BACKGROUND
hiv-associated dementia  is the most common dementia type in young adults less than  <dig> years of age.
to date, the cumulative incidence of had is 25-38%, and the prevalence is around 37%  <cit> .
although the highly active antiretroviral therapy  has had considerable success in preventing virus-mediated immune collapse end-stage complications, the prevalence of hiv-associated cognitive impairment appears to be on the rise due to the increased life span of hiv+ population.
it is well recognized that host-virus interactions play a crucial role in the occurrence and pathogenesis of had.
microarray and high throughput genomic technologies have greatly facilitated the examination of this interaction.
a panoply of host genes have been shown to be influenced by hiv infection that facilitate subversion and manipulation of the host immune system during hiv infection of the brain.
to date, most studies exploring had pathogenesis  <cit>  have largely been confined to cultured cells, cerebrospinal fluid  and animal models, which can’t mimic and reveal the breadth of in vivo cellular responses subverted and manipulated as a consequence of hiv infection.
recently, micrornas , a class of small  non-coding rnas, have been recognized as master post-transcriptional regulators of mrnas  <cit>  due to their numerous targeting capabilities along with their ability to form non-linear functionally viable gene regulatory networks, which together have wide-ranging effects on the control of host gene expression.
in addition, mirnas are also involved in diverse processes, which include neuronal development, cell differentiation, synapse formation and neuronal plasticity  <cit> .
thus, not surprisingly, mirnas are significantly involved in neurodegenerative diseases, such as parkinson disease   <cit> , alzheimer’s disease   <cit> , schizophrenia  <cit> , aggressive behaviour  <cit>  and depression  <cit> .
further, mirnas also play an important role in hiv-host interaction  <cit> .
hiv can even manipulate the expression of host neuronal genes, such as snap <dig> and cell death-related genes, including caspase- <dig>  via the modulation of expression of selected micrornas, which might serve as key elements in gene regulatory networks in hiv-associated neurobehavioral disorder  <cit> .
to date, a joint study of the genome wide mrna and mirna profiling based on hiv-infected brain tissue with and without dementia is still lacking, although some individual mrna  <cit>  and mirna  <cit>  profiling studies based on human brain have been done.
therefore, our innovative approach has studied the utility of parallel genome-wide mrna and mirna analysis in the native frontal lobe post-mortem brain tissue from hiv patients with and without dementia.
this carries enormous value in understanding gene expression in the context of hiv-mediated neurodegeneration and its regulation through mirnas.
this study has been designed with an objective to comprehensively delineate host transcriptional programming that occurs in concert with the regulatory mirnas and to find how this interaction between mrna and mirna tampers with neurodegenerative pathways and dictates neurological manifestation of hiv disease.
here, we examined the gene ontology and pathways of differentially expressed  mrna in parallel with the global gene targets of de mirnas.
moreover, we derived paired functional correlation between mrna and mirna expressions using splitting-averaging strategy  to demonstrate intrinsic functional relationship between gene expression and its regulation.
in light of these data we believe that these results will not only facilitate a greater understanding of hiv pathogenesis of the brain and its neurological manifestation, but will also help define potential candidates for early detection and future therapy for neurodegeneration in hiv patients and related disorders.
RESULTS
a snapshot of de mrna and mirna profiles between had and hiv non-demented brains
in order to identify the mrna and mirna, which may contribute to the pathogenesis of had, a parallel genome-wide mrna and mirna profiling of the frontal cortex from hiv patients with and without dementia was performed.
genomestudio was used to analyse the normalized mrna dataset.
we observed  <dig> statistically significant candidate genes that were differentially expressed between two groups  .
among them,  <dig> genes were down regulated and  <dig> genes were up regulated , and  <dig> of  <dig> genes dysregulated greater than  <dig> -fold change.
to determine the similarity of global gene expression between samples and also in relation to the de genes, hierarchical clustering was performed and generated using genomestudio .
the had group formed an independent cluster away from the hiv non-dementia group, with the exception of one sample with dementia  which clustered together with the hiv non-dementia group.
not surprisingly, the hiv non-dementia group clustered together with hiv negative control due to the absence of neuropathological changes and the absence of actively replicating hiv, which is consistent with a previous study  <cit>  and our own study  <cit> .
the analysis of mirna data using genespring identified  <dig> mirna that were significantly differentially expressed in had and hiv non-dementia cortex .
among them,  <dig> mirnas were down regulated and  <dig> were upregulated.
27/ <dig> mirnas were dysregulated greater than  <dig> fold.
hierarchical clustering was carried out to demonstrate patterns of mirna expression profiling between two groups , which was consistent with our mrna hierarchical clustering.
targetscan was used to predict the gene targets of de mirnas, because it is the most advanced, respected, widely used and relatively conservative database in comparison to other databases .
in addition, we have carried out a low and high stringency g-seed search for mir- <dig>  mir- <dig> and mir- <dig> targets based on a minimal free energy  ¾ - <dig> and ¾- <dig>  respectively .
furthermore, these predictions were made in the context of changes in gene expression observed in the same rna.
functional annotation of the mrna de genes and mirna target genes was carried out in database for annotation, visualization and integrated discovery  together with extensive literature search.
these mrna de genes were significantly associated with biological processes, such as cell death/cell cycle, neuronal processes, metabolism, transcriptional regulation, protein modification, signal transduction, and response to virus/stress, as shown in figure 1a.
in addition, according to cellular components distribution, 29% of genes fell into neuronal-related components, such as axon, neuron projection, and dendrite .
furthermore, gene set enrichment analysis  was also used to examine significantly enriched go  gene sets comparing the normalized data of the entire  <dig>  gene transcripts from had and hiv non-dementia brains to  <dig> go gene sets in gsea molecular signatures database   <cit> .
eight gene sets were found statistical significantly enriched  in hiv non-dementia group while no gene sets were significantly enriched in the had group  .
of the eight enriched gene sets in the non-dementia group, four were closely related to neurological and/or hiv disease: dendrite  , atpase activity coupled to transmembrane movement of ions  , atpase activity coupled to transmembrane movement of ions phosphorylative mechanism , and cytoskeleton dependent intracellular transport  .
