Background: Runt-related transcription factor 3 (RUNX3) is a developmental regulator, and methylation of the RUNX3 is significantly associated with the occurrence and development of carcinogenesis. Previous studies have identified an association of increased methylation level of RUNX3 in prostate cancer (PCa); however, the role and molecular mechanism underlying aberrant methylation of the RUNX3 gene in prostate tumorigenesis remain elusive. In this study, we will investigate the role of RUNX3 promoter methylation and its gene expression in PCa cells.
Methods: The methylation of the RUNX3 in the promoter region in PCa cells was detected by bisulfite-sequencing PCR (BSP). Following treatment of the PCa cells with DNA methylation transferase inhibitor 5-AZA-2'-deoxycytidine (AZA), the effect on methylation level and expression of RUNX3 were analyzed by qRT-PCR, Western blot, and BSP assays. Furthermore, we investigated the effect of the demethylated RUNX3 on proliferation, cell cycle and apoptosis of PCa cells using CCK-8 and flow cytometry assays. Using the DNA methylation transferase (DNMT3b) knockout or overexpression models, the relationship between DNMT3b and RUNX3 methylation was further assessed by qRT-PCR, Western blot and methylation-specific PCR (MSP).
Results: The results indicated that the methylation level of RUNX3 in PCa cell lines was significantly higher than that of normal prostate epithelial (RWPE-1) cells. Furthermore, treatment with AZA not only promoted the demethylation of RUNX3 but also restored the mRNA and protein expression of RUNX3, and the reactivation of expression of the later exhibited its anti-tumor effects through regulation of the cycle progression in PCa cells. Moreover, DNMT3b could regulate the expression level of RUNX3 by altering the DNA methylation of the RUNX3 in PCa cells.
Conclusion: RUNX3 is hypermethylated in a panel of PCa cell lines; Inhibits DNA methylation of RUNX3 could restored its gene expression, which in turn induced its anti-cancer effects. Thus, RUNX3 may serve as a novel putative molecular target gene for PCa therapy.