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Published on: October 30, 2013
[5-aza-2'-deoxycytidine induces changes of histone H3-lysine 9 methylation in bladder tumor cells]
1Department of Clinical Laboratory Medicine, Shanghai Tenth People's Hospital, Tongji University, Shanghai 200072, China.
Objective:
To investigate the effect of DNA methyltransferases inhibitor 5-aza-2'-deoxycytidine (5-Aza-CdR) on the histone H3-lysine 9 methylation status and gene expression of RUNX3 in human bladder tumor cells.
Methods:
Human bladder tumor cells of the line EJ were cultured and treated with 5-Aza-CdR for 24 h. MTT test was used to observe the proliferation and growth of the EJ cells. Other EJ cells were cultured and treated with 5-Aza-CdR and then chromatin immunoprecipitation assay was used to analyze the histone H3-lysine 9 methylation status of RUNX3 promoter and the second exon. The expression of RUNX3 was measured by RT-PCR.
Results:
The survival rate of the EJ cells treated with 5-Aza-CdR of the concentrations of 0.1, 0.5, 1.0, 2.0, 5.0, and 10.0 micromol/L were 98.1%, 95.3%, 75.9%, 52.3%, 16.2%, and 7.7% respectively. And the survival rates of the EJ cells treated with 5-Aza-CdR for 12, 24, 36, 48, 72, and 96 hours were 89.4%, 85.2%, 78.6%, 37.1%, 8.9%, and 7.1% respectively. The survival rates of the 1.0, 2.0, 5.0, and 10.0 micromol/L group were significantly lower than that of the control group (all P < 0.05). Before the intervention, the amplified bands of the histone H3-lysine 9 methylation status of RUNX3 gene promoter and the second exon zone were obvious, but both disappeared after the treatment with 2.0 and 5.0 micromol/L 5-Aza-CdR. Before the intervention, RUNX3 gene was not expressed, and was expressed after treatment with 2.0 micromol/L 5-Aza-CdR.
Conclusion:
5-Aza-CdR not only obviously inhibits the proliferation of human bladder cancer cells but also reactivates RUNX3 gene through demethylation of histone H3-lysine9 at RUNX3 promoter and the second exon. H3-lysine 9 trimethylation may be one of the most important reasons for gene inactivation of the RUNX3 gene.
Insights
The DNA methyltransferases inhibitor 5-aza-2'-deoxycytidine (5-Aza-CdR) reduces bladder cancer cell proliferation. It also reactivates the RUNX3 gene by demethylating histone H3-lysine 9 at its promoter and second exon.
Area of Science:
- Oncology
- Epigenetics
- Molecular Biology
Background:
- Bladder cancer is a significant health concern.
- RUNX3 gene expression is often silenced in bladder tumors.
- Epigenetic modifications, such as histone methylation, play a role in gene regulation.
Purpose of the Study:
- To investigate the effect of 5-aza-2 '-deoxycytidine (5-Aza-CdR) on human bladder tumor cells.
- To analyze the impact of 5-Aza-CdR on histone H3-lysine 9 methylation status of the RUNX3 gene.
- To determine if 5-Aza-CdR can restore RUNX3 gene expression.
Main Methods:
- Human bladder tumor cell line EJ was treated with varying concentrations and durations of 5-Aza-CdR.
- MTT assay was used to assess cell proliferation and survival.
- Chromatin immunoprecipitation (ChIP) assay was performed to analyze histone H3-lysine 9 methylation at the RUNX3 promoter and second exon.
- RT-PCR was employed to measure RUNX3 gene expression.
Main Results:
- 5-Aza-CdR significantly inhibited EJ cell proliferation in a dose- and time-dependent manner.
- Treatment with 5-Aza-CdR led to the disappearance of histone H3-lysine 9 methylation marks at the RUNX3 promoter and second exon.
- RUNX3 gene expression, initially absent, was detected after 5-Aza-CdR treatment.
Conclusions:
- 5-Aza-CdR effectively inhibits human bladder cancer cell proliferation.
- 5-Aza-CdR reactivates RUNX3 gene expression by demethylating histone H3-lysine 9.
- Histone H3-lysine 9 trimethylation is implicated as a key mechanism for RUNX3 gene silencing in bladder cancer.