functional annotation results of mirna target genes were consistent with that of mrna results, but noteworthy is that the mirna target genes showed relatively more comprehensive biological processes and cellular components, which could be attributed to the ability of single mirna to have numerous mrna targets, therefore further validation might be needed to precise the specificity.
cellular components of mirna target genes are mainly distributed in cellular  and neuronal related-structures , but they display scattering into ion channel, actin cytoskeleton, tight junction, transcription factor complex and chromatin, which concur with our mrna results.
for instance, we found significant dysregulation of genes in microtubule assembly , microtubule nucleation , cytoskeleton movement  and microtubule stabilization .
in regards to ion channel genes, we found the upregulation of genes related to ion transport or ion channel, including ca2+, cl-, na+, k+, cu2+, zn2+ and glutamine transport.
especially, the genes involved in ca2+, na+ and glutamine transport changed greater than  <dig>  fold, the dysregulation of which is known to play a significant role in pathophysiology of neurodegenerative diseases as discussed in subsequent sections.
we also found more than  <dig> genes dysregulated in transcriptional regulation process, and similarly, the biological process of small percentage of mirna target genes fall into cell adhesion, tissue/embryonic development, learning, and chromatin modification etc.
.
although they were not dominant, they all can play a very important role in neurological degeneration  <cit> .
quantitative real time rt-pcr corroboration of mrna and mirna array dataset
in order to confirm and validate the data obtained by both data sets, we analysed  <dig> de genes and  <dig> de mirnas and  <dig> non-changed mirna using quantitative real time rt-pcr on the same study sample set.
for mrna, the quantitative real time rt-pcr for  <dig> of the  <dig> genes was fully consistent with their microarray expression profiles and trends  .
the two genes  for which the rt-pcr was not consistent with the microarray were excluded from further analysis.
this, in no way, compromises the interpretation of our dataset.
for mirna, the quantitative rt-pcr for  <dig> of  <dig> mirnas was consistent with the trend seen in microarray analysis  , which enhances the confidence and shows the validity of our mirna data set and their gene targets defined herein.
pathway analysis of de mrnas and mirnas
de gene list and de mirna target gene list were uploaded to david to explore functionally relevant pathways.
for mrna, long-term potentiation, axon guidance and signalling pathways  stood out significantly.
not surprisingly, we found  <dig> genes significantly dysregulated in long-term potentiation pathway  .
among them, itpr <dig> and ppp3ca down regulated greater than  <dig> -fold change.
axon guidance pathway was significantly down regulated in had versus non-demented patients with  <dig> down regulated genes  .
among them, epha <dig> was down regulated > <dig> fold, while plxnd <dig>  srgap <dig>  ppp3ca were down regulated > <dig>  fold.
there were a total of  <dig> genes involved in signalling pathway.
we found that all the 12/ <dig> genes in the mapk pathway were down regulated, including the key members of the mapk signalling pathway, such as map2k <dig>  map2k <dig>  map3k <dig>  rras <dig>  rps6ka <dig> and taok <dig>  among them, taok <dig> was down regulated greater than  <dig> fold, while map2k <dig>  map3k <dig>  pla2g4a and rras <dig> was dysregulated greater than  <dig>  fold .
two key proteins in the mapk pathway, map2k <dig>  and map2k <dig> , were also further validated by western blotting analysis using  <dig> samples from had and  <dig> samples from hiv non-dementia group, respectively.
both proteins showed down-regulation in had brains, which followed the same trend observed in our microarray analysis.
mek <dig> was recognized by p-mek-1/ <dig> antibody at  <dig> kda .
it was expressed slightly weaker in had brains as opposed to hiv non-dementia patients .
interestingly, this difference was more prominent when severe dementia and non-dementia patients were compared , which indicates its significance in had pathogenesis.
map2k <dig> was recognized by jnk1/ <dig> antibody at  <dig> kda .
it was downregulated in the had brain as well .
there are  <dig> genes dysregulated in the calcium signalling pathway,  <dig> in jak-stat signalling pathway and  <dig> in vegf signalling pathway .
the details were listed in additional file  <dig>  additional file  <dig> and additional file  <dig>
it is worth mentioning that most of the core-enriched genes contributing to each individual gene set significantly enriched in gsea analysis fell into neurodegenerative disease related pathways, such as tight junction kegg pathway, neurodegenerative disease pathway, mapk signalling pathway, axon guidance pathway, and phosphorylative mechanisms/signalling pathway .
these results are consistent with our previous observations  <cit>  and functional annotation analysis, therefore further confirmed the significant involvement of these pathways in had pathogenesis.
for mirna, a number of significantly involved pathways  were revealed, including signalling pathways, adhesion/junction, axon guidance, depression/potentiation, apoptosis/cell cycle, inflammation-related pathways, ubiquitin mediated proteolysis, and regulation of actin cytoskeleton .
notable was that several pathways were targeted by more than  <dig> de mirnas.
for instance, the wnt-signalling pathway was targeted by  <dig> de mirnas, the axon guidance pathway and endocytosis pathway by  <dig> de mirnas, insulin signalling pathway, long-term potentiation pathway and focal adhesion pathway by  <dig> de mirnas.
interestingly, the de hsa-mir-19a targeted all  <dig> pathways listed above, whereas the de hsa-mir- <dig>  hsa-mir- <dig> and hsa-mir- <dig> targeted  <dig> pathways, and the de hsa-mir- <dig> and hsa-mir- <dig> targeted  <dig> pathways .
following the incorporation of mrna pathway and gsea analysis results, this is a highly comprehensive dataset in the context of neurodegeneration and pathogenesis.
in addition, we also found several cancer-related pathways significant as well, which were consistent with the fact that viruses can trigger or be co-factor of cancers and that a number of cancer genes are pro-inflammation, a scenario also seen in hiv infection and in neurodegenerative process, where hiv initiates cascade of pro-inflammatory mediators  and upregulation of their respective genes during infection  <cit> .
