MicroRNA Expression and Regulation in Human, Chimpanzee, and Macaque Brains
[['Phenotypic differences between species , including human-specific features such as language and tool-making , are thought to have arisen , to a large extent , through changes in gene expression [1] .', 'Indeed , humans and the closest living primate relatives , chimpanzees , display substantial gene expression divergence in all tissues including the brain [2] , [3] .', 'Mechanistically , this divergence might have been caused by mutations in regulatory elements proximal to genes ( cis- effects ) , or changes in expression or sequence of distal regulators ( trans- effects ) .', 'Previous studies focusing on transcription factors ( TFs ) have indicated an excess of human-specific expression divergence for several TFs in the liver [4] and the brain [5] .', 'These findings suggest that changes in TF expression might explain some of human-chimpanzee gene expression divergence .', 'In this study , we investigated the contribution of another type of gene expression regulator , miRNA , to human-specific gene expression divergence .', 'miRNA are short ( 20–23-nucleotide ) , endogenous , single-stranded RNA involved in post-transcriptional gene expression silencing .', 'Mature miRNA function as part of the RNA-induced silencing complex ( RISC ) , mediating post-transcriptional gene expression inhibition [6]–[8] .', 'In animals , the predominant mechanism of miRNA-mediated gene silencing employs complementary base-pairing between the miRNA seed region and the mRNA 3′ UTR region [9] , [10] .', 'This interaction guides RISC to target transcripts , which are consequently degraded , destabilized or translationally inhibited , causing an inverse expression relationship between miRNA and its cognate targets [8]–[12] .', 'miRNA-mediated gene expression silencing has previously been shown to be important for a variety of physiological and pathological processes , ranging from developmental patterning to cancer progression , as well as important neural functions and dysfunctions [7] , [13]–[15] .', 'The roles of miRNA in determining gene expression divergence between species and , in particular , their contribution to expression differences specific to the human brain remains , however , largely unknown .'], ['To assess miRNA expression divergence between human brains and brains of closely related primate species , we measured miRNA levels in two distinct brain regions , the prefrontal cortex ( dorsal-lateral prefrontal region ) and the cerebellum ( lateral cerebellar cortex ) , of humans ( age: 14–58 years ) , chimpanzees ( age: 12–40 years ) and rhesus macaques ( age: 6–15 years ) using high-throughput sequencing ( Illumina ) .', 'In the prefrontal cortex , a brain region known to play a part in the control of high-level cognitive functions , such as abstract thinking and planning [16]–[18] , we measured miRNA expression samples containing RNA pooled from multiple individuals , for each species ( Table S1 ) .', 'To assess technical variation of the sequencing measurements , we prepared and sequenced small RNA libraries twice .', 'In the cerebellum , we sequenced two human samples , one chimpanzee sample and one rhesus macaque sample , all composed from RNA pooled from multiple individuals ( Table S1 ) .', 'We obtained an average of 7 . 6 million sequencing reads per sample , approximately 49% of which could be perfectly mapped to the corresponding reference genome ( Table S2 ) .', 'Based on these data , we detected expression of 413 miRNA covered by least 10 sequence reads in the human prefrontal cortex or cerebellum .', 'To obtain the corresponding miRNA expression estimates for chimpanzees and rhesus macaques , we mapped all annotated human miRNA precursors to the chimpanzee and rhesus macaque genomes , using a combination of reciprocal BLAT , BLAST and liftOver [19]–[21] and extracted mature miRNA sequences using ClustalW2 precursor sequence alignment [22] ( Materials and Methods ) .', 'For 413 miRNA expressed in the human brain , we could unambiguously identify 385 and 390 corresponding genomic locations in the chimpanzee and rhesus macaque genomes , respectively .', 'The vast majority of these miRNA were also detected in chimpanzee ( 375 ) and rhesus macaque ( 366 ) brains ( Table S3 ) .', 'Due to lower quality of the chimpanzee and the rhesus macaque genomes as well as low expression levels of human miRNA with no chimpanzee or macaque orthologs , we omitted these miRNA from further analyses .', 'In all three species , high-throughput sequencing generated highly reproducible miRNA expression measurements , with good positive correlation between technical replicates ( Pearson correlation , r>0 . 