BACKGROUND
down syndrome   is the result of an extra copy of chromosome  <dig> .
the phenotype of ds is characterized by a cognitive impairment and a plethora of symptoms affecting a wide range of organs  <cit> .
it is likely that most of the ds phenotypes are related to alterations in gene expression.
changes in the transcriptome of ds have shown that about 29 % of the expressed chromosome  <dig> transcripts are overexpressed, the remaining being either compensated or highly variable between individuals  <cit> .
this suggests the role of a selective group of genes to be central in ds phenotype.
supporting this view, it was recently proposed that one of the contributing mechanisms relies on the overexpression of few hsa <dig> genes modifying the chromatin environment of the nuclear compartment what leads to a general perturbation of the transcriptome  <cit> .
however, little is known about the contribution of hsa <dig> overexpressed mirnas to variations in the transcriptome.
mouse models are valuable tools to study the impact of gene expression variation on the ds phenotype  <cit> .
in particular, the ts65dn is a model of ds that carries an extra chromosome spanning most of the region of mmu <dig> that is homologous to hsa <dig>  the trisomic region extends from few kb upstream mrpl <dig> to zfp <dig> and contains roughly  <dig> genes and two mirnas, mir- <dig> and mir- <dig>  <cit> .
robust differences in the expression of thousands of genes in the cerebella of trisomic and euploid mice have been described to create a variable trisomic transcriptome  <cit> .
gene expression analysis of ca <dig> pyramidal neurons from the hippocampus of ts65dn and euploid mice identified alterations in transcripts relevant to neurodegeneration  <cit> .
mirna profiles in ts65dn mice identified  <dig> mirnas differentially expressed between ts65dn and euploid suggesting their potential role in regulating a plethora of target genes  <cit> .
in the current work, we propose a novel approach to study the contribution of overexpressed mirnas to transcriptomic perturbations.
RESULTS
to investigate the contribution of overexpressed mirnas to the ds transcriptome, we have focused our study on the effects of mir- <dig>  and mir- <dig> , since both mirnas are found in triple copy in the ts65dn mouse genome .
a bioinformatic analysis of mir- <dig> and mir- <dig> predicted target genes, using mirwalk database, that combines information from  <dig> established mirna predition programs  <cit> , identified  <dig> and  <dig> potential targets, respectively, with an overlap of  <dig> genes .
gene ontology analysis of these putative commonly regulated transcripts using david software   identified an enrichment in genes involved in relevant go terms such as phosphoproteins, neuron projections, synapses and dendrites, being all them key elements in neuronal physiology .
this is interesting as ts65dn mice exhibit behavioral and cognitive dysfunction with alterations in different processes involved in memory and learning , features similar to those present in ds individuals.fig.
<dig> predicted mir- <dig> and mir- <dig> targets are enriched in neuronal functions by gene ontology analysis.
a schematic representation of human chromosome  <dig> long-arm comprised of mouse chromosomal segments from chromosomes  <dig>   <dig>  and  <dig>  the portion of chromosome  <dig> that is found in trisomy in the ts65dn mouse expands from mrpl <dig>  to zfp <dig>  and harbors mir- <dig> and mir- <dig>  b venn diagram of hsa-mir- <dig> and hsa-mir- <dig> mirwalk predicted targets.
c functional profiling of the resulting list of  <dig> commonly predicted mir- <dig> and mir- <dig> targets in brain was analyzed using david bioinformatic resources.
left panel shows enrichment in gene ontology terms related to sp pir keywords.
right panel shows enrichment in gene ontology terms related to cellular compartment
to study the impact of mir- <dig> and mir- <dig> overexpression we have developed a strategy that consists in the use of mirna sponge technology to sequester mir- <dig> and mir- <dig> and thereby prevent their binding to endogenous targets, resulting in normalization of gene expression of target genes in trisomic cells.
the mirna sponge engineered in viral vectors offer several advantages over the use of mirna inhibitors oligonucleotides, such as good cellular delivery, sustained effects with no requirement of repeated injections and the potential of tissue specificity.
we generated lentiviral vectors expressing egfp with a 3′utr containing  <dig> incomplete complementary binding sites recognizing mir- <dig>  or both mir- <dig> and mir- <dig>  .