correlation between expression levels of de mirnas and de mrnas
we evaluated the significance levels of all possible correlations between de mrnas and mirnas using sa-bns  <cit> .
we found  <dig> interactions with high confidence in total.
among them,  <dig> were statistically significant , including  <dig> mirna and  <dig> mrna, whose expression levels correlated with each other according to pearson’s correlation  .
the pearson's correlation of mirna-mrna pairs vs. significant confidence of interaction discovered by sa-bns has been shown in figure  <dig>  interestingly, most of them correlated positively although mirnas were downregulated.
among them, hsa-mir- <dig>  hsa-mir- <dig>  hsa-mir-299-5p, hsa-mir- <dig> and hsa-mir-376a were outstanding due to their functional correlation with numerous genes.
it is interesting to see that most of the mirna are down regulated in had versus hiv non-demented brains, and positively correlated with their mrna target, which is supported by previous findings  <cit> .
to validate this correlation further, mirna mimic of mir- <dig> and negative control treatments were carried out.
that led a significant decrease in expression levels of nufib <dig>  slc, rnf, bag <dig>  spred, zranb at  <dig> h after transfection , which are all the genes negatively regulated by mir <dig> according to the correlation network we found.
this result added extra confidence to our correlation network.
discussion
this is the first joint study of whole-genome mrna and mirna profiling using individual human brain rna from autopsies of had and hiv non-dementia patients.
in this study, we initially compared mrna and mirna data at the clustering, gene ontology , and pathway levels.
following that, sa-bns correlating mirnas and mrnas by their expression levels were performed to validate the accurate prediction of genes potentially targeted by dysregulated mirnas.
the clustering and gene ontology results showed excellent functional concordance between mrna and mirna, demonstrating the significant involvement of neuronal cellular components and biological processes such as: signal transduction, transcriptional regulation, metabolism, response to stimuli, cell cycle/apoptosis, protein modification, neuronal processes and ion transport, respectively.
this intrinsic functional consistency between mirna and mrna has given an extra power to our findings in relation to understanding the genomic basis of hiv neuropathogenesis and hiv-mediated neurodegeneration.
moreover, the de mirnas were more robust than their mrna counterparts in providing a comprehensive snapshot of cellular components and biological processes involved in neuropathology and neurodegeneration.
compared to de mirnas, de mrnas could only predict elemental functional pathways and processes related to neuropathology.
de mirnas revealed the participation of additional cellular components  and biological processes , which also concurs with biological processes of mrna.
interestingly, these findings are consistent with study, which has been done using csf of hive patients  <cit> .
the most plausible explanation for the comprehensiveness of mirna coverage as compared to their mrna counterpart is that a single mirna or the mirnas belonging to the same family in the cluster can target several hundred genes within a biological process or pathway.
therefore, it is not surprising that mirna gives broader information compared to mrna.
secondly, there are many other gene regulational mechanisms apart from mirna and together they can compensate the effect of mirna dysregulation to some degree, consequently the mirna regulation effect can only be seen after certain stage of disease progression when other mechanisms are not sufficient any more  <cit> .
pathway analysis of the joint mrna and mirna results provided the first in vivo evidence of significant involvement of axon guidance pathway and its downstream signalling pathways  on both transcriptional level and regulation level.
axon guidance pledges precise path finding and defines their termination zones and synaptic partners  <cit> , which is fundamental to neuronal development and networks.
in addition, misrouted fibers have been shown in ad and pd’s brains  <cit> .
furthermore, it is well known that hiv envelope glycoprotein  can cause axonal degeneration  <cit>  and recently axon damage has been claimed as a key predictor of outcome in multiple neurological disorders, including had  <cit> .
axon guidance pathway contains four prominent families of ligands , their receptors and downstream signalling proteins.
the role of axon guidance pathway molecules in the maintenance and plasticity of neural circuits has been reported  <cit> .
moreover, the variations  in axon guidance pathway genes have been reported to predict pd outcomes  <cit> .
significantly,  <dig> of our de mirnas have been found targeting this pathway according to targetscan results; and more importantly these results support previous observations with  <dig> out of  <dig> ligands/receptors being dysregulated in our mrna studies, including ephrin receptor a <dig> , netrin g <dig> , and semaphoring 3a , strongly suggesting the impairment of axon guidance pathway in had brains in vivo.
moreover, our results highlight axon guidance downstream signalling pathways, which allow precise patterns of connectivity within the cns.
for instance, the mapk pathway comes out significant in both our mrna and mirna profiling.
studies have shown that the activation of mapk is necessary for axon guidance  <cit> , and it contributes to netrin signalling in axon guidance  <cit> .
besides, netrin-dependent axon outgrowth and orientation can be antagonized by inhibition of erk-1/ <dig>  <cit> .
the role of mapk pathway in hiv infection has also been well documented.
for instance, it has been reported that the mapk pathway plays a crucial role in hiv- <dig> replication and virulence  <cit>  and is one of the transcriptional signatures in hiv+ long-term non-progressors  <cit> .
in addition, the binding of hiv- <dig> gp <dig> to cd <dig> receptors on t cells can activate the mapk pathway and induce transcription of cytokine and chemokine genes  <cit> .
interestingly, mapk pathway was targeted by  <dig> de mirnas and it includes  <dig> of our de genes, such as rps6ka <dig>  flna, rras <dig>  and map2k <dig> etc., each of which play an important role in mapk signalling.
mapk signalling cross-talks with the jak-stat signalling pathway at multiple levels.
in mammals, the jak-stat signalling pathway is the principal signalling mechanism for cytokines and growth factors and therefore plays a key role in cell proliferation, differentiation, cell migration and apoptosis.
supporting this relationship, jak-stat was highlighted in our analysis as well.
we found  <dig> genes significantly dysregulated in that category  and it was targeted by de mir-19a.
dysregulation of jak signalling might result in inflammation  <cit> , which is commonly accepted as an important mediator in the pathogenesis of neurodegeneration.
