UCLA

Non-coding RNAs play a pivotal role in the regulation of a myriad of cellular processes. Among these RNAs, microRNAs (miRNAs) – short, single-stranded, RNAs about 20 nucleotides long – have been shown to be key regulators of various oncogenes, and dysregulation of their expression is strongly correlated with the cancerous state. In particular, the exogenous reintroduction of these miRNAs (via short-hairpin RNAs) has been shown by many groups to reduce the ability of cancer cells to spread in vitro. However, there are important limitations to this existing approach: delivery of short-hairpin RNA is inherently transient, as the RNA is exogenously produced and transfected into the cell; and shRNA-based gene silencing relies on the activity of the DICER complex, which often has reduced efficiency in cancerous cells. Here, we utilize the modified, self-replicating, genome of the single-stranded RNA virus Nodamura to deliver mature, self-amplifying, miRNAs cells in vitro. This autonomously replicating RNA, known as a replicon, replicates readily in the cytoplasm, synthesizing up to a million copies of itself per cell in 48 hours. By inserting into this replicon a cassette consisting of a miRNA of interest (here miR-34A) flanked by two ribozymes, which serve to splice the miRNA out of the viral genome, we have created a means to up-regulate the expression of particular miRNAs in a persistent manner. We have quantified the levels of both the replicon and miRNA, and demonstrate correlation of their expressions. By 72 hours post-transfection, the miRNAs outnumber the parent replicon molecules by 400-fold. Finally, we show that these replicon-derived microRNAs are capable of inducing apoptosis in human prostate cancer cells.