99 , p<10−15 ) ( Figure S1 ) .', 'Furthermore , in both brain regions , miRNA expression divergence among species was evidently greater than variation within species ( Figure 1A-1B ) .', 'The extent of miRNA expression divergence followed the phylogenetic relationship among species in both prefrontal cortex and cerebellum , i . e . human and chimpanzee samples clustered as sister species , with macaque samples forming an outgroup .', 'In the human or chimpanzee prefrontal cortex , 325 miRNA were represented by at least 10 sequence reads in at least one technical replicate of one species .', 'All 325 miRNA had orthologs in the chimpanzee genome ( Table S3 ) .', "Of these , 37 were differently expressed between species in both technical replicates ( Fisher's exact test , p<0 . 01 & fold-change>2 ) .", 'Using an alternative procedure , based on the assumption that sequence read follow a negative binomial distribution , implemented in the edgeR package [23] , 35 miRNA were differently expressed between humans and chimpanzees ( p<0 . 001 & FDR<0 . 01 ) ( Table S4 ) .', 'Thirty one overlapped between the two methods ( binomial test , p<0 . 0001 ) .', "Using the same criteria , 106 out of 338 miRNA detected in human and rhesus macaque prefrontal cortex were differently expressed between the two species , according to Fisher's test .", 'Eighty-eight out of these 106 miRNA were also classified by edgeR as differently expressed ( Table S4 ) .', 'The vast majority of miRNA expression differences that were found between species in the prefrontal cortex could be reproduced in the cerebellum .', "Specifically , out of 37 miRNA differently expressed between humans and chimpanzees in the prefrontal cortex , according to Fisher's exact test , 31 ( 84% ) showed consistent expression differences between species in both brain regions ( Figure S2A-S2B ) .", 'Similarly , out of 106 miRNA differently expressed between humans and macaques in the prefrontal cortex , 82 ( 77% ) showed consistent expression differences between the two species in both brain regions ( Figure S2C-S2D ) .', 'In both cases , the agreement between the two brain regions was far greater than expected by chance ( binomial test , p<0 . 0001 ) .', 'Although the prefrontal cortex and the cerebellum are histologically different , previous studies have shown that mRNA expression differences between humans and chimpanzees are largely shared between these two brain regions [24] .', 'Our results indicate that miRNA divergence is similarly shared between the prefrontal cortex and the cerebellum .', 'Furthermore , good agreement of miRNA divergence estimates between the two brain regions supports robustness of our measurements .', 'To further test the robustness of the miRNA divergence estimates obtained using high-throughput sequencing and to overcome potential problems caused by pooling samples from multiple individuals , we measured miRNA expression in the prefrontal cortex of three human , three chimpanzee and two rhesus macaque individuals using miRNA microarrays ( Agilent ) .', 'To exclude possible hybridization artefacts , array probes corresponding to 150 miRNA with sequence differences between humans and chimpanzees and to 313 miRNA with sequence differences between humans and rhesus macaques were masked prior to further analyses .', 'Overall , microarray were less sensitive than sequencing , with a total of 287 miRNA detected as expressed above the default threshold in the human or chimpanzee prefrontal cortex ( Table S5 ) .', 'Concordant with results obtained using high-throughput sequencing , intra-species variation of the microarray miRNA expression measurements was lower than between-species divergence .', 'Further , as in case of the sequencing data , miRNA expression divergence measured by arrays followed the phylogenetic relationship among species ( Figure 1C ) .', 'Further supporting the authenticity of our miRNA divergence estimates , the differences found between humans and chimpanzees or macaques , using high-throughput sequencing , were largely reproduced in microarray experiments .', 'Due to the lower sensitivity of microarray experiments , out of 37 miRNA that were classified as differently expressed between human and chimpanzee prefrontal cortex using sequencing , 12 were detected reliably on the microarrays and 9 showed consistent direction of expression divergence ( Figure 1D ) .', "Analyzing miRNA expression divergence based on microarray data alone and applying statistical criteria similar to the ones used in sequencing data analysis ( Student's t-test , p<0 . 