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Epigenetic alterations, such as histone methylations, affect the pathogenesis of tumors including prostate cancer (PCa). Previously, we reported that metformin reduced SUV39H1, a histone methyltransferase of H3 Lys9, to inhibit the migration of PCa cells. Since histone methylation is functionally linked to DNA methylation, we speculate that the knockout of the SUV39H1 gene will affect the genomic DNA methylation profile to regulate PCa cell migration and invasion. The genome-wide DNA methylation level is lower in SUV39H1 knockout (KO) cells than wild-type (WT) ones. However, the methylation levels in functional regions of CpG Islands (CGI), 5′ untranslated region (UTR5), and exon regions are higher in KO cells than WT cells. Analysis of differentially methylated regions (DMRs) identified 1241 DMR genes that have differential methylation on CG sites when comparing the KO and WT samples. Gene ontology enrichment and Kyoto Encyclopedia of Genes and Genomes Pathways analysis showed that knockout of SUV39H1 affects gene sets and pathways that are heavily involved in cell shapes, cell recognition, adhesion, motility, and migration. Our study suggests that SUV39H1 plays an important role in PCa migration via the epigenetic regulation of methylation on CG sites, and is a novel and legitimate target to inhibit PCa cell migration. [ABSTRACT FROM AUTHOR]