the sponge constructs were generated to contain bulged sites that are mispaired at positions 9– <dig>  since it has been reported that such designs cannot mediate ago <dig> cleavage of the transcript, allowing the mirna binding to the transcript for longer, thus reducing its availability to regulate other transcripts  <cit> .
on the other hand this binding can stimulate the target-directed destabilization of the mirna by the tailor and trimming processes leading to mirna decay  <cit> .
despite the presence of mispaired nucleotides, the recognition of the designed target sites for the mirnas was highly efficient since the minimum free energy calculated for the bulged sites was within − <dig> and −35 kcal/mol  <cit>  .
the affinity for this incomplete binding showed to be far above from target sites of validated endogenous genes and close to the perfect pairing.
to validate the capacity and specificity of the designed lentiviral vectors to modulate mir- <dig> and mir- <dig>  hela cells generated to overexpress mir- <dig> and mir- <dig>  were transduced with different moi of lv-mir155t.
mirna content was analyzed by rt-qpcr taqman assays and a dose-dependent reduction in mir- <dig> levels was observed whereas no changes were detected in mir- <dig> indicating the specificity of the effect .
moreover, hela cells overexpressing mir- <dig> and mir- <dig> when transduced with lv-mir155-802t resulted in reduced expression of both mir- <dig> and mir- <dig> .
furthermore, the number of gfp expressing cells was substantially reduced in hela cells expressing mir- <dig> and mir- <dig> with respect to hela control cells   when transduced with lv-mir155t or lv-mir155-802t.
these results demonstrate that lv-mir155-802t is a good tool to modulate mir- <dig> and mir- <dig> expression.fig.
<dig> mir- <dig> and mir- <dig> expression are specifically reduced in cells infected with mirna-sponge lentiviruses.
a top panel shows the sequence alignment of mmu-mir- <dig> and mmu-mir- <dig> to the corresponding mirna sponge sites engineered in the lentiviral vectors.
bottom panel shows lv-mirt constructs containing the cmv promoter to drive destabilized green fluorescent protein expression  bearing  <dig> or  <dig> mirna sponge sites in the 3′utr.
b minimum free energy and complementarity displayed in the mirna:target sites hybridizations: engineered mirt sites , validated seeds , rand seq , rand seq + fixed seed .
energies were calculated using rna hybrid algorithm.
c hela cells stably expressing both mmu-mir- <dig> and mmu-mir- <dig> were transduced with  <dig> and  <dig> moi of lv-mir155t or lv-control.
expression of both mirnas was analyzed 72-h later.
rq values represent mean ± sem of four independent samples; *p <  <dig>  and **p <  <dig>  .
d hela cells stably expressing both mmu-mir- <dig> and mmu-mir- <dig> were transduced with  <dig> moi of lv-mir155-802t or lv-control.
expression of both mirnas was analyzed 72-h later.
rq values represent mean ± sem of four independent experiments; *p <  <dig>
to validate mirna sponges as a strategy to identify mirna targets in vivo, we stereotaxically infused in the ventral hippocampus of young adult  euploid and ts65dn mice lv-mir155-802t and lv-control viral particles.
three weeks upon infusion animals were sacrificed and mir- <dig> and mir- <dig> expression was analyzed .
increased mir- <dig> and mir- <dig> levels were detected in the hippocampus of ts65dn mice injected with lv-control with respect to euploid animals confirming previous findings  <cit> .
interestingly, the administration of lv-mir155-802t resulted in a reduction in mir- <dig> and mir- <dig> content in ts65dn to levels similar to those of euploid mice.
no effect was detected in control animals injected with the lentiviral sponge .
in addition, we analyzed the expression levels of mecp <dig>  a previously validated target gene for both mirnas  <cit> , ship <dig> a mir- <dig> target gene  <cit>  and foxm <dig> a mir- <dig> target  <cit> .
expression analysis of mecp <dig>  ship <dig> and foxm <dig> showed a reduction in the hippocampus of trisomic mice that were rescued to wild type levels in lv-mir155-802t injected animals .fig.
