Sponges are artificial nucleotide sequences which are complementary to a miRNA of interest.
This miRNA decoys were developed in 2007 by M. Ebert and introduced as a means to create loss-of-function phenotypes for miRNA families in cell culture and in virally infected tissue and transgenic animals, so vectors that express miRNA target sites have been used to inhibit the function of endogenous miRNA. The transcripts containing the miRNA targets are designed to accumulate in a cell and act in order to soak up the corresponding miRNA and prevent its association with its natural targets ( Exploiting and antagonizing microRNA regulation for therapeutic and experimental applications, 2009 ; MicroRNA: An emerging therapeutic target and intervention tool, 2008 ).
In the absence of the sponge treatment, target mRNA for a miRNA seed family are repressed. After introduction of the sponge transgene, sponge mRNA are expressed at high level and sequester the miRNA complexes, rescuing the expression of the endogenous targets ( Emerging roles for natural microRNA sponges, 2010 ).
METHODS FOR miRNA LOSS-OF-FUNCTION
There are three methods for miRNA loss-of-function studies: genetic knockouts, antisense oligonucleotide inhibitors and sponges. The sponge mRNA contains multiple target sites complementary to a miRNA of interest. The sponges are a dominant negative method and offers several advantages. The first one is the convenience of making dominant negative transgenics over knockouts and the applicability to a broader range of organisms and cell lines. Many miRNAs have seed family members encoded at multiple distant loci, so these miRNAs would have to be knocked out individually and the animals bred to generate the complete knockout strain. Targeting of an entire miRNA family occurs because all family members have the same seed sequence. Some miRNA precursor are transcribed in clusters and this may make it difficult to delete one miRNA without affecting the processing of the other miRNAs, since the sponges interact only with the mature miRNA, their effectiveness is unaffected by the clustering of miRNA precursors. Moreover the generation of knockout mice is time consuming, costly and technically challenging, so the miRNA sponges are a valid alternative method to the generation of knockouts.
This method offer advantages also over antisense oligonucleotide inhibitors: the inhibitors are specific for one miRNA as they depend upon extensive sequence complementarity beyond seed region, so in order to neutralize a family of miRNA it is needed to deliver a mixture of perfectly complementary oligonucleotides. Another problem is the fact that many cells in vivo and in vitro are resistant to the uptake of oligonucleotides. Antagomir require repeated administration in large doses to inhibit a miRNA over a long durations ( MicroRNA sponges: progress and possibility, 2010 ). Finally oligonucleotides are effective for short term (24-72h) expreriments and they are expensive ( Generation of miRNA sponge constructs, 2012 ).
SPONGE DESIGN
Example of a sponge
Typical sponge constructs contain 4 to 10 binding sites separated by few nucleotides each (linker regions). Increasing the number of binding sites is not useful because each site increases the probability of sponge RNA degradation. Sites are normally placed in an unstructured, noncoding region of the RNA usually in the 3’UTR of a reporter gene. ( MicroRNA sponges: competitive inhibitors of small RNAs in mammalian cells, 2007 ).
The miRNA binding sites in sponges’ contrsucts are either perfectly antisense or contain mismatches in the middle positions, which if perfectly base-paired would be vulnerable to Ago2-mediated endonucleolytic cleavage. (MicroRNA sponges: progress and possibility, 2010). By introducing a bulge at position of 9-12 in analogy to natural miRNA binding rather than perfect complementarity, these molecules achieved stronger derepressive effect, possibly due to prevention of mRNA cleavage and increased retention of miRNAs ( MicroRNA: An emerging therapeutic target and intervention tool, 2008 ).
The efficacy of a miRNA sponge depends not only on the affinity and avidity of binding sites, but also on the concentration of the sponge RNAs relative to the concentration of the miRNA. In order to maximize sponge expression, it should be used the strongest promoter for the cell type of interest, for example a CMV promoter for mammalian cell lines. Sponges delivered in vivo can also have tissue specific promoters. RNA polymerase II or III promoters have also been used to express transcripts bearing miRNA target sites, and were found to mediate effective miRNA inhibition ( Exploiting and antagonizing microRNA regulation for therapeutic and experimental applications, 2009 ).
A reporter gene is placed directly upstream of the miRNA binding sites, such that the protein expression directly represents sponge RNA expression. Fluorescent reporters enable quantitative analysis and sorting of individual cell and can be diversified with different colors representing different miRNA sponges. (MicroRNA sponges: progress and possibility, 2010 ). The use of the reporter is important because it can provide a useful readout of miRNA saturation as its accumulation indicates that the miRNA activity has been overwhelmed by an effective decoy vector ( Exploiting and antagonizing microRNA regulation for therapeutic and experimental applications, 2009 ). The most used reporters are eGFP, luciferase and mCherry.
One could include regulatory elements in the sponge promoter to make it drug- inducible or tissue-specific for the tissue of choice. . ( MicroRNA sponges: progress and possibility, 2010 ).
LIMITATIONS OF THE TECHNOLOGY OF THE SPONGES
Despite the advantages of the technology, a decoy vector has some limitation.
A high vector copy number is needed and it can be difficult to achieve in some cell or tissue types. Where miRNA concentration is very high , complete titration demands a very high and possibly unachievable dose of sponge RNA. On the other hand, in cells expressing a large pool of endogenous targets for the miRNA family of interest, there should be less free miRNA available, so a lower dose of sponge RNA should suffice to give strong inhibition.
Moreover the overexpression of the reporter gene can be toxic or create a off-target effect.
