LNA-modified oligonucleotides mediate specific inhibition of microRNA function

Abstract

microRNAs are short, endogenous non-coding RNAs that act as post-transcriptional modulators of gene expression. Important functions for microRNAs have been found in the regulation of development, cellular proliferation and differentiation, while perturbed miRNA expression patterns have been observed in many human cancers. Here we present a method for specific inhibition of miRNA function through interaction with LNA-modified antisense oligonucleotides and report the specificity of this application. We show that LNA-modified oligonucleotides can inhibit exogenously introduced miRNAs with high specificity using a heterologous reporter assay, and furthermore demonstrate their ability to inhibit an endogenous miRNA in Drosophila melanogaster cells, leading to up-regulation of the cognate target protein. The method shows stoichiometric and reliable inhibition of the targeted miRNA and can thus be applied to studies of miRNA functions and validation of putative target genes.

Introduction

miRNAs are a class of short, non-coding RNAs that post-transcriptionally regulate gene expression in organisms ranging from nematodes to plants and humans by interaction with complementary sites in the 3′ UTR of target mRNAs (Bartel, 2004). miRNAs are expressed as long endogenous primary transcripts and processed sequentially by the RNase III endonucleases Drosha and Dicer to 21–23 nt mature miRNAs (Nakahara and Carthew, 2004). The majority of plant miRNAs have perfect or near-perfect complementarity with their target sites and direct RISC-mediated target mRNA cleavage, whereas most animal miRNAs recognize their target sites located in 3′ UTRs by incomplete base-pairing, resulting in translational repression of the target genes (Bartel, 2004), and in some cases degradation of the messenger (Bagga et al., 2005, Lim et al., 2005). miRNAs exhibit diverse biological functions with roles in cell growth and apoptosis (Brennecke et al., 2003), fat metabolism (Xu et al., 2003), hematopoietic lineage differentiation (Chen et al., 2004), homeobox gene regulation (Yekta et al., 2004), neuronal asymmetry (Johnston and Hobert, 2003), insulin secretion (Poy et al., 2004), and brain morphogenesis (Giraldez et al., 2005). In fact, recent bioinformatic predictions of miRNA targets in vertebrates indicate that hundreds of miRNAs are responsible for regulating up to 30% of the human protein-coding genes (Krek et al., 2005, Lewis et al., 2005).

Perturbed miRNA expression patterns have been reported in many human cancers. For example, the human miRNA genes miR15a and miR16-1 are deleted or down-regulated in the majority of B-cell chronic lymphocytic leukemia cases (Calin et al., 2002). The role of miRNAs in cancer is further supported by the fact that more than 50% of the human miRNA genes are located in cancer-associated genomic regions or at fragile sites (Calin et al., 2004). Recently, systematic expression analysis of a diversity of human cancers revealed a general down-regulation of miRNAs in tumors compared to normal tissues (Lu et al., 2005) and miRNAs have been shown to be deregulated in breast (Iorio et al., 2005) and lung cancer (Johnson et al., 2005), while the miR-17-92 cluster, which is amplified in human B-cell lymphomas, has been implicated as a potential human oncogene (He et al., 2005).

The challenge of establishing miRNA function and understanding the biological processes they regulate calls for robust and improved technologies for miRNA detection and functional studies. Recently, LNA (locked nucleic acid)-modified oligonucleotides have been used as sensitive and specific miRNA detection probes in Northern blots (Valoczi et al., 2004). Compared to DNA probes, the sensitivity was reportedly increased by at least 10-fold using LNA probes, without compromising probe specificity. Besides being efficient as Northern probes, LNA-modified oligonucleotide probes have proven highly useful for in situ localization of miRNAs in cells and tissues (Wienholds et al., 2005). Here, we report that LNA-modified oligonucleotides can also be used to inhibit miRNA function in cultured cells and describe the efficacy and specificity of the LNA inhibitors by using the well-characterized interaction between the Drosophila melanogaster bantam miRNA and its target gene hid as a model (Hipfner et al., 2002, Brennecke et al., 2003).