miR-125b precursor was ordered from Ambion. SUV39H1 3′ untranslated region (UTR) sequences, containing wild-type (WT) or

mutated miR-125b-binding sites, were cloned into the dual-luciferase miRNA target expression vector, Z-IETD-FMK in vitro pmirGLO (Promega, Madison, WI). HCC cells (5 × 104) were seeded into each well of a 24-well plate the day before transfection. miR-125b precursor (15 ρmole) was first transfected into BEL7402 cells using X-tremeGene (Roche, Basel, Switzerland). Twenty-four hours later, 0.5 µg of pmirGLO, containing WT or mutated miR-125b-targeted SUV39H1 3′ UTR sequence, was transfected into BEL7402 cells using FuGENE 6 (Roche). Firefly and Renilla luciferase activity of transfected cells were determined 48 hours after transfection by using the Dual-Luciferase Assay Kit (Promega), according to the manufacturer’s protocol. Renilla luciferase activity was used as the internal control for normalization. Three

independent experiments were performed. Epigenetics allows differential gene expression without altering the underlying DNA sequence and is controlled by various epigenetic modifiers. Recently, we analyzed the expression profile of a total of 90 epigenetic regulators in 38 paired human HCC samples.17 selleck products We found that the epigenetic regulators′ expression profile could clearly distinguished cancerous tissue from the adjacent non-tumorous

liver,17 suggesting that epigenetic alternation is common in HCC development. Interestingly, among the aberrantly expressed epigenetic modifiers, the prototype of SET-domain-containing histone methyltransferase SUV39H1 was one of the most significantly elevated in the primary HCC samples, relative to the non-tumorous liver and normal liver controls (P < 0.001; Fig. 1A). SUV39H1 expression level was also positively associated with proliferation second marker Ki67 expression (R = 0.693, P < 0.001; Fig. 1B). This finding suggested that deregulation of SUV39H1 may be implicated in human hepatocarcinogenesis and thus prompted us to further investigate the roles of SUV39H1 in human HCC. In this study, we first confirmed the up-regulation of SUV39H1 by performing qRT-PCR in an additional 67 paired HCCs and 7 normal liver samples. Combining the data from profiling and validation cohorts, up-regulation of SUV39H1 was frequently found in human primary HCC (59 of 105; 56.2%) (Fig. 1C). Importantly, up-regulation of SUV39H1 was significantly associated with an aggressive HCC pathological feature: the presence of venous invasion in patients’ livers (P = 0.017; Fig. 1D; Supporting Tables 1 and 2). These observations highlighted the clinical relevance of SUV39H1 in hepatocarcinogenesis, particularly in the aspect of cancer cell proliferation and metastasis.