Determing whether a sponge treatment is successful in inhibiting the miRNA of interest is very challenging. It can be validated in cell culture by reporter assay or assays fot the expression of known target genes. A luciferase reporter fused to miRNA binding sites or a confirmed target 3’UTR is measured in the presence of the miRNA sponge or a negative control sponge containing no binding sites or nonspecific sites. In the presence of the miRNA of interest, the luciferase reporter should be depressed by the sponge ( Exploiting and antagonizing microRNA regulation for therapeutic and experimental applications, 2009 MicroRNA sponges: progress and possibility, 2010 ).
TRANSIENT OR STABLE miRNA SPONGE EXPRESSION
Different strategies have been described for cloning of miRNA sponges containing multiple miRNA antisense binding sites (MBS). One approach is based on the non-directional concatemerization of oligo duplexes followed by the ligation of 5’ and 3’ adapters, then the products are gel-purified , digested with restriction enzymes and cloned to the vector. A second approach uses ling oligos that allow 2 or 4 MBS designed with the appropriate overhangs to allow direct directional cloning ( Generation of miRNA sponge constructs, 2012 ).
Sponges were transfected or transduced into human, mouse, and rat cell lines such as melanoma ( microRNA-214 contributes to melanoma tumour progression through suppression of TFAP2C, 2010 ), retina ( Sponge transgenic mouse model reveals important roles for the microRNA-183 (miR-183)/96/182 cluster in postmitotic photoreceptors of the retina, 2011 ), nonsmall cell lung cancer ( Suppression of non-small cell lung tumor development by the let-7 microRNA family, 2008 ), B cell lymphoma ( Reticuloendotheliosis virus strain T induces miR-155, which targets JARID2 and promotes cell, 2009 ), embryonic neural stem cells ( A feedback loop comprising lin-28 and let-7 controls pre-let-7 maturation during neural stem-cell commitment, 2008 ); motor neuron ( An Optimized Sponge for microRNA miR-9 Affects Spinal Motor Neuron Development in vivo, 2012 ).
This technology has utilized a variety of delivery systems such as plasmids or vectors based on adenoviruses, retroviruses and lentiviruses. Saturating a highly expressed miRNA using a vector-based approach requires high expression levels of the target-containing transcript in a cell. Supraphysiological target expression can be achieved with high vector copy, strong promoters and stable transcripts ( Exploiting and antagonizing microRNA regulation for therapeutic and experimental applications, 2009 ). As vectors, the most commonly used were plasmids, and also retroviruses, lentiviruses or adenovirus.
Cellular assays or target validation assays were performed 24-72 h after trasfection.
Continuous expression of the sponge makes it possible to perform long-term miRNA loss-of-function studies in cell culture and in vivo assays, such as bone marrow reconstitution and cancer xenografts.
The application of stable sponge expression are to probe the roles of miRNAs in differentiation pathways, to mimic the down-regulation of specific miRNAs that are aberrantly expressed in certain disease states, to mimic the genetic state of patients with a genomic deletion of a particular miRNA or miRNA cluster ( MicroRNA sponges: progress and possibility, 2010 ).
IN VIVO APPLICATION FOR miRNA SPONGES
The first transgenic organism made to express miRNA sponger were plants ( Target mimicry provides a new mechanism for regulation of microRNA activity, 2007 ).
It could be possible to generate germline transgenic sponge-expressing animals to continuously inhibit the miRNA of interest for the lifetime of the animal and the first animal model created was Drosophila. Other group worked with transgenic vertebrate: Asakawa et al. introduced sponge transgenes for tissue-specific expression in Zebrafish ( Targeted gene expression by the Gal4-UAS system in zebrafish, 2008 ), in the mouse, an inducible sponge could be created by means of the Cre-lox system or with tet-responsive element driving the sponge and tissue-specific reverse tet transactivator (rtTA) expression in combination with feeding the animal doxycycline ( MicroRNA sponges: progress and possibility, 2010 ). Zhu et al. generated a sponge transgenic mouse model that distrupted the activities of miR-183, miR-96 and miR-182 simultaneously and selectively in the retina ( Sponge transgenic mouse model reveals important roles for the microRNA-183 (miR-183)/96/182 cluster in postmitotic photoreceptors of the retina, 2011 ).
NATURAL miRNA SPONGES
The first endogenous sponge RNA was discovered in plants and found to attenuate a miRNA-mediated response to an environmental stress.
There are also hints that a viral miRNA sponge may be produced in cells lytically infected with murine cytomegalovirus ( Emerging roles for natural microRNA sponges, 2010 ).
Recently, a mammalian cellular non-coding RNA was proposed as a miRNA sponge. PTENP1 is a pseudogene of PTEN derived from retrotransposition and containing a mutated start codon such that its mRNA does not produce protein. The pseudogene RNA has a sponge activity, it regulates PTEN expression by competing for the same combination of miRNAs (Poliseno et al., 2010). Salameno et al., started calling this natural sponges “competivive endogenous RNA” ceRNA), they hypothesize that all types of RNA transcripts communicate through a new language mediated by microRNA-binding sites ( A ceRNA hypothesis: the Rosetta Stone of a hidden RNA language?, 2011 ).
This technology provides a versatile way to investigate miRNA biology and it is used to study the role of miRNA in cancer, cardiac function, hematopoiesis and viral infection, and because in some cases the desease is partly due to overexpression of a specific miRNA, it may also be possible to use a decoy vector to treat pathological conditions.