01 , fold-change>2 ) , four miRNA differed significantly between humans and chimpanzees in the prefrontal cortex .", 'Three of these showed consistent direction of expression change and one passed the significance cutoff level in the sequencing data .', 'Thus , out of 15 miRNA classified as being differently expressed between humans and chimpanzees by at least one methodology , 12 showed consistent direction of expression change ( binomial test , p<0 . 05 ) ( Figure 1D ) .', 'Similarly , out of 106 miRNA differently expressed between human and macaque prefrontal cortex , according to sequencing , 61 were detected by microarrays and 55 showed consistent direction of expression change ( binomial test , p<0 . 01 ) ( Figure 1E ) .', 'Despite overall agreement between the two methodologies , some of the human-chimpanzee miRNA expression differences identified using sequencing were not confirmed by the microarrays .', 'To further test the validity of our results , we measured expression of 6 miRNA in three human and three chimpanzee individuals using a third methodology: quantitative RT-PCR .', 'We chose three types of miRNA differences: ( 1 ) consistent by both methodologies: miR-383 and miR-34c-5p; ( 2 ) significant according to sequencing , but unconfirmed in the microarray experiment: miR-143 and miR-499; ( 3 ) significant according to sequencing , but not detected or masked on the microarrays: miR-184 and miR-299-3p .', 'Quantitative RT-PCR results confirmed expression differences for all miRNA in the first and the third categories , but not for the miRNA in the second category ( Figure S3 ) .', 'Thus , miRNA expression differences that were consistent across methodologies , or large differences that were identified by the sequencing , but masked or undetected on the arrays , are both likely to reflect actual miRNA expression divergence between human and chimpanzee brains .', 'In the prefrontal cortex , we identified 25 miRNA with the human-chimpanzee expression divergence estimates consistent across methodologies or showing large divergence in the sequencing data , but masked or undetected on the arrays ( Table S6 ) .', 'Using rhesus macaque miRNA expression as an outgroup , 13 of the 25 could be assigned to the human evolutionary lineage and 8 to the chimpanzee evolutionary lineage ( Table S6 ) .', 'Requiring significant support by at least two out of three methodologies ( sequencing , microarrays and Q-PCR ) , expression changes in five miRNA ( miR-184 , miR-299-3p , miR-487a , miR-383 and miR-34c-5p ) could be assigned to the human evolutionary lineage and two ( miR-375 and miR-154* ) to the chimpanzee evolutionary lineage ( Figure 2 ) .', 'Six out of 7 miRNA assigned to the human- and the chimpanzee-evolutionary lineages in the prefrontal cortex also showed human- and chimpanzee-specific expression patterns in cerebellum ( Figure 2 ) .', 'Do miRNA expression differences between human and chimpanzee brains contribute to gene expression divergence between these species ?', 'To estimate this , we measured mRNA and protein expression in human and chimpanzee prefrontal cortex: mRNA expression in five individuals of each species using Affymetrix Exon arrays , protein expression in four individuals of each species with two technical replicates using a label-free 2D-MS/MS Thermo-LTQ proteomics methodology ( Table S1 and Table S7 , Materials and Methods ) .', 'Identified miRNA expression differences indeed had a significant negative effect on mRNA and protein expression in the human and chimpanzee prefrontal cortex , i . e . , targets of highly expressed miRNA were down-regulated in the corresponding species ( Figure 3 and Table S8 ) .', 'This effect was significant for differentially expressed miRNA that were identified using both sequencing and microarray methodologies , as well as for miRNA that were identified by sequencing alone ( Figure 3 and Table S8 ) .', 'The significance level of the effect did not depend on the choice of the miRNA target prediction algorithm: In brief , we obtained similar results using TargetScan5 