<dig> decoy activity of lv-mir155-802t significantly reduces hippocampal mir- <dig> and mir- <dig> content in ts65dn mice.
a schematic representation of the experimental design for local lentiviral infusion and sample collection.
b expression analysis by rt-qpcr  of mmu-mir- <dig>  and mmu-mir- <dig>  in rna extracts from hippocampus of euploid  and ts65dn mice treated with lv-control or lv-mir155-802t.
rq values represent mean ± sem of six independent samples; *p <  <dig>  and **p <  <dig>  .
c expression analysis by rt-qpcr  of mmu-mir- <dig> and mmu-mir- <dig> validated target gene mecp <dig>  the mmu-mir- <dig> target ship <dig> and the mmu-mir- <dig> validated target foxm <dig> in rna extracts from hippocampus of euploid  and trisomic  adult mice treated with lv-control or lv-mir155-802t.
values represent mean ± sem of six independent samples; ns, non significant and * p <  <dig>  and ** p <  <dig>
to assess the transcriptomic impact of mir- <dig> and mir- <dig> overexpression we performed gene-expression profiling of euploid and ts65dn hippocampi treated with lv-control or lv-mir155-802t in an agilent microarray platform .
cluster analysis by principle component analysis  discriminated between genotypes but not between treatments , suggesting that the dosage imbalance effects on the steady state levels of thousands of genes was superior than the modulation by the lv-mir155-802t sponge within each genotype.
to assess the effects of lv-mir-155-802t sponge we performed a gene set enrichment analysis  <cit>   of array data on the set of mir- <dig> and mir- <dig> predicted target genes from targetscan.
we found enrichment in genes that negatively correlate with mir- <dig> or mir- <dig>  consistent with mirna activity .
further analysis on this enrichment was performed using mircomb r package, which calculated the correlation coefficients between signal intensity of transcripts in the microarray and mir- <dig> or mir- <dig> expression levels.
this regression analysis considered all the treatment groups with variable levels of mir- <dig> or mir- <dig>  involving genotype and treatment-based differences.
the distributions of correlation scores showed that those genes with increased number of target sites display stronger negative correlation .
this analytical approach combines sequence conservation parameters and experimental expression data, what refines mirna target predictions integrating data from biological samples .
moreover, the topological analysis  <cit>  of predicted genes with a significant negative correlation showed a genome-wide impact of mir- <dig> and mir- <dig> imbalance .
interestingly, when we constructed a topological map, based on the gedds defined by letourneau and collaborators  <cit>  plotted with our expression data, we could identify regions of coincidence from gedds to the expression associated to mir- <dig> and mir- <dig> deregulation .
a 47 % of pairwise changes were estimated between the two studies.
these data highlight that some of the epigenetic events can also be the consequence of the deregulation of epigenetic modulators such as mecp <dig>  a direct target of mir- <dig> and mir- <dig>  <cit> .
these results point that the in vivo approach of lentiviral mirna sponge is a good strategy to highlight the link of mir- <dig> and mir- <dig> levels regulating mrna predicted targets.
of notice, lentivirus integration into the genome allows the analysis of steady-state transcriptomic variation derived from mirna modulation.
interestingly, we performed a compared in silico analysis between our results and microarray data from geo gse <dig>  <cit> , in which the authors achieved a chromosome silencing of the extra hsa <dig> copy in trisomy  <dig> cells.
in such experiments, presumably mir- <dig> and mir- <dig> expression should be normalized and impact to the global transcriptome.
the results of this comparative analysis showed that 61–53 % genes predicted to be mir- <dig> and mir- <dig> targets based on our negative correlations showed downregulation in trisomy  <dig> cells, and more interestingly from the same mirna prediction lists 59–58 % presented a normalization of its expression upon extra hsa <dig> silencing .
interestingly, the targetscan platform facilitated further analyses to show the number and the conservation of mirna target sites within the transcript and to infer regulatory networks between mouse and human cells.fig.
<dig> transcriptome analysis reveals an enriched downregulation of mir- <dig> and mir- <dig> predicted targets.
a gene set enrichment analysis of array data was performed on the set of mouse predicted targets for mir- <dig>  and mir- <dig>  in targetscan <dig> .
b analysis of gene expression explained by the number of predicted mirna sites.
box plots represent the distribution of mircomb pearson correlation coefficients among the targets sorted according to the predicted number of target sites .
