitro cell migration and invasion assays.
- In vivo tumorigenesis experiments using nude mouse xenografts.
Main Results:
- 5-azaC inhibited Eca109 cell viability in a dose- and time-dependent manner.
- 5-azaC treatment significantly decreased RUNX3 gene methylation and increased RUNX3 mRNA and protein expression in Eca109 cells.
- 5-azaC treatment suppressed the migration and invasion capabilities of Eca109 cells.
- In vivo, 5-azaC significantly inhibited the growth of Eca109 xenografts in nude mice.
Conclusions:
- The RUNX3 gene is epigenetically silenced by hypermethylation in esophageal carcinoma Eca109 cells.
- 5-azaC effectively induces RUNX3 gene expression through demethylation, thereby inhibiting proliferation, migration, and invasion of Eca109 cells.
- Targeting RUNX3 demethylation with agents like 5-azaC represents a potential therapeutic strategy for esophageal cancer.
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