predictions [10] , [25] - based on the presence of conserved miRNA binding sites in mRNA 3′ UTR regions and reported to have good sensitivity and specificity [26] ( Figure 3A-3C and Table S8 ) - as we obtained using PITA ( TOP ) predictions - based on the free energy gained from the formation of the miRNA-target duplex [27] ( Figure 3D-3F and Table S8 ) .', 'Further , the negative effect of miRNA expression differences on mRNA and protein expression could be observed at various miRNA expression level cutoffs .', "For highly expressed miRNA , the negative effect on their targets' expression levels tended to be more significant ( Table S8 ) .", 'Finally , the negative effect of miRNA on mRNA expression divergence between human and chimpanzee brains could also be reproduced at various mRNA expression divergence cutoffs ( Figure S4E-S4F ) .', 'To assess an overall contribution of miRNA regulation to mRNA and protein expression divergence between human and chimpanzee brains , we calculated the proportion of significant mRNA and protein expression differences that could be negatively associated with miRNA expression differences .', 'Since some of these associations might be caused by factors other than miRNA regulation , we used a number of significant mRNA and protein expression differences showing positive association between miRNA and target genes , as a background .', 'At p<0 . 001 mRNA divergence cutoff ( FDR<2% ) , 68 out of 479 ( 14% ) mRNA , with significant expression differences between human and chimpanzee prefrontal cortex , could be negatively associated with miRNA expression differences .', 'By contrast , 58 ( 12% ) mRNA showed positive association .', 'Thus , 2% of mRNA expression differences between human and chimpanzee brains could be assigned to miRNA regulation .', 'Although this effect appears small , it can be observed consistently at all mRNA expression divergence cutoffs ( Figure S4A-S4B ) .', 'Further , at more stringent mRNA divergence cutoffs , the miRNA regulatory effect became more apparent reaching 4% at p\u200a=\u200a0 . 0005 .', 'At the protein level , 26 out of 117 ( 22% ) proteins with significant expression differences between humans and chimpanzees ( FDR<5% ) were negatively associated , and 21 ( 18% ) - positively , with the miRNA expression divergence .', 'Thus , we estimate that 4% of protein expression differences between human and chimpanzee brains could be caused by miRNA .', 'Similarly , the miRNA regulatory effect could be consistently detected at all protein expression divergence cutoffs , and increased to 6% at p\u200a=\u200a0 . 001 ( Figure S4C-S4D ) .', 'These estimates are based on the assumptions that negative relationship between miRNA and target gene expression levels in the two species indicates regulation , while a positive relationship does not .', 'Both assumptions might be incorrect .', 'An excess of negative associations between miRNA and their predicted targets might be caused by yet unknown factors , rather than miRNA regulation .', 'On the other hand , positive regulatory relationship between miRNA and target gene expression has been reported [28] , [29] .', 'Further , a positive correlation between miRNA and target gene expression could be caused by indirect regulatory effects [30] .', 'Thus , the actual extent of the effect that miRNA have on gene expression divergence between adult human and chimpanzee brains remains to be estimated .', 'Nevertheless , the consistent and significant negative relationship between miRNA expression , and the expression of their target genes , on both mRNA and protein levels ( Figure 3 and Figure S4 ) , and the consistent excess of negative associations between miRNA and their targets ( Figure S4 ) , demonstrates that miRNA expression divergence does contribute to gene expression divergence between humans and chimpanzees .', 'To assess whether miRNA with expression divergence on the human lineage might be associated with human cognitive functions , we investigated the expression of genes targeted by five miRNA showing human-specific expression , according to multiple methodologies: miR-184 , miR-487a , miR-383 , miR-34c-5p and miR-299-3p ( Figure 2 ) .', 'On the DNA sequence level , these miRNA tend to be conserved: miR-184 mature miRNA sequence is evolutionarily conserved from insects to humans , with only one nucleotide different at 3′end of mature sequence , while miR-383 and miR-34c-5p are classified as broadly conserved