* and *** denote p <  <dig>  and p <  <dig> , respectively.
c representation of the correlations between predicted target genes and mir- <dig>  and mir- <dig>  according to its topology in mouse genome.
the layer over the chromosomes represents in graphic bars the regulation of genes within subchromosomic segments of 1000 kb.
representation was performed using circos visualisation package.
represented correlations are those that presented negative coefficients for a predicted target and a p-value <  <dig>  in mircomb analysis
further analyses of transcriptomic changes associated to mir- <dig> and mir- <dig> expression were conducted to identify those predicted mrnas more likely to be directly regulated.
to this end gene sets were sorted according to the number of predicted mirna target sites in the 3′utr and the correlation score .
a subset of five genes, on the top of that list, were selected for validation .
quantitative rt-qpcr data confirmed downregulation of rufy <dig>  nova <dig>  nav <dig>  thoc <dig> and sumo <dig> in the hippocampi of ts65dn mice and their restored expression in ts65dn lv-mir-155-802t treated mice .
interestingly, all five genes are involved in neuronal function and cellular homeostasis, with relevant implications to neurological diseases.
for instance, rufy <dig>  belongs to rufy family, which is involved in the regulation of neuronal polarity and membrane trafficking  <cit> .
moreover, rufy <dig> has been implicated in the reduction of beta-amyloid secretion in late-onset alzheimer’s disease  <cit> .
similarly, sumo <dig>  has also been linked to alzheimer’s disease, since endogenous protein sumoylation activity reduces beta-amyloid production  <cit> .
nova <dig>  is a neuron-specific splicing factor that controls the alternative processing of a large set of mrnas important for the synaptic activity  <cit> .
the defect in neuronal viability observed in nova- <dig> null mice suggests either a loss of function or a gain of function derived splicing defects in inhibitory receptors gabargamma <dig>  glyralpha <dig>  that could trigger imbalances between inhibition and excitation signals  <cit> .
in this line, treatment of ts65dn mice with gaba antagonists have shown a rescue of cognitive defects and improved long-term potentiation  <cit> .
nav <dig>  is a microtubule-associated protein involved in neuronal migration  <cit>  and reorganization of the cytoskeleton to induce neurite-like extensions  <cit> .
accordingly, reduced neurite length of neuronal precursors was observed in ts65dn mice  <cit> .
finally, thoc <dig>  is a ribonucleoprotein involved in mrna elongation essential for early embryonic development  <cit> , however its contribution to neuronal homeostasis is still unclear.
whether the transcriptomic rescue of the perturbed gene set in the hippocampi of ts65dn mice upon lentiviral lv-mir-155-802t infusion may have any impact in brain physiology or the cognitive deficits of ts65dn mice remains to be studied.
however, it could be speculated that normalization of the expression levels of the key target genes identified will be of functional relevance.
in fact, normalization of dyrk1a or snx <dig> expression in the hippocampi of adult ts65dn mice by strategies based on viral vector administration has shown rescue of synaptic deficits and partial recovery of behavioural abnormalities  <cit> .fig.
<dig> validation of novel target genes contributing to t565dn phenotype by rt-pcr.
a upper panel shows a table with five predicted target genes selected based on the criteria of predicted number of target sites and strong correlation coefficient .
table shows correlation values and the number of predicted mirna target sites classified according its evolutionary conservancy.
below panel shows the correlation plots calculated using mircomb package for the previously selected target genes.
b expression analysis by rt-qpcr  of mir- <dig> and mir- <dig> target genes in rna extracts from hippocampus of euploid  and trisomic  adult mice treated with lv-control or lv-mir155-802t.
data normalized to the expression of gdx  housekeeping gene.
values represent mean ± sem of six independent samples.
* and ns denote p <  <dig>  and not significant, respectively
the potential therapeutic effect of the sponge approach could also be of interest to be explored in other diseases by which functional mirna-disease associations have been identified such as cancer, type ii diabetes, asthma or alzheimer disease  <cit> .
