Upregulation of miR-21 by Ras in vivo and its role in tumor growth

miR-21 is a microRNA (miRNA) frequently over-expressed in human cancers. Here we show that miR-21is upregulated both in vitro and in vivo by oncogenic Ras,thus linking this miRNA to one of the most frequentlyactivated oncogenes in human cancers. Ras regulation ofmiR-21 occurs with a delayed kinetic and requires at leasttwo Ras downstream pathways. A screen of humanthyroid cancers and non-small-cell lung cancers for theexpression of miR-21 reveals that it is overexpressedmainly in anaplastic thyroid carcinomas, the mostaggressive form of thyroid cancer, whereas in lung itsoverexpression appears to be inversely correlated withtumor progression. We also show that a LNA directedagainst miR-21 slows down tumor growth in mice.Consistently, a search for mRNAs downregulated bymiR-21 shows an enrichment for mRNAs encoding cellcycle checkpoints regulators, suggesting an important rolefor miR-21 in oncogenic Ras-induced cell proliferation.Oncogene (2011) 30, 275–286; doi:10.1038/onc.2010.416;published online 18 October 2010Keywords: microRNA; miR-21; Ras; anaplastic thyroidcarcinoma; non-small-cell lung cancerIntroductionThe involvement of microRNAs (miRNAs) in canceremerged from several reports showing that they areaberrantly expressed in neoplastic tissues (Lu et al.,2005; Volinia et al., 2006) The identification of severaltargets of miRNAs involved in cancer has led to thewidely accepted idea that networks comprising classicaloncogenes or tumor suppressors and miRNAs havepivotal roles in cancer initiation, progression andmetastatization (Esquela-Kerscher and Slack, 2006;Zhang et al., 2007). Deregulation of miRNAs expressionin cancer can occur by genetic mechanisms includingamplification, deletion and mutation, as well as byepigenetic silencing (Calin et al., 2004; Zhang et al.,2006). Moreover, it has recently been demonstrated thatoncogenes and tumor suppressors exert their action alsoby regulating the expression of specific miRNAs.Oncogenic activation of Myc is able to induce thetranscription of the oncogenic miRNA cluster miR-17-92, even though its genome-wide effect is a widespreadrepression of miRNA expression contributing to thetumorigenic potential of lymphoma cells (O’Donnell,2005; He et al., 2007; Chang et al., 2008). Also, miR-34family miRNAs are direct transcriptional targets of p53,and act in concert with other p53 effectors to down-regulate a set of genes involved in cell cycle progression(O’Donnell, 2005; He et al., 2007). One of the oncogenesmost frequently activated in human cancers is Ras,which, because of its central role in the transduction ofextracellular signals, is able to re-programme thetranscriptome of cells when constitutively activated.We have recently demonstrated that Ras is able toinduce aberrant expression of miRNAs during thetransformation of thyroid epithelial cells (Landgrafet al., 2007). The top-scored upregulated miRNA ismiR-21, a miRNA found overexpressed in several solidand hematopoietic malignancies (Krichevsky and Gab-riely, 2009). In vitro studies have demonstrated thatmiR-21 knockdown in tumor cell lines leads to increasedapoptotic cell death (Seike et al., 2009). Furthermore,miR-21 depletion reduces the growth of tumor cell linesxenografts in mice (Si et al., 2007). Several targetmRNAs have already been validated for miR-21, amongwhich there are at least four known tumor suppressors(PTEN, TPM1, PDCD4 and maspin) and the transcrip-tion factor NFIB. The genomic locus encoding miR-21is not amplified in most cancers, including thoseexpressing very high levels of miR-21, such as glioblas-toma and chronic lymphocytic leukemia, suggesting thatthe deregulated expression of this miRNA occurs ateither the transcriptional or the post-transcriptionallevel or both. The transcription of miR-21 primaryRNA is controlled by a conserved upstream enhancer,