Epigenetic silencing of ARNTL, a circadian gene and potential tumor suppressor in ovarian cancer.

Ovarian cancer is the fifth leading cause of cancer death and the most deadly gynecological malignancy in women. Epigenetic modifications play an important role in regulating gene transcription. Specifically, aberrant promoter hypermethylation has been implicated as a hallmark of cancer. In order to identify genes that are differentially methylated in ovarian cancer, we performed meDIP-chip in various ovarian cancer cell lines using Agilent 244K CpG island microarray. One of the targets, ARNTL which is a core component of the circadian clock is methylated in a sub-set of ovarian cancer cell lines. Combined bisulfite restriction analysis (COBRA) confirmed the results of the microarray. Additional analysis using ChIP-PCR revealed that promoter of ARNTL is enriched with the repressive histone mark H3K27me3 in CP70 and MCP2 ovarian cancer cells. Treatment with the EZH2 inhibitor (GSK126) significantly restored ARNTL expression in these cells (CP70 and MCP2). Further functional analysis demonstrated that overexpression of ARNTL inhibited cell growth and enhanced chemosensitivity of cisplatin in ovarian cancer cells. Finally, overexpression of ARNTL restored the rhythmic activity of c-MYC in ovarian cancer cells. These results suggested that ARNTL may be a tumor suppressor and is epigenetically silenced in ovarian cancer.

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