CONCLUSIONS
the present study establishes a link between overexpression of mir- <dig> and mir- <dig> and downregulation of a new set of candidate genes with potential contribution to ds neuronal function.
the mirna sponge developed strategy provides a useful tool for the identification of subtle transcriptomic changes, often masked for interindividual variability.
these results also highlight that ds hippocampal-dependent phenotypes have connections with alterations present in neurological-associated diseases such as alzheimer and suggests that modulation of mirnas could be envisioned as a novel pharmacotherapy approach in ds.

 down syndrome is the result of an extra copy of chromosome. the phenotype of ds is characterized by a cognitive impairment and a plethora of symptoms affecting a wide range of organs. it is likely that most of the ds phenotypes are related to alterations in gene expression. changes in the transcriptome of ds have shown that about 29 % of the expressed chromosome transcripts are overexpressed, the remaining being either compensated or highly variable between individuals. this suggests the role of a selective group of genes to be central in ds phenotype. supporting this view, it was recently proposed that one of the contributing mechanisms relies on the overexpression of few hsa genes modifying the chromatin environment of the nuclear compartment what leads to a general perturbation of the transcriptome. however, little is known about the contribution of hsa overexpressed mirnas to variations in the transcriptome. mouse models are valuable tools to study the impact of gene expression variation on the ds phenotype. in particular, the ts65dn is a model of ds that carries an extra chromosome spanning most of the region of mmu that is homologous to hsa the trisomic region extends from few kb upstream mrpl to zfp and contains roughly genes and two mirnas, mir- and mir-. robust differences in the expression of thousands of genes in the cerebella of trisomic and euploid mice have been described to create a variable trisomic transcriptome. gene expression analysis of ca pyramidal neurons from the hippocampus of ts65dn and euploid mice identified alterations in transcripts relevant to neurodegeneration. mirna profiles in ts65dn mice identified mirnas differentially expressed between ts65dn and euploid suggesting their potential role in regulating a plethora of target genes. in the current work, we propose a novel approach to study the contribution of overexpressed mirnas to transcriptomic perturbations.  to investigate the contribution of overexpressed mirnas to the ds transcriptome, we have focused our study on the effects of mir- and mir-, since both mirnas are found in triple copy in the ts65dn mouse genome. a bioinformatic analysis of mir- and mir- predicted target genes, using mirwalk database, that combines information from established mirna predition programs, identified and potential targets, respectively, with an overlap of genes. gene ontology analysis of these putative commonly regulated transcripts using david software identified an enrichment in genes involved in relevant go terms such as phosphoproteins, neuron projections, synapses and dendrites, being all them key elements in neuronal physiology. this is interesting as ts65dn mice exhibit behavioral and cognitive dysfunction with alterations in different processes involved in memory and learning, features similar to those present in ds individuals.fig. predicted mir- and mir- targets are enriched in neuronal functions by gene ontology analysis. a schematic representation of human chromosome long-arm comprised of mouse chromosomal segments from chromosomes and the portion of chromosome that is found in trisomy in the ts65dn mouse expands from mrpl to zfp and harbors mir- and mir- b venn diagram of hsa-mir- and hsa-mir- mirwalk predicted targets. c functional profiling of the resulting list of commonly predicted mir- and mir- targets in brain was analyzed using david bioinformatic resources. left panel shows enrichment in gene ontology terms related to sp pir keywords. right panel shows enrichment in gene ontology terms related to cellular compartment to study the impact of mir- and mir- overexpression we have developed a strategy that consists in the use of mirna sponge technology to sequester mir- and mir- and thereby prevent their binding to endogenous targets, resulting in normalization of gene expression of target genes in trisomic cells. the mirna sponge engineered in viral vectors offer several advantages over the use of mirna inhibitors oligonucleotides, such as good cellular delivery, sustained effects with no requirement of repeated injections and the potential of tissue specificity. we generated lentiviral vectors expressing egfp with a 3′utr containing incomplete complementary binding sites recognizing mir- or both mir- and mir-. the sponge constructs were generated to contain bulged sites that are mispaired at positions 9– since it has been reported that such designs cannot mediate ago cleavage of the transcript, allowing the mirna binding