and miR-299-3p - as conserved among animal species [25] , [31] .', 'High sequence conservation indicates the functional importance of these miRNA and shows that expression divergence on the human evolutionary lineage is unlikely to be caused by lack of a selection constraint .', 'Notably , genes targeted by these five miRNA were enriched in neural functions .', 'By contrast , no neural-related enrichment was observed for targets of the two miRNA showing chimpanzee-specific expression .', 'Specifically , based on a functional analysis using DAVID [32] , combined targets of the five miRNA with human-specific expression were significantly enriched in terms “signal transduction” , “synaptic transmission” , “cell surface receptor mediated signal transduction” , “neuronal activities” and “cell proliferation and differentiation” ( Bonferroni-corrected p<0 . 05 ) ( Table S9 ) .', 'Targets of miRNA with chimpanzee-specific expression were significantly enriched in terms “nucleoside , nucleotide and nucleic acid metabolism” , “mRNA transcription” and “mRNA transcription regulation” ( Table S9 ) .', 'Similarly , based on the DIANA-mirPath algorithm [33] , targets of miR-184 , miR-487a and miR-299-3p were significantly enriched in KEGG pathways that are related to neural functions ( Table S10 ) .', 'Finding three out of five miRNA with significant target gene enrichment in neural functions was unexpected ( permutation test , p\u200a=\u200a0 . 067 ) .', 'Furthermore , miR-184 targets were significantly enriched in “long-term potentiation” pathway – one of the few pathways directly connected to learning and memory formation [34] , [35] .', 'Recent studies have also shown that miR-184 is involved in regulation of neural stem cell proliferation and differentiation [36] .', 'Similarly , targets of miR-299-3p were significantly enriched in the “axon guidance” pathway , which is associated with neuronal cell differentiation and functions .', 'To further test association of miR-184 and miR-299-3p with neuronal functions , we determined their expression patterns in the human and macaque prefrontal cortex by in situ hybridization with specific LNA-probes ( Table S11 ) .', 'Expression of the two miRNA co-localized with expression of NeuN protein , an established vertebrate neuronal-specific marker [37] , [38] ( Figure 4 ) .', 'Thus , even though pathway enrichment results were based on a limited number of miRNAs with species-specific expression in the prefrontal cortex , and although they relied on predicted miRNA-target relationships , both miRNA , with significant target gene enrichment in neuron-related pathways , are indeed preferentially expressed in neurons .', 'Due to the fact that miRNA functions cannot be tested in human or chimpanzee brains , we used two human neuroblastoma cell lines , SH-SY5Y and SK-N-SH , to verify miRNA target predictions and test their functions , as well as their expression changes on the human evolutionary linage .', 'In order to achieve this , we transfected the two cell lines with double-stranded oligonucleotides , which mimic human mature miRNA sequences ( Table S1 , Materials and Methods ) .', 'We tested the effects of all five miRNA showing human-specific expression in brain , as well as effects of the chimpanzee version of miR-299-3p sequence .', 'The effects of each miRNA were assayed 24 hours after transfection using Affymetrix Human Genome U133 Plus 2 . 0 arrays .', 'For each cell line , miRNA regulatory effects were calculated as the difference in expression levels between cells transfected with miRNA analogue and cells transfected with negative control oligonucleotides .', 'For each cell line , transfection with negative control oligonucleotides was carried out in two independent replicates .', 'In both cell lines we observed significant expression inhibition of predicted miRNA targets for all 6 miRNA sequences ( Figure 5 ) .', 'The results were highly consistent between two independent negative control replicates ( Figure S5 and Figure S6 ) and showed significant overlap between the two cell lines ( Figure S7 ) .', 'Further , in both cell lines we observed highly correlated target effects after transfection with human and chimpanzee versions of miR-299-3p .', 'The mature sequence of miR-299-3p contains human-specific C to T substitution at position 10 .', 'While this substitution might affect relative stand selection efficiency during miRNA procession , as well as target selection , we did not find any significant differences in target effects between the human and the chimpanzee versions of miR-299-3p in our experiment ( Figure S8 ) .', 'To capture the majority of possible miRNA targets , we used 9 common target prediction algorithms .', 'In agreement with a previous report [26] , among the 9 algorithms , TargetScan resulted in better agreement between experimental results and target predictions ( Table S12 ) .', 'It is noted , however , that targets predicted by other algorithms showed significant inhibition in transfection experiments .', 'Thus , we used either TargetScan or we combined target predictions and down-regulation in cell line experiments to identify experimentally verified miRNA targets ( Table S13 and Table S14 , Materials and Methods ) .', 'Consistent with results based on computational predictions , the experimentally verified targets of miRNA with human-specific expression showed significant enrichment in certain neuronal functions .', "Specifically , based on a functional analysis using DAVID [32] , experimentally verified targets of the five miRNA were significantly enriched , compared to genes expressed in the brain and in at least one of the two cell lines ( Fisher's exact test , p<0 . 05 ) , in the following biological processes and KEGG pathways associated with neural functions: “signal transduction” , “synaptic transmission” , “neurotransmitter release” , “adherens junction” and “axon guidance” ( Tables S15 ) .", 'Notably , miRNA-target relationships experimentally verified in the two cell lines was also observed in brain .', 'For each of the five human-specifically expressed miRNA we found inverse correlation between miRNA and target gene expression on the human evolutionary lineage ( Figure 6 ) .', 'This effect was significant for combined targets of the five miRNA , as well as for targets of miR-184 and miR-383 analyzed individually .', 'For the remaining miRNA regulatory effects were not significant , but they did show target expression inhibition .', 'Thus , miRNA-target relationship identified in cell line experiments did allow us to capture miRNA-target relationship , thus explaining some of gene expression changes that took place in the brain on the human evolutionary lineage .', 'While human and chimpanzee evolutionary lineages separated approximately 6–7 million years ago , humans and Neanderthals shared a common ancestor less than half a million years ago [39] .', 'Thus , using Neanderthal data it might be possible to date miRNA expression change more precisely .', 'Although miRNA expression in Neanderthal brain cannot be estimated , signature of positive selection spanning miRNA promoter , or the regulatory region in the human genome , would indicate that expression change might have taken place after human and Neanderthal linage separation [40] .', "We indeed found a significant excess of human derived SNPs , indicating the presence of positive selection on the human evolution linage after the human-Neanderthal split , in the upstream regions of one out of five miRNA with human-specific gene expression: miR-34c-5p ( Fisher's exact test , Bonferroni corrected p<0 . 05 , Materials and Methods ) .", 'Genome-wide , the possibility of finding a signature of positive selection at this significance level within the upstream region of five randomly chosen miRNA is low ( 1000 permutations , p<0 . 05 ) .', 'Notably , for miR-34c-5p signature of positive selection was located in the putative enhancer region approximately 100kb upstream of the miRNA gene ( Figure 7 ) .', 'Thus , although indirectly , these results indicate that the change in miR-34c-5p with human-specific expression might have taken place after the separation of the human and the Neanderthal evolutionary lineages .', 'Furthermore , positive selection on changes in regulatory regions of this miRNA indicates their potential adaptive significance .', 'Functionally , miR-34c-5p was previously shown to be down-regulated in cancer and Parkinson disease [41]–[44] .', 'We further characterized possible functions of miR-34c-5p in the human brain , based on target genes experimentally verified in cell lines .', "Compared to the genes expressed in brain , these target genes were significantly enriched , among others , in biological processes “neurotransmitter secretion” and “behaviour” , as well as cellular components “dendrite cytoplasm” , “synapse” and “cell junction” ( Fisher's exact test p<0 . 