Hypoxic silencing of tumor suppressor RUNX3 by histone modification in gastric cancer cells.

RUNX3 is a tumor suppressor that is silenced in cancer following hypermethylation of its promoter. The effects of hypoxia in tumor suppressor gene (TSG) transcription are largely unknown. Here, we investigated hypoxia-induced silencing mechanisms of RUNX3. The expression of RUNX3 was downregulated in response to hypoxia in human gastric cancer cells at the transcriptional level. This downregulation was abolished following treatment with the histone deacetylase (HDAC) inhibitor trichostatin A (TSA) and cytosine methylation inhibitor 5-aza-2-deoxycytidine (5-Aza), suggesting that an epigenetic regulatory mechanism may be involved in RUNX3 silencing by hypoxia. DNA methylation PCR and bisulfite-sequencing data revealed that hypoxia did not affect the methylation of RUNX3 promoter. A chromatin immunoprecipitation (ChIP) assay revealed increased histone H3-lysine 9 dimethylation and decreased H3 acetylation in the RUNX3 promoter following hypoxia. Hypoxia resulted in the upregulation of G9a histone methyltransferase (HMT) and HDAC1; additionally, overexpression of G9a and HDAC1 attenuated RUNX3 expression. The overexpression of G9a and HDAC1, but not their mutants, inhibited the nuclear localization and expression of RUNX3. Diminished mRNA expression and nuclear localization of RUNX3 during hypoxia was abolished by siRNA-mediated knockdown of G9a and HDAC1. This study suggests that hypoxia silences RUNX3 by epigenetic histone regulation during the progression of gastric cancer.