to the transcript for longer, thus reducing its availability to regulate other transcripts. on the other hand this binding can stimulate the target-directed destabilization of the mirna by the tailor and trimming processes leading to mirna decay. despite the presence of mispaired nucleotides, the recognition of the designed target sites for the mirnas was highly efficient since the minimum free energy calculated for the bulged sites was within − and −35 kcal/mol. the affinity for this incomplete binding showed to be far above from target sites of validated endogenous genes and close to the perfect pairing. to validate the capacity and specificity of the designed lentiviral vectors to modulate mir- and mir- hela cells generated to overexpress mir- and mir- were transduced with different moi of lv-mir155t. mirna content was analyzed by rt-qpcr taqman assays and a dose-dependent reduction in mir- levels was observed whereas no changes were detected in mir- indicating the specificity of the effect. moreover, hela cells overexpressing mir- and mir- when transduced with lv-mir155-802t resulted in reduced expression of both mir- and mir-. furthermore, the number of gfp expressing cells was substantially reduced in hela cells expressing mir- and mir- with respect to hela control cells when transduced with lv-mir155t or lv-mir155-802t. these results demonstrate that lv-mir155-802t is a good tool to modulate mir- and mir- expression.fig. mir- and mir- expression are specifically reduced in cells infected with mirna-sponge lentiviruses. a top panel shows the sequence alignment of mmu-mir- and mmu-mir- to the corresponding mirna sponge sites engineered in the lentiviral vectors. bottom panel shows lv-mirt constructs containing the cmv promoter to drive destabilized green fluorescent protein expression bearing or mirna sponge sites in the 3′utr. b minimum free energy and complementarity displayed in the mirna:target sites hybridizations: engineered mirt sites, validated seeds, rand seq, rand seq + fixed seed. energies were calculated using rna hybrid algorithm. c hela cells stably expressing both mmu-mir- and mmu-mir- were transduced with and moi of lv-mir155t or lv-control. expression of both mirnas was analyzed 72-h later. rq values represent mean ± sem of four independent samples; *p <  and **p < . d hela cells stably expressing both mmu-mir- and mmu-mir- were transduced with moi of lv-mir155-802t or lv-control. expression of both mirnas was analyzed 72-h later. rq values represent mean ± sem of four independent experiments; *p <  to validate mirna sponges as a strategy to identify mirna targets in vivo, we stereotaxically infused in the ventral hippocampus of young adult euploid and ts65dn mice lv-mir155-802t and lv-control viral particles. three weeks upon infusion animals were sacrificed and mir- and mir- expression was analyzed. increased mir- and mir- levels were detected in the hippocampus of ts65dn mice injected with lv-control with respect to euploid animals confirming previous findings. interestingly, the administration of lv-mir155-802t resulted in a reduction in mir- and mir- content in ts65dn to levels similar to those of euploid mice. no effect was detected in control animals injected with the lentiviral sponge. in addition, we analyzed the expression levels of mecp a previously validated target gene for both mirnas, ship a mir- target gene and foxm a mir- target. expression analysis of mecp ship and foxm showed a reduction in the hippocampus of trisomic mice that were rescued to wild type levels in lv-mir155-802t injected animals.fig. decoy activity of lv-mir155-802t significantly reduces hippocampal mir- and mir- content in ts65dn mice. a schematic representation of the experimental design for local lentiviral infusion and sample collection. b expression analysis by rt-qpcr of mmu-mir- and mmu-mir- in rna extracts from hippocampus of euploid and ts65dn mice treated with lv-control or lv-mir155-802t. rq values represent mean ± sem of six independent samples; *p <  and **p < . c expression analysis by rt-qpcr of mmu-mir- and mmu-mir- validated target gene mecp the mmu-mir- target ship and the mmu-mir- validated target foxm in rna extracts from hippocampus of euploid and trisomic adult mice treated with lv-control or lv-mir155-802t. values represent mean ± sem of six independent samples; ns, non significant and * p <  and ** p <  to assess the transcriptomic impact of mir- and mir- overexpression we performed gene-expression profiling of euploid and ts65dn hippocampi treated with lv-control or lv-mir155-802t in an agilent microarray platform. cluster analysis by principle component analysis discriminated between genotypes but not between treatments, suggesting that the dosage imbalance effects on the steady state levels of thousands of genes was superior than the modulation by the lv-mir155-802t sponge within each genotype. to assess the effects of lv-mir-155-802t sponge we performed a gene set enrichment analysis of array data on the set of mir- and mir- predicted target genes from targetscan. we found enrichment in genes