01 , Tables S16 ) .", 'These findings indicate that changes in miR-34c-5p expression on the human evolutionary linage might have resulted in gene expression changes affecting cognitive functions .', 'In conclusion , despite high sequence conservation of 325 miRNA expressed in the prefrontal cortex , 11% were expressed at significantly different levels in humans and chimpanzees .', 'The vast majority of these differences were also found in cerebellum and were confirmed by microarray and Q-PCR experiments .', 'Importantly , we observed significant inverse relationship between human-chimpanzee miRNA expression divergence and expression divergence of the predicted target genes at both mRNA and protein levels .', 'This indicates that miRNA expression divergence plays an important role in shaping gene expression divergence among species .', 'Approximately half of the miRNA expression differences found in the prefrontal cortex could be assigned to the human evolutionary lineage .', 'These miRNA , as well as their target genes , were conserved at the sequence level .', 'Thus , their expression divergence is unlikely to be explained by a lack of selective constraints .', 'Instead , targets of miRNA with human-specific expression were enriched in neural functions associated with learning and memory pathways , such as “axon guidance” and “long term potentiation” .', 'Potential influence of miRNA divergence on neuronal functions was further confirmed by preferential expression of the corresponding miR-299-3p and miR-184 in cortical neurons , as well as verification of the predicted miRNA-target relationship in two human neuroblastoma cell lines .', 'Based on miRNA-target relationships verified in cell lines , we further demonstrated the effect of miRNA regulation on gene expression changes in brain , on the human evolutionary lineage .', 'Finally , we show that at least one out of five human-specific miRNA expression changes found in brain might have occurred after separation of the human and the Neanderthal evolutionary lineages .', 'Signature of positive selection found in the enhancer region of the miRNA , miR-34c-5p , further indicates that this change might have had adaptive significance .', 'Although these findings do not provide direct evidence that miRNA regulation resulted in human-specific phenotypic adaptations , taken together they indicate that miRNA regulation did contribute to gene expression changes on the human evolutionary lineage and that it affected genes involved in neuronal functions .', 'Further studies are needed to evaluate functional significance of the miRNA-driven transcriptome changes .'], ['Informed consent for the use of human tissues for research was obtained in writing from all donors or their next of kin .', 'All non-human primates used in this study suffered sudden deaths for reasons other than their participation in this study and without any relation to the tissue used .', 'Biomedical Research Ethics Committee of Shanghai Institutes for Biological Sciences completed the review of the use and care of the animals in the research project ( approval ID: ER-SIBS-260802P ) .', 'Human tissue was obtained from the NICHD Brain and Tissue Bank for Developmental Disorders at the University of Maryland , Baltimore , MD .', 'The role of the NICHD Brain and Tissue Bank is to distribute tissue and , therefore , cannot endorse the studies performed or the interpretation of results .', 'All subjects were defined as normal controls by forensic pathologists at the NICHD Brain and Tissue Bank .', 'No subjects who suffered a prolonged agonal state were used .', 'For the prefrontal cortex , samples were taken from the frontal part of the superior frontal gyrus: a cortical region approximately corresponding to Brodmann Area 9 .', 'For all samples , similar proportions of grey and white matter were dissected .', 'Total RNA was isolated from the frozen prefrontal cortex tissue using the Trizol ( Invitrogen , USA ) protocol with no modifications .', 'Prior to low molecular weight RNA isolation , the total RNA from 20 male individuals aged between 14 and 58 years was combined in equal amounts .', 'Low molecular weight RNA was isolated , ligated to the adapters , amplified and sequenced following the Small RNA Preparation Protocol ( Illumina , USA ) with no modifications .', 'Technical replication was completed by independently processing the mixed sample of 20 individuals starting from the low molecular