Purpose: To investigate the role of DNA methylation in the regulation of Runt-related transcription factor 3 (RUNX3 ) and the effect of such mechanism on the proliferation of prostate cancer (PCa) cells.
Materials and 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-2ʹ-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; inhibition of DNA methylation of RUNX3  could restore its gene expression, which could promote its anticancer effect. Thus, RUNX3  may serve as a novel putative molecular target gene for PCa therapy.
Keywords: prostate cancer, RUNX3 , DNA methylation, AZA, DNMT3b