that negatively correlate with mir- or mir- consistent with mirna activity. further analysis on this enrichment was performed using mircomb r package, which calculated the correlation coefficients between signal intensity of transcripts in the microarray and mir- or mir- expression levels. this regression analysis considered all the treatment groups with variable levels of mir- or mir- involving genotype and treatment-based differences. the distributions of correlation scores showed that those genes with increased number of target sites display stronger negative correlation. this analytical approach combines sequence conservation parameters and experimental expression data, what refines mirna target predictions integrating data from biological samples. moreover, the topological analysis of predicted genes with a significant negative correlation showed a genome-wide impact of mir- and mir- imbalance. interestingly, when we constructed a topological map, based on the gedds defined by letourneau and collaborators plotted with our expression data, we could identify regions of coincidence from gedds to the expression associated to mir- and mir- deregulation. a 47 % of pairwise changes were estimated between the two studies. these data highlight that some of the epigenetic events can also be the consequence of the deregulation of epigenetic modulators such as mecp a direct target of mir- and mir-. these results point that the in vivo approach of lentiviral mirna sponge is a good strategy to highlight the link of mir- and mir- levels regulating mrna predicted targets. of notice, lentivirus integration into the genome allows the analysis of steady-state transcriptomic variation derived from mirna modulation. interestingly, we performed a compared in silico analysis between our results and microarray data from geo gse, in which the authors achieved a chromosome silencing of the extra hsa copy in trisomy cells. in such experiments, presumably mir- and mir- expression should be normalized and impact to the global transcriptome. the results of this comparative analysis showed that 61–53 % genes predicted to be mir- and mir- targets based on our negative correlations showed downregulation in trisomy cells, and more interestingly from the same mirna prediction lists 59–58 % presented a normalization of its expression upon extra hsa silencing. interestingly, the targetscan platform facilitated further analyses to show the number and the conservation of mirna target sites within the transcript and to infer regulatory networks between mouse and human cells.fig. transcriptome analysis reveals an enriched downregulation of mir- and mir- predicted targets. a gene set enrichment analysis of array data was performed on the set of mouse predicted targets for mir- and mir- in targetscan. b analysis of gene expression explained by the number of predicted mirna sites. box plots represent the distribution of mircomb pearson correlation coefficients among the targets sorted according to the predicted number of target sites. * and *** denote p <  and p < , respectively. c representation of the correlations between predicted target genes and mir- and mir- according to its topology in mouse genome. the layer over the chromosomes represents in graphic bars the regulation of genes within subchromosomic segments of 1000 kb. representation was performed using circos visualisation package. represented correlations are those that presented negative coefficients for a predicted target and a p-value <  in mircomb analysis further analyses of transcriptomic changes associated to mir- and mir- expression were conducted to identify those predicted mrnas more likely to be directly regulated. to this end gene sets were sorted according to the number of predicted mirna target sites in the 3′utr and the correlation score. a subset of five genes, on the top of that list, were selected for validation. quantitative rt-qpcr data confirmed downregulation of rufy nova nav thoc and sumo in the hippocampi of ts65dn mice and their restored expression in ts65dn lv-mir-155-802t treated mice. interestingly, all five genes are involved in neuronal function and cellular homeostasis, with relevant implications to neurological diseases. for instance, rufy belongs to rufy family, which is involved in the regulation of neuronal polarity and membrane trafficking. moreover, rufy has been implicated in the reduction of beta-amyloid secretion in late-onset alzheimer’s disease. similarly, sumo has also been linked to alzheimer’s disease, since endogenous protein sumoylation activity reduces beta-amyloid production. nova is a neuron-specific splicing factor that controls the alternative processing of a large set of mrnas important for the synaptic activity. the defect in neuronal viability observed in nova- null mice suggests either a loss of function or a gain of function derived splicing defects in inhibitory receptors gabargamma glyralpha that could trigger imbalances between inhibition and excitation signals. in this line, treatment of ts65dn mice with gaba antagonists have shown a rescue of