The research and field of microRNA (miRNA) is relatively young in molecular biology. Researchers are only beginning to ascertain the essential functional impact that miRNA serve in tissue development and disease progression. For example, miRNA can act as both oncogenic ‘oncomirs’ or as tumor-suppressor genes in cancer biology. Amazingly, those same miRNA genes can also be critical to the function and cellular homeostasis of normal progenitor and mature cells. These findings illustrate the incredible promiscuity of miRNA functionality as well as their broad target specificity. Novel methods and techniques are needed not only to validate miRNA gene function; but also to identify pathway-related genes specific to both miRNA gene families as well as miRNA gene targets.
Dr. Phillip Sharp’s laboratory at MIT first proposed the concept of miRNA ‘sponges’ back in 20071. MiRNA sponges are plasmid constructs either transiently or stably transfected into mammalian cells containing multiple miR-binding sites for a chosen miRNA gene. The binding sites are located in tandem along an expressed transcript (see Figure 1). The ‘sponges’ are expressed via a strong promoter element and the endogenous, targeted miRNA of interest is soaked up by the ‘sponge’ transcript. MiRNA sponges mimic the effects of miRNA inhibitors; the endogenous miRNA’s function is lost and researchers can ascertain varied mRNA targets and other downstream effects resulting from this targeting inhibition.
Figure 1: (from Ebert et al.)
Kluiver et al. recently published a Methods paper outlining detailed protocols for the generation of stably-expressed miRNA sponges that can contain upwards of 20+ miRNA binding sites. This protocol can also generate varying binding sites to target several different miRNA genes2. The article takes the ideas of Ebert et al. and expands upon their initial findings. The construct of tandem-repeated miRNA binding sites using this latest methodology can allow researchers to tailor their experiments to study miRNA genes of similar sequence, or entire miRNA families. These new constructs can also allow for long-term functional analysis of miRNA repression in a host of cell lines or animal models. Kluiver et al. uses a GFP reporter to ascertain transfection efficiency and mark for the expression of the sponge transcript. Kluiver et al. also provides an analysis for the generation of the miRNA binding sites themselves: the authors argue that a 3-4 nucleotide bulge in the center of the miRNA binding site, as opposed to complete complemtarity of sponge sequences to the miRNA of interest, will prevent cleavage (via RISC proteins) of the sponge transcript. This should preserve the long-term effects of the miRNA sponge and protect them from quick degradation.
Just as the original article from the Sharp group predicted, these miRNA sponges can now be constructed via a relatively straight-forward methodology to include tissue-specific promoters which would allow for selected expression of these constructs in animal models, or under specific temporal-selection in both animal models and cell culture. These methodologies introduce a new tool for researchers to use when studying their microRNA of interest and these techniques will undoubtedly be updated in the near future to allow for other dynamic experiments.
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