Background

microRNAs (miRNAs) are short, non-coding RNAs with a size of about 21-24 nucleotides that post-transcriptionally silence the majority of all protein-coding genes in mammals. Since their discovery, more and more studies have clearly identified this regulatory layer as a crucial element for almost all physiological processes. Not surprising in this context, aberrant expression of miRNAs has also been causally linked to several human malignancies including cancer.

Using classic gain-of-function approaches, early studies have often utilized overexpression to evaluate the function of a particular miRNA. However, this can reach miRNA levels up to 100-fold or higher compared to the physiological context and may generate a phenotype that is not necessarily linked to the miRNA’s normal function.

To avoid this obvious problem with possible overexpression artifacts, in the last years several techniques for miRNA inhibition have been developed both for in vitro and in vivo use, thereby allowing the analysis of a specific miRNA or a group of miRNAs in a loss-of-function approach. These include e.g., the expression of miRNA decoys or sponges, long transcripts that interfere with miRNA function by sequestering miRNA:RISC complexes in a sequence-specific manner (Ebert et al., 2007; Gentner et al., 2009). Another frequently used strategy to interfere with miRNA function are antagomirs, small antisense RNAs complementary to specific miRNAs that can be brought into cells by different means such as by transfection or by viral transduction (Krützfeldt et al., 2005; Scherr et al., 2007). Notably, antagomirs have also been modified in a way that facilitates their cellular uptake, making them an attractive therapeutic tool. More recently, CRISPR/Cas9-mediated genome editing has been shown to be able to abolish miRNA expression on the level of its gene (Chang et al., 2016).

Here I provide a detailed protocol for the generation and functional validation of microRNA sponges as recently described (Lindner et al., 2017). Compared to synthesized small RNAs such as antagomirs, miRNA sponges are cheap and can be stably expressed by retroviral integration. This allows the long-term miRNA knockdown in almost every type of cell and tissue, even in specimen that are difficult to transfect. Moreover, the expression of the sponge RNA can be linked to a fluorescent and/or genetic marker, allowing the easy tracking of sponge-positive cells e.g., by microscopy or flow cytometry. Last, miRNA sponges have been shown to sequester not single miRNAs, but rather all miRNAs of the same family as defined by their seed sequence, i.e., sponges may uncover redundant roles of miRNAs expressed in a particular tissue.