cognitive defects and improved long-term potentiation. nav is a microtubule-associated protein involved in neuronal migration and reorganization of the cytoskeleton to induce neurite-like extensions. accordingly, reduced neurite length of neuronal precursors was observed in ts65dn mice. finally, thoc is a ribonucleoprotein involved in mrna elongation essential for early embryonic development, however its contribution to neuronal homeostasis is still unclear. whether the transcriptomic rescue of the perturbed gene set in the hippocampi of ts65dn mice upon lentiviral lv-mir-155-802t infusion may have any impact in brain physiology or the cognitive deficits of ts65dn mice remains to be studied. however, it could be speculated that normalization of the expression levels of the key target genes identified will be of functional relevance. in fact, normalization of dyrk1a or snx expression in the hippocampi of adult ts65dn mice by strategies based on viral vector administration has shown rescue of synaptic deficits and partial recovery of behavioural abnormalities.fig. validation of novel target genes contributing to t565dn phenotype by rt-pcr. a upper panel shows a table with five predicted target genes selected based on the criteria of predicted number of target sites and strong correlation coefficient. table shows correlation values and the number of predicted mirna target sites classified according its evolutionary conservancy. below panel shows the correlation plots calculated using mircomb package for the previously selected target genes. b expression analysis by rt-qpcr of mir- and mir- target genes in rna extracts from hippocampus of euploid and trisomic adult mice treated with lv-control or lv-mir155-802t. data normalized to the expression of gdx housekeeping gene. values represent mean ± sem of six independent samples. * and ns denote p <  and not significant, respectively the potential therapeutic effect of the sponge approach could also be of interest to be explored in other diseases by which functional mirna-disease associations have been identified such as cancer, type ii diabetes, asthma or alzheimer disease.  the present study establishes a link between overexpression of mir- and mir- and downregulation of a new set of candidate genes with potential contribution to ds neuronal function. the mirna sponge developed strategy provides a useful tool for the identification of subtle transcriptomic changes, often masked for interindividual variability. these results also highlight that ds hippocampal-dependent phenotypes have connections with alterations present in neurological-associated diseases such as alzheimer and suggests that modulation of mirnas could be envisioned as a novel pharmacotherapy approach in ds. 
five chromosome mirnas have been found overexpressed in ds individuals and could function as key elements in the pathophysiology. in the current work, we interrogated the impact of mir- and mir- upregulation on the transcriptome of ts65dn brains. hippocampal injections of lentiviral sponges in ts65dn mice normalized the expression of mir- and mir- and rescued the levels of their targets methyl-cpg-binding protein gene, sh -containing inositol phosphatase- and forkhead box protein m.
 down syndrome or trisomy is the result of a genetic dosage imbalance that translates in a broad clinical spectrum. a major challenge in the study of ds is the identification of functional genetic elements with wide impact on phenotypic alterations. recently, mirnas have been recognized as major contributors to several disease conditions by acting as post-transcriptional regulators of a plethora of genes. five chromosome mirnas have been found overexpressed in ds individuals and could function as key elements in the pathophysiology. interestingly, in the trisomic ts65dn ds mouse model two of these mirnas are also triplicated and overexpressed in brain.  in the current work, we interrogated the impact of mir- and mir- upregulation on the transcriptome of ts65dn brains. we developed a lentiviral mirna-sponge strategy to identify in vivo relevant mir- and mir- target mrnas. hippocampal injections of lentiviral sponges in ts65dn mice normalized the expression of mir- and mir- and rescued the levels of their targets methyl-cpg-binding protein gene, sh -containing inositol phosphatase- and forkhead box protein m. transcriptomic data of lv-mir155-802t mirna-sponge treated hippocampi correlated with candidate targets highlighting mirna dosage-sensitive genes. significant associations were found in a subset of genes that could be experimentally validated.  the lentiviral mirna-sponge strategy demonstrated the genome-wide regulatory effects of mir- and mir- furthermore, the analysis combining predicted candidates and experimental transcriptomic data proved to retrieve genes with potential significance in ds-hippocampal phenotype bridging with ds other neurological-associated diseases such as alzheimer’s disease. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords mirnastranscriptomespongesdown syndromeissue-copyright-statement© the author 2015 