weight RNA isolation step .', 'We carried out the sample preparation and deep sequencing by choosing 5 adult chimpanzee individuals and 5 rhesus macaque individuals following the protocols used for human samples .', 'Details of all samples are given in Table S1 .', 'All original deep sequencing data is deposited in the NCBI GEO database [GSE26545] .', 'Total RNA was isolated using the mirVana miRNA isolation kit ( Ambion ) .', '100 ng of each RNA sample were hybridized to Agilent Human microRNA Microarray ( G4471A , Agilent Technologies ) .', "MicroRNA labelling , hybridization and washing were carried out following Agilent's instructions [45] .", 'Agilent microRNA assays integrate eight individual microarrays on a single glass slide .', 'Each microarray includes approximately 15 k features containing probes sourced from the miRBase public database .', 'The probes are 60-mer oligonucleotides directly synthesized on the array .', 'We used Human miRNA Microarray Version3 , which contains probes for 866 human and 89 human viral microRNAs from the Sanger miRBase v12 . 0 .', 'All samples used in the prefrontal cortex comparison among three species were hybridized to one array .', 'Data from samples hybridized on a single array were processed and analyzed separately to avoid possible batch effects .', 'Images of hybridized microarrays were acquired with a DNA microarray scanner ( Agilent G2565BA ) ; Feature Extract
['Introduction', 'Results/Discussion', 'Materials and Methods']
['Among other factors , changes in gene expression on the human evolutionary lineage have been suggested to play an important role in the establishment of human-specific phenotypes .', 'However , the molecular mechanisms underlying these expression changes are largely unknown .', 'Here , we have explored the role of microRNA ( miRNA ) in the regulation of gene expression divergence among adult humans , chimpanzees , and rhesus macaques , in two brain regions: prefrontal cortex and cerebellum .', 'Using a combination of high-throughput sequencing , miRNA microarrays , and Q-PCR , we have shown that up to 11% of the 325 expressed miRNA diverged significantly between humans and chimpanzees and up to 31% between humans and macaques .', 'Measuring mRNA and protein expression in human and chimpanzee brains , we found a significant inverse relationship between the miRNA and the target genes expression divergence , explaining 2%–4% of mRNA and 4%–6% of protein expression differences .', 'Notably , miRNA showing human-specific expression localize in neurons and target genes that are involved in neural functions .', 'Enrichment in neural functions , as well as miRNA–driven regulation on the human evolutionary lineage , was further confirmed by experimental validation of predicted miRNA targets in two neuroblastoma cell lines .', 'Finally , we identified a signature of positive selection in the upstream region of one of the five miRNA with human-specific expression , miR-34c-5p .', 'This suggests that miR-34c-5p expression change took place after the split of the human and the Neanderthal lineages and had adaptive significance .', 'Taken together these results indicate that changes in miRNA expression might have contributed to evolution of human cognitive functions .']
['Humans are remarkably similar to apes and monkeys on the genome sequence level but remain remarkably distinct with respect to cognitive abilities .', 'How could human cognition evolve within such a short evolutionary time ?', 'Among many hypotheses , evolution in expression of a few key regulators affecting hundreds of their target genes was proposed as one possible solution .', 'Here , we tested this notion by studying expression divergence of a specific type of regulatory RNA , microRNA ( miRNA ) , and its effect on gene expression profiles in brains of humans , chimpanzees , and rhesus macaques .', 'Our results indicate that changes in miRNA expression have played a considerable role in the establishment of gene expression divergence between human brains and brains of non-human primates at both mRNA and protein expression levels .', 'Furthermore , we find indications that some of the human-specific gene expression profiles caused by miRNA expression divergence might be associated with evolution of human-specific functions .']
['microarrays', 'sequence analysis', 'organismal evolution', 'genomics', 'evolutionary biology', 'human evolution', 'biology', 'computational biology']