55E-06). Additionally, HCCP showed a much higher frequency of PLK2 methylation than HCCB (18/40 [45%] versus 7/35 [20%]; P < 0.03) (Fig. 2B). PLK2 promoter methylation resulted in significantly reduced PLK mRNA (data not shown) and protein levels in HCC (22.3 ± 2.2 versus 48.0 ± 12.3; P = 2.03E-07). A similar trend was detected when assessing the frequency of PLK3 promoter methylation. Indeed, the PLK3 gene was silenced by promoter hypermethylation almost exclusively in HCC (28/75 [37.3%]) (Fig. 2C), whereas it was epigenetically inactivated by promoter hypermethylation in only

two nonneoplastic surrounding livers (2.7%; P = 3.04E-08) (Fig. 2A). In HCC, frequency of promoter hypermethylation was significantly higher in HCCP (23/40 [57.5%]) than in HCCB (5/35 [14.3%]; P = 6.14E-05). Similar to PLK2, PLK3 levels were significantly reduced in HCCs

with promoter hypermethylation (protein: 42.0 ± 7.8 versus 90.5 ± 13.3; P = 6.82E-10). In selleck products contrast, no PLK4 promoter hypermethylation was detected in any of the samples tested (Fig. 2D). Genomic status of PLK2, PLK3, and PLK4 was further investigated through loss of heterozygosity (LOH) analysis of PLK2, PLK3, and PLK4 loci by comparing each HCC with respective SL. The LOH rates at PLK2, PLK3, and PLK4 gene loci were 20%, 24%, and 45.3%, respectively, and were always significantly more frequent in HCCP versus HCCB (Fig. 2B-D). Although LOH at the PLK2, PLK3, and PLK4 EPZ-6438 in vivo gene loci was statistically associated

with reduced expression levels (15.9 ± 4.1 上海皓元 versus 45.4 ± 5.9 [P = 2.03E-07 for PLK2]; 42.2 ± 9.7 versus 81.8 ± 10.9 [P = 1.00E-05] for PLK3; 34.0 ± 6.3 versus 126 ± 18.6 [P = 3.82E-15] for PLK4, respectively), it showed a significant correlation with promoter hypermethylation for PLK2 (11/15 HCCs; Spearman’s rho = 0.64; P = 8.45E-10) and PLK3 (13/19 HCCs; rho = 0.42; P = 1.84E-04), respectively, suggesting the inactivation of both alleles in these cases. The role of methylation on PLK2 and PLK3 expression was further investigated in vitro. First, we screened 11 HCC cell lines for PLK2 and PLK3 promoter methylation. PLK2 methylation was detected in HepG2, HuH7, and Hep3B cell lines, whereas PLK3 methylation was detected in HepG2, HuH7, Hep3B, and SNU-387 cells (Supporting Fig. 1A). Subsequent treatment with the demethylating agent 5-AZA-cytidine caused a dose-dependent up-regulation of PLK2 and PLK3 mRNA in HepG2 and Hep3B (harboring PLK2 and PLK3 methylated promoter), but not in PLC (harboring PLK2 and PLK3 unmethylated promoter; Supporting Fig. 1B,C) cells. The role of PLK family members in HCC cell growth was investigated by assessing the consequence of their inactivation by siRNA in HCC cell lines. Suppression of PLK1 expression resulted in a marked decrease of cell viability in HepG2 (p53 wild-type) and Hep3B (p53 deleted) cell lines (≈60% and 80%, respectively) when compared with untreated cells (Fig. 3A).