vegf signalling pathway is another significant pathway revealed by our results, and it closely links to mapk signalling pathway as well.
via activating mapk signalling pathway, vegf can exert direct effect on multiple types of neuronal cells, including neurons, astrocytes, and microglias  <cit> .
vegf also has been reported to be involved in vascular permeability  <cit>  and several studies have shown the potential utility of inhibiting vegf signalling pathway in reducing bbb disruption  <cit> .
besides, ca2+ can mediate guidance receptor signalling in vitro <cit>  and change in ca2+ concentration can signal growth cone turning  <cit> .
equivalently, guidance cues can also trigger ca2+ influx and alteration in ca2+ concentration or slope its gradient, thereby influencing the outcome of growth cone behavior   <cit> .
our studies have demonstrated several genes related to ca2+ transport/signalling dysregulated, including atp2b <dig>  which play a critical role in intracellular calcium homeostasis.
in addition, endocytosis is another critical aspect of guidance receptor activation and signalling  <cit> .
nine of our de mirnas were found targeting this pathway and several key genes were found dysregulated.
efficient cell detachment needs the endocytosis of the ephrin-eph complex, or even bidirectional endocytosis for ephrinb-ephb-induced repulsive guidance.
in addition, endocytosis also plays a role in regulating the senstivity of the growth cone correspondent to a repulsive cue.
again,  <dig> of  <dig> of our de mirnas targeted endocytosis pathway.
our mrna study also revealed dysregulation of ras related protein  and ehd protein , which are important components of endocytosis pathway.
we also found adam <dig> dysregulated, whose family member adam <dig> has been reported to play a role in converting initial adhesive interaction into repulsion and therefore providing an effective strategy for axon detachment and attenuation of signalling  <cit> .
further, our mirna and mrna bayesian correlation analysis has provided an unambiguous snapshot of mirna and mrna functional interactions and their biological significance.
sophisticated bayesian structure learning approach defines mirna-mrna interactions based on their relative expression of all of these molecules in each condition.
this network-based approach identified these key interactions with very high confidence.
these interactions define the network topography that is provided by bayesian statistics and is substantially more rigorous than individual correlations that can be defined conventionally.
these relationships, therefore, are more likely to be meaningful at the system level compared to reporter assay.
we identified  <dig> positive and negative statistically significant correlations  between our de mrnas and mirnas.
it strengthens the correlation and shows the credibility of bayesian analysis we have performed for establishing this intrinsic functional relationship between mrna and mirna.
among them, two thirds of functional correlations are from  <dig> de mirnas: mir- <dig>   <dig>   <dig>  376a and 299-5p.
they all changed greater than  <dig> fold.
since all of them have been reported to be involved in classical neurodegeneration, for instance, mir-376a has been reported to mutate in huntington’s disease  brains  <cit> ; mir-299-5p has been reported to be dysregulated in multiple sclerosis  brains  <cit>  and more interestingly, all of them have been shown to be dysregulated in the ad brains  <cit> .
in particular, mir- <dig>   <dig> and  <dig>  which can target more than  <dig> neurodegeneration-related pathways, implying their functional relevance to the observations noted in this study.
the mir- <dig> has been shown to be enriched in neurons, especially within the dentate gyrus and the molecular layer of adult hippocampus  <cit>  and studies have shown that it plays an important role in modulating neuronal cell proliferation and differentiation  <cit> .
moreover, it has been shown to be genetically associated with schizophrenia  <cit> .
recently, it has been shown dysregulated in the csf of hive patients  <cit> , which is consistent with the expression of mirna- <dig> in the frontal cortex in our study in had patients.
in addition, the mir- <dig> is enriched in hippocampus  <cit>  and altered mir- <dig> expression has been reported in hd and ms brains  <cit> .
the mir- <dig> has been shown to play important role in ad and pd pathogenesis  <cit> .
it can downregulate the expression of app protein in vivo, suggesting its possible role in ad pathogenesis  <cit> .
moreover, it can regulate α-synuclein, which is the primary structural components of lewy bodies, indicating its role in pd pathophysiological process  <cit> .
the genes that correlated to are all involved in several significant pathways discussed above.
in particular, spred <dig>  map2k <dig> and diras <dig>  they all correlated with  <dig> out of  <dig> mirnas  and they all appear to be involved in mapk signalling pathway, which strongly indicate the participation of mapk pathway in had pathogenesis and is consistent with our previous proteomic studies  <cit> .
although our study is the first comprehensive parallel genome-wide mrna and mirna profiling of hiv infected human brains, there are still limitations:  <dig>  in future, a bigger sample size, blood and csf samples will be needed to further validate these findings, and confirm the clinical value of this findings;  <dig>  these findings are based on the whole human brain cortex rather than specific cell types  due to lack of plausible and effective methodologies for perfect cell separation.
although laser capture microdissection  is currently used in studying cell types, there are significant limitations in profiling single cell type  <cit> : a. due to the minimum laser spot size , it poses a limit to the precision of single cell micro-dissection without contaminating fragments of adjacent cells.
b. due to its suboptimal optical resolution on uncovered sections, it will compromise cell borders distinction and result in cytoplasmic compartment loss, which is crucial for our mrna analysis.
although immunohistochemical  staining will circumvent this problem to some degree, it is impossible to recover cytoplasmic compartment precisely without contamination.
moreover, ihc procedures, tissue fixation and lcm capturing of cells dramatically affect rna yield.
c. sectioning will generate large number of attached fragments, which might alter expression profiles greatly.
in addition, due to the lack of the cytoplasm or even the nucleus, the genomic information will be considerably compromised.
overall, our study provides a strong foundation and durable framework for systematic large-scale studies on hiv-infected adult brain to define functional genomic phenotypes of neurodegenerative diseases and functional networks between mirna and mrna, which may lead to the development of new generation of prognostic and diagnostic markers and therapeutic intervention strategies for viral and non-viral neurodegenerative diseases.
