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Objective To evaluate whether mono (2-ethylhexyl) phthalate (MEHP) affects genomic DNA methylation and the methylation status of some specific genes such as patched gene ( PTCH ) and smoothened gene ( SMO ) in LNCaP cells. Methods LNCaP cells were treated with MEHP (0, 1, 5, 10, and 25 μmol/L) for 3 days. An ELISA assay was preformed to detect genomic methylation, including 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) content. A pyrosequencing assay was applied to assess DNA methylation in PTCH and SMO gene promoters. The correlation between DNA methylation and gene expression was assessed. Results The proportion of cytosines with 5-mC methylation in LNCaP cells was significantly decreased by MEHP (1, 5, 10, and 25 μmol/L) in a dose-dependent manner ( P < 0.01). For genes in the Hedgehog pathway, there was no significant MEHP concentration-dependent difference in the DNA methylation of PTCH and SMO . Conclusion MEHP might affect the progression of prostate cancer through its effect on global DNA methylation. [ABSTRACT FROM AUTHOR]