P140 MicroRNA-29 regulation of RNase-L reveals a novel function in oncogenesis

Abstract

The endoribonuclease RNase-L is the terminal component of an interferon-regulated RNA cleavage pathway known as the 2′-5′-oligoadenylate (2-5A) system whose established functions include antimicrobial and tumor suppressive activities. RNase-L enzymatic activation requires the binding of the small molecule 2-5A, leading to RNase-L dimerization and cleavage of single-stranded RNA. RNase-L expression is controlled post-transcriptionally by its 3′-untranslated region (3′UTR), which exerts a strong negative regulatory effect on RNase-L levels. miRNAs are a class of small noncoding RNAs that repress expression of target genes by binding to regions of complementarity in their 3′UTRs. The miR-29 family has been shown play a tumor suppressive role in several cancers, including acute and chronic myelogenous leukemia (CML), and has many known oncogenic targets. miR-29 dependent regulation of endogenous RNase-L and a 3′UTR-luciferase reporter was examined following manipulation of miR-29 expression. The biological consequences of RNase-L regulation by miR-29 were analyzed by measuring proliferation and tumorigenesis in control or RNase-L knockdown K562 CML cells. Here, we report that the miR-29 family represses expression of RNase-L protein across several cell types. Repression occurs with no change in RNASEL mRNA. Using a luciferase reporter, we showed that miR-29 acts via four target sites within the RNASEL 3′UTR. Mutation of all sites is required for abrogation of miR-29 repression. In light of the reported tumor suppressive role of miR-29 in K562 CML cells and miR-29 repression of RNase-L in these cells, we generated K562 cells with stable RNase-L knockdown and demonstrated that loss of RNase-L expression inhibits proliferation in vitro as well as tumor growth in a nude mouse xenograft model. Our findings identify a previously unknown miRNA regulator of RNase-L expression and support a novel oncogenic role for RNase-L in CML and potentially other hematopoietic malignancies.