CONCLUSIONS
this study is the first report on whole-genome joint mrna and mirna profile analysis from individual native brain tissue from hiv+ patients with and without dementia and it underscores the important role of intrinsic functional correlation between mrna-mirna, which is closely tied to hiv-mediated neurodegeneration.
through mrna and mirna joint profiling this study has provided the first thorough in vivo evidence on the genomic basis of hiv-mediated neurodegeneration and its correlation with mirna.
this provided a firm support to intrinsic functional relationship that exists between mrna and mirna in guiding neurodegeneration in hiv-infected brains.
from the concordance between mirna and mrna, it demonstrates the significant involvement of axon guidance and its downstream signalling pathways in hiv-mediated neurodegeneration and development of had.
most importantly, the most significant dysregulated and highly biological relevant  <dig> mirnas identified here, mir- <dig>   <dig> and  <dig>  cumulatively targeted the axon guidance pathway as well as its downstream signalling pathways, which may find potential use as diagnostic/prognostic biomarkers and for developing new generation of therapeutic intervention strategies for hiv-associated and possibly other neurodegenerative diseases.

 hiv-associated dementia is the most common dementia type in young adults less than years of age. to date, the cumulative incidence of had is 25-38%, and the prevalence is around 37%. although the highly active antiretroviral therapy has had considerable success in preventing virus-mediated immune collapse end-stage complications, the prevalence of hiv-associated cognitive impairment appears to be on the rise due to the increased life span of hiv+ population. it is well recognized that host-virus interactions play a crucial role in the occurrence and pathogenesis of had. microarray and high throughput genomic technologies have greatly facilitated the examination of this interaction. a panoply of host genes have been shown to be influenced by hiv infection that facilitate subversion and manipulation of the host immune system during hiv infection of the brain. to date, most studies exploring had pathogenesis have largely been confined to cultured cells, cerebrospinal fluid and animal models, which can’t mimic and reveal the breadth of in vivo cellular responses subverted and manipulated as a consequence of hiv infection. recently, micrornas, a class of small non-coding rnas, have been recognized as master post-transcriptional regulators of mrnas due to their numerous targeting capabilities along with their ability to form non-linear functionally viable gene regulatory networks, which together have wide-ranging effects on the control of host gene expression. in addition, mirnas are also involved in diverse processes, which include neuronal development, cell differentiation, synapse formation and neuronal plasticity. thus, not surprisingly, mirnas are significantly involved in neurodegenerative diseases, such as parkinson disease, alzheimer’s disease, schizophrenia, aggressive behaviour and depression. further, mirnas also play an important role in hiv-host interaction. hiv can even manipulate the expression of host neuronal genes, such as snap and cell death-related genes, including caspase- via the modulation of expression of selected micrornas, which might serve as key elements in gene regulatory networks in hiv-associated neurobehavioral disorder. to date, a joint study of the genome wide mrna and mirna profiling based on hiv-infected brain tissue with and without dementia is still lacking, although some individual mrna and mirna profiling studies based on human brain have been done. therefore, our innovative approach has studied the utility of parallel genome-wide mrna and mirna analysis in the native frontal lobe post-mortem brain tissue from hiv patients with and without dementia. this carries enormous value in understanding gene expression in the context of hiv-mediated neurodegeneration and its regulation through mirnas. this study has been designed with an objective to comprehensively delineate host transcriptional programming that occurs in concert with the regulatory mirnas and to find how this interaction between mrna and mirna tampers with neurodegenerative pathways and dictates neurological manifestation of hiv disease. here, we examined the gene ontology and pathways of differentially expressed mrna in parallel with the global gene targets of de mirnas. moreover, we derived paired functional correlation between mrna and mirna expressions using splitting-averaging strategy to demonstrate intrinsic functional relationship between gene expression and its regulation. in light of these data we believe that these results will not only facilitate a greater understanding of hiv pathogenesis of the brain and its neurological manifestation, but will also help define potential candidates for early detection and future therapy for neurodegeneration in hiv patients and related disorders.  a snapshot of de mrna and mirna profiles between had and hiv non-demented brains in order to identify the mrna and mirna, which may contribute to the pathogenesis of had, a parallel genome-wide mrna and mirna profiling of the frontal cortex from hiv patients with and without dementia was performed. genomestudio was used to analyse the normalized mrna dataset. we observed statistically significant candidate genes that were differentially expressed between two groups. among them, genes were down regulated and genes were up regulated, and of genes dysregulated greater than -fold change. to determine the similarity of global gene expression between samples and also in relation to the de genes, hierarchical clustering was performed and generated using genomestudio. the had group formed an independent cluster away from the hiv non-dementia group, with the exception of one sample with dementia which clustered together with the hiv non-dementia group. not surprisingly, the hiv non-dementia group clustered together with hiv negative control due to the absence of neuropathological changes and the absence of actively replicating hiv, which is consistent with a previous study and our own study. the analysis of mirna data using genespring identified mirna that were significantly differentially expressed in had and hiv non-dementia cortex. among them, mirnas were down regulated and were upregulated. 27/ mirnas were dysregulated greater than fold. hierarchical clustering was carried out to demonstrate patterns of mirna expression profiling between two groups, which was consistent with our mrna hierarchical clustering. targetscan was used to predict the gene targets of de mirnas, because it is the most advanced, respected, widely used and relatively conservative database in comparison to other databases. in addition, we have carried out a low and high stringency g-seed search for mir- mir- and mir- targets based on a minimal free energy ¾ - and ¾- respectively. furthermore, these predictions were made in the context of changes in gene expression observed in the same rna. functional annotation of the mrna de genes and mirna target genes was carried out in database for annotation, visualization and integrated discovery together with extensive literature search. these mrna de genes were significantly associated with biological processes, such as cell death/cell cycle, neuronal processes, metabolism, transcriptional regulation, protein modification, signal transduction, and response to virus/stress, as shown in figure 1a. in addition, according to cellular components distribution, 29% of genes fell into neuronal-related components, such