Short double-stranded RNA induces transcriptional gene silencing in human cancer cells in the absence of DNA methylation

Double-stranded RNA molecules targeted to gene promoter regions can induce transcriptional gene silencing in a DNA cytosine methylation-dependent manner in plants (RNA-dependent DNA methylation)(1-3). Whether a similar mechanism exists in mammalian systems is a vital and controversial issue(4-6). DNA methylation is an important component in mammalian gene silencing for normal processes such as gene imprinting and X-chromosome inactivation(7-9), and aberrant CpG island hypermethylation at tumor-suppressor promoters is associated with transcriptional silencing and loss of gene function in cancer(10). Hence, we investigated whether RNA-dependent DNA methylation might operate in human cancers to mediate epigenetic silencing using the endogenous gene CDH1 as a potential target. The loss of this cell-cell adhesion factor facilitates the metastatic process, and its promoter is frequently hypermethylated in breast and other cancers(11-13). We found that, although small double-stranded RNAs targeted exclusively to the CDH1 promoter could effectively induce transcriptional repression with chromatin changes characteristic of inactive promoters, this was entirely independent of DNA methylation. Moreover, we could accomplish such silencing in a cancer cell line genetically modified to lack virtually any capacity to methylate DNA.