Enhancer of Zeste Homolog 2 Silences MicroRNA-218 in Human Pancreatic Ductal Adenocarcinoma Cells by Inducing Formation of Heterochromatin

Abstract

Enhancer of zeste homolog 2 (EZH2) is a histone methyltransferase that is overexpressed by pancreatic ductal adenocarcinoma (PDAC) cells and increases their aggressiveness. We identified microRNAs (miRs) that are regulated by EZH2 and studied their functions in PDAC cells. We performed miR profile analysis of PDAC cells incubated with EZH2 inhibitor 3-deazaneplanocin A, and pancreatic ductal epithelial cells that overexpressed EZH2. Expression levels of miRs and the targets of miRs were analyzed by quantitative reverse transcription polymerase chain reaction and immunohistochemistry. We expressed different forms of EZH2 to analyze functional domains and used small interfering RNAs to reduce its level in PDAC cells. Expression of miR-218 was repressed by EZH2 in PDAC cells. Levels of miR-218 were significantly reduced in primary PDAC tumor samples compared with paired, adjacent nontumor tissue. Overexpression of miR-218 in SW1990 cells reduced their proliferation and tumor formation and metastasis in nude mice. Loss of miR-218 from SW1990 cells increased levels of UDP-glycosyltransferase 8 and miR-218 was found to bind to its 3'-UTR. Levels of UDP-glycosyltransferase protein and messenger RNA were associated with the metastatic potential of PDAC cell lines and progression of tumors in patients. EZH2 was found to silence miR-218 by binding to its promoter, promoting heterochromatin formation, and recruiting the DNAs methyltransferase 1, 3A, and 3B. EZH2 is up-regulated in PDAC samples from patients and silences miR-218. MicroRNA-218 prevents proliferation of PDAC cells in culture, and tumor growth and metastasis in nude mice. MicroRNA-218 reduces levels of UDP-glycosyltransferase, which is associated with the metastatic potential of PDAC tumors in mice and progression of human PDAC.