as axon, neuron projection, and dendrite. furthermore, gene set enrichment analysis was also used to examine significantly enriched go gene sets comparing the normalized data of the entire gene transcripts from had and hiv non-dementia brains to go gene sets in gsea molecular signatures database. eight gene sets were found statistical significantly enriched in hiv non-dementia group while no gene sets were significantly enriched in the had group. of the eight enriched gene sets in the non-dementia group, four were closely related to neurological and/or hiv disease: dendrite, atpase activity coupled to transmembrane movement of ions, atpase activity coupled to transmembrane movement of ions phosphorylative mechanism, and cytoskeleton dependent intracellular transport. functional annotation results of mirna target genes were consistent with that of mrna results, but noteworthy is that the mirna target genes showed relatively more comprehensive biological processes and cellular components, which could be attributed to the ability of single mirna to have numerous mrna targets, therefore further validation might be needed to precise the specificity. cellular components of mirna target genes are mainly distributed in cellular and neuronal related-structures, but they display scattering into ion channel, actin cytoskeleton, tight junction, transcription factor complex and chromatin, which concur with our mrna results. for instance, we found significant dysregulation of genes in microtubule assembly, microtubule nucleation, cytoskeleton movement and microtubule stabilization. in regards to ion channel genes, we found the upregulation of genes related to ion transport or ion channel, including ca2+, cl-, na+, k+, cu2+, zn2+ and glutamine transport. especially, the genes involved in ca2+, na+ and glutamine transport changed greater than fold, the dysregulation of which is known to play a significant role in pathophysiology of neurodegenerative diseases as discussed in subsequent sections. we also found more than genes dysregulated in transcriptional regulation process, and similarly, the biological process of small percentage of mirna target genes fall into cell adhesion, tissue/embryonic development, learning, and chromatin modification etc.. although they were not dominant, they all can play a very important role in neurological degeneration. quantitative real time rt-pcr corroboration of mrna and mirna array dataset in order to confirm and validate the data obtained by both data sets, we analysed de genes and de mirnas and non-changed mirna using quantitative real time rt-pcr on the same study sample set. for mrna, the quantitative real time rt-pcr for of the genes was fully consistent with their microarray expression profiles and trends. the two genes for which the rt-pcr was not consistent with the microarray were excluded from further analysis. this, in no way, compromises the interpretation of our dataset. for mirna, the quantitative rt-pcr for of mirnas was consistent with the trend seen in microarray analysis, which enhances the confidence and shows the validity of our mirna data set and their gene targets defined herein. pathway analysis of de mrnas and mirnas de gene list and de mirna target gene list were uploaded to david to explore functionally relevant pathways. for mrna, long-term potentiation, axon guidance and signalling pathways stood out significantly. not surprisingly, we found genes significantly dysregulated in long-term potentiation pathway. among them, itpr and ppp3ca down regulated greater than -fold change. axon guidance pathway was significantly down regulated in had versus non-demented patients with down regulated genes. among them, epha was down regulated > fold, while plxnd srgap ppp3ca were down regulated > fold. there were a total of genes involved in signalling pathway. we found that all the 12/ genes in the mapk pathway were down regulated, including the key members of the mapk signalling pathway, such as map2k map2k map3k rras rps6ka and taok among them, taok was down regulated greater than fold, while map2k map3k pla2g4a and rras was dysregulated greater than fold. two key proteins in the mapk pathway, map2k and map2k, were also further validated by western blotting analysis using samples from had and samples from hiv non-dementia group, respectively. both proteins showed down-regulation in had brains, which followed the same trend observed in our microarray analysis. mek was recognized by p-mek-1/ antibody at kda. it was expressed slightly weaker in had brains as opposed to hiv non-dementia patients. interestingly, this difference was more prominent when severe dementia and non-dementia patients were compared, which indicates its significance in had pathogenesis. map2k was recognized by jnk1/ antibody at kda. it was downregulated in the had brain as well. there are genes dysregulated in the calcium signalling pathway, in jak-stat signalling pathway and in vegf signalling pathway. the details were listed in additional file additional file and additional file it is worth mentioning that most of the core-enriched genes contributing to each individual gene set significantly enriched in gsea analysis fell into neurodegenerative disease related pathways, such as tight junction kegg pathway, neurodegenerative disease pathway, mapk signalling pathway, axon guidance pathway, and phosphorylative mechanisms/signalling pathway. these results are consistent with our previous observations and functional annotation analysis, therefore further confirmed the significant involvement of these pathways in had pathogenesis. for mirna, a number of significantly involved pathways were revealed, including signalling pathways, adhesion/junction, axon guidance, depression/potentiation, apoptosis/cell cycle, inflammation-related pathways, ubiquitin mediated proteolysis, and regulation of actin cytoskeleton. notable was that several pathways were targeted by more than de mirnas. for instance, the wnt-signalling pathway was targeted by de mirnas, the axon guidance pathway and endocytosis pathway by de mirnas, insulin signalling pathway, long-term potentiation pathway and focal adhesion pathway by de mirnas. interestingly, the de hsa-mir-19a targeted all pathways listed above, whereas the de hsa-mir- hsa-mir- and hsa-mir- targeted pathways, and the de hsa-mir- and hsa-mir- targeted pathways. following the incorporation of mrna pathway and gsea analysis results, this is a highly comprehensive dataset in the context of neurodegeneration and pathogenesis. in addition, we also found several cancer-related pathways significant as well, which were consistent with the fact that viruses can trigger or be co-factor of cancers and that a number of cancer genes are pro-inflammation, a scenario also seen in hiv infection and in neurodegenerative process, where hiv initiates cascade of pro-inflammatory mediators and upregulation of their respective genes during infection. correlation between expression levels of de mirnas and de mrnas we evaluated the significance levels of all possible correlations between de mrnas and mirnas using sa-bns. we found interactions with high confidence in total. among them, were statistically significant, including mirna and mrna, whose expression levels correlated with each other according to pearson’s correlation. the pearson's correlation of mirna-mrna pairs vs. significant confidence of interaction discovered by sa-bns has been shown in figure interestingly, most of them correlated positively although mirnas were downregulated. among them, hsa-mir- hsa-mir- hsa-mir-299-5p, hsa-mir- and hsa-mir-376a were outstanding due to their functional correlation with numerous genes. it