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Polycomb group protein EZH2 and Bmi1 are reportedly involved in the progression of malignant tumours. We examined the participation of EZH2 in multi-step cholangiocarcinogenesis in hepatolithiasis with respect to tumour suppressor genep16INK4a. We examined 20 hepatolithiatic livers with intrahepatic cholangiocarcinoma (ICC) and 10 histologically normal livers. Neoplastic biliary lesions were classified into biliary intraepithelial neoplasm (BilIN-1, 2 and 3) and invasive carcinoma. We selected 15 foci of invasive carcinoma, 8 BilIN-3 (carcinomain situ), 12 BilIN-2 (high-grade dysplasia), 32 BilIN-1 (low-grade dysplasia) and 37 non-neoplastic biliary epithelia from these livers. Expression of p16INK4a, EZH2 and Bmi1 were surveyed in these foci. P16INK4a promoter methylation was examined in microdissected tissues. Taking advantage of two cell lines of CC (HuCTT-1 and TFK-1) and small interfering RNA (siRNA), the effects of the knockdown of EZH2 on p16INK4a methylation of CC cells were examined. Expression of p16INK4a, which was frequent in BilIN1, was decreased in BilIN-2/3 and invasive carcinoma, while EZH2 expression showed step-wise increase from BilIN-1, -2 and -3 to invasive carcinoma (p< 0.01). P16INK4a promoter hypermethylation was related to aberrant expression of EZH2. The knockdown of EZH2 in cultured CC cells decreased p16INK4a methylation and decreased binding of EZH2 to the p16INK4a gene promoter. The latter suggested that direct binding of EZH2 is involved in the regulation of the p16INK4a gene. Our data suggest that over-expression of EZH2 may induce hypermethylation of p16INK4a promoter followed by decreased expression of p16INK4a in the multi-step cholangiocarcinogenesis through intraepithelial neoplasm in hepatolithiasis. Copyright © 2008 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.