is interesting to see that most of the mirna are down regulated in had versus hiv non-demented brains, and positively correlated with their mrna target, which is supported by previous findings. to validate this correlation further, mirna mimic of mir- and negative control treatments were carried out. that led a significant decrease in expression levels of nufib slc, rnf, bag spred, zranb at h after transfection, which are all the genes negatively regulated by mir according to the correlation network we found. this result added extra confidence to our correlation network. discussion this is the first joint study of whole-genome mrna and mirna profiling using individual human brain rna from autopsies of had and hiv non-dementia patients. in this study, we initially compared mrna and mirna data at the clustering, gene ontology, and pathway levels. following that, sa-bns correlating mirnas and mrnas by their expression levels were performed to validate the accurate prediction of genes potentially targeted by dysregulated mirnas. the clustering and gene ontology results showed excellent functional concordance between mrna and mirna, demonstrating the significant involvement of neuronal cellular components and biological processes such as: signal transduction, transcriptional regulation, metabolism, response to stimuli, cell cycle/apoptosis, protein modification, neuronal processes and ion transport, respectively. this intrinsic functional consistency between mirna and mrna has given an extra power to our findings in relation to understanding the genomic basis of hiv neuropathogenesis and hiv-mediated neurodegeneration. moreover, the de mirnas were more robust than their mrna counterparts in providing a comprehensive snapshot of cellular components and biological processes involved in neuropathology and neurodegeneration. compared to de mirnas, de mrnas could only predict elemental functional pathways and processes related to neuropathology. de mirnas revealed the participation of additional cellular components and biological processes, which also concurs with biological processes of mrna. interestingly, these findings are consistent with study, which has been done using csf of hive patients. the most plausible explanation for the comprehensiveness of mirna coverage as compared to their mrna counterpart is that a single mirna or the mirnas belonging to the same family in the cluster can target several hundred genes within a biological process or pathway. therefore, it is not surprising that mirna gives broader information compared to mrna. secondly, there are many other gene regulational mechanisms apart from mirna and together they can compensate the effect of mirna dysregulation to some degree, consequently the mirna regulation effect can only be seen after certain stage of disease progression when other mechanisms are not sufficient any more. pathway analysis of the joint mrna and mirna results provided the first in vivo evidence of significant involvement of axon guidance pathway and its downstream signalling pathways on both transcriptional level and regulation level. axon guidance pledges precise path finding and defines their termination zones and synaptic partners, which is fundamental to neuronal development and networks. in addition, misrouted fibers have been shown in ad and pd’s brains. furthermore, it is well known that hiv envelope glycoprotein can cause axonal degeneration and recently axon damage has been claimed as a key predictor of outcome in multiple neurological disorders, including had. axon guidance pathway contains four prominent families of ligands, their receptors and downstream signalling proteins. the role of axon guidance pathway molecules in the maintenance and plasticity of neural circuits has been reported. moreover, the variations in axon guidance pathway genes have been reported to predict pd outcomes. significantly, of our de mirnas have been found targeting this pathway according to targetscan results; and more importantly these results support previous observations with out of ligands/receptors being dysregulated in our mrna studies, including ephrin receptor a, netrin g, and semaphoring 3a, strongly suggesting the impairment of axon guidance pathway in had brains in vivo. moreover, our results highlight axon guidance downstream signalling pathways, which allow precise patterns of connectivity within the cns. for instance, the mapk pathway comes out significant in both our mrna and mirna profiling. studies have shown that the activation of mapk is necessary for axon guidance, and it contributes to netrin signalling in axon guidance. besides, netrin-dependent axon outgrowth and orientation can be antagonized by inhibition of erk-1/. the role of mapk pathway in hiv infection has also been well documented. for instance, it has been reported that the mapk pathway plays a crucial role in hiv- replication and virulence and is one of the transcriptional signatures in hiv+ long-term non-progressors. in addition, the binding of hiv- gp to cd receptors on t cells can activate the mapk pathway and induce transcription of cytokine and chemokine genes. interestingly, mapk pathway was targeted by de mirnas and it includes of our de genes, such as rps6ka flna, rras and map2k etc., each of which play an important role in mapk signalling. mapk signalling cross-talks with the jak-stat signalling pathway at multiple levels. in mammals, the jak-stat signalling pathway is the principal signalling mechanism for cytokines and growth factors and therefore plays a key role in cell proliferation, differentiation, cell migration and apoptosis. supporting this relationship, jak-stat was highlighted in our analysis as well. we found genes significantly dysregulated in that category and it was targeted by de mir-19a. dysregulation of jak signalling might result in inflammation, which is commonly accepted as an important mediator in the pathogenesis of neurodegeneration. vegf signalling pathway is another significant pathway revealed by our results, and it closely links to mapk signalling pathway as well. via activating mapk signalling pathway, vegf can exert direct effect on multiple types of neuronal cells, including neurons, astrocytes, and microglias. vegf also has been reported to be involved in vascular permeability and several studies have shown the potential utility of inhibiting vegf signalling pathway in reducing bbb disruption. besides, ca2+ can mediate guidance receptor signalling in vitro and change in ca2+ concentration can signal growth cone turning. equivalently, guidance cues can also trigger ca2+ influx and alteration in ca2+ concentration or slope its gradient, thereby influencing the outcome of growth cone behavior. our studies have demonstrated several genes related to ca2+ transport/signalling dysregulated, including atp2b which play a critical role in intracellular calcium homeostasis. in addition, endocytosis is another critical aspect of guidance receptor activation and signalling. nine of our de mirnas were found targeting this pathway and several key genes were found dysregulated. efficient cell detachment needs the endocytosis of the ephrin-eph complex, or even bidirectional endocytosis for ephrinb-ephb-induced repulsive guidance. in addition, endocytosis also plays a role in regulating the senstivity of the growth cone correspondent to a repulsive cue. again, of of our de mirnas targeted endocytosis pathway. our mrna study also revealed dysregulation of ras related protein and ehd protein, which are important components of endocytosis pathway. we also found adam dysregulated, whose family member adam has been reported to play a role in converting initial adhesive interaction into repulsion and therefore providing an effective strategy for axon detachment and attenuation of signalling. further, our mirna and mrna bayesian correlation analysis has provided an unambiguous snapshot of mirna and mrna functional interactions and their biological significance. sophisticated bayesian structure learning approach defines mirna-mrna interactions based on their relative expression of all of these molecules in each condition. this network-based approach identified these key interactions with very high confidence. these interactions define the network topography that is provided by bayesian statistics and is substantially more rigorous than individual correlations that can be defined conventionally. these relationships, therefore, are more likely to be meaningful at the system level compared to reporter assay. we identified positive and negative statistically significant correlations between our de mrnas and mirnas. it strengthens the correlation and shows the credibility of bayesian analysis we have performed for establishing this intrinsic functional relationship between mrna and mirna. among them, two thirds of functional correlations are from de mirnas: mir- 376a and 299-5p. they all changed greater than fold. since all of them have been reported to be involved in classical neurodegeneration, for instance, mir-376a has been reported to mutate in huntington’s disease brains; mir-299-5p has been reported to be dysregulated in multiple sclerosis brains and more interestingly, all of them have been shown to be dysregulated in the ad brains. in particular, mir- and which can target more than neurodegeneration-related pathways, implying their functional relevance to the observations noted in this study. the mir- has been shown to be enriched in neurons, especially within the dentate gyrus and the molecular layer of adult hippocampus and studies have shown that it plays an important role in modulating neuronal cell proliferation and differentiation. moreover, it has been shown to be genetically associated with schizophrenia. recently, it has been shown dysregulated in the csf of hive patients, which is consistent with the expression of mirna- in the frontal cortex in our study in had patients. in addition, the mir- is enriched in hippocampus and altered mir- expression has been reported in hd and ms brains. the mir- has been shown to play important role in ad and pd pathogenesis. it can downregulate the expression of app protein in vivo, suggesting its possible role in ad pathogenesis. moreover, it can regulate α-synuclein, which is the primary structural components of lewy bodies, indicating its role in pd pathophysiological process. the genes that correlated to are all involved in several significant pathways discussed above. in particular, spred map2k and diras they all correlated with out of mirnas and they all appear to be involved in mapk signalling pathway, which strongly indicate the participation of mapk pathway in had pathogenesis and is consistent with our previous proteomic studies. although our study is the first comprehensive parallel genome-wide mrna and mirna profiling of hiv infected human brains, there are still limitations: in future, a bigger sample size, blood and csf samples will be needed to further validate these findings, and confirm the clinical value of this findings; these findings are based on the whole human brain cortex rather than specific cell types due to lack of plausible and effective methodologies for perfect cell separation. although laser capture microdissection is currently used in studying cell types, there are significant limitations in profiling single cell type: a. due to the minimum laser spot size, it poses a limit to the precision of single cell micro-dissection without contaminating fragments of adjacent cells. b. due to its suboptimal optical resolution on uncovered sections, it will compromise cell borders distinction and result in cytoplasmic compartment loss, which is crucial for our mrna analysis. although immunohistochemical staining will circumvent this problem to some degree, it is impossible to recover cytoplasmic compartment precisely without contamination. moreover, ihc procedures, tissue fixation and lcm capturing of cells dramatically affect rna yield. c. sectioning will generate large number of attached fragments, which might alter expression profiles greatly. in addition, due to the lack of the cytoplasm or even the nucleus, the genomic information will be considerably compromised. overall, our study provides a strong foundation and durable framework for systematic large-scale studies on hiv-infected adult brain to define functional genomic phenotypes of neurodegenerative diseases and functional networks between mirna and mrna, which may lead to the development of new generation of prognostic and diagnostic markers and therapeutic intervention strategies for viral and non-viral neurodegenerative diseases.  this study is the first report on whole-genome joint mrna and mirna profile analysis from individual native brain tissue from hiv+ patients with and without dementia and it underscores the important role of intrinsic functional correlation between mrna-mirna, which is closely tied to hiv-mediated neurodegeneration. through mrna and mirna joint profiling this study has provided the first thorough in vivo evidence on the genomic basis of hiv-mediated neurodegeneration and its correlation with mirna. this provided a firm support to intrinsic functional relationship that exists between mrna and mirna in guiding neurodegeneration in hiv-infected brains. from the concordance between mirna and mrna, it demonstrates the significant involvement of axon guidance and its downstream signalling pathways in hiv-mediated neurodegeneration and development of had. most importantly, the most significant dysregulated and highly biological relevant mirnas identified here, mir- and cumulatively targeted the axon guidance pathway as well as its downstream signalling pathways, which may find potential use as diagnostic/prognostic biomarkers and for developing new generation of therapeutic intervention strategies for hiv-associated and possibly other neurodegenerative diseases. 
these data provide an excellent avenue for the development of new generation of diagnostic/prognostic biomarkers and therapeutic intervention strategies for hiv-associated neurodegeneration. the gene ontology and pathway analysis of the two data sets showed high concordance between mirna and mrnas, revealing significant interference with the host axon guidance and its downstream signalling pathways in had brains. although the neurotoxins, oxidative/metabolic stress and impaired activity of neurotrophic factors are believed to be underlying reasons for the development of had, the genomic basis, which ultimately defines the virus-host interaction and leads to neurologic manifestation of hiv disease is lacking. furthermore, this relationship between de mirnas and de mrnas was also reflected in correlation analysis using bayesian networks by splitting-averaging strategy, which revealed statistically significant correlated mirna-mrna pairs according to pearson’s correlation test. our study provides the first evidence on unambiguous support for intrinsic functional relationship between mrna and mirna in the context of hiv-mediated neurodegeneration, which shows that neurologic manifestation in hiv patients possibly occurs through the interference with the host axon guidance and its downstream signalling pathways. hiv-associated dementia is the most common dementia type in young adults less than years of age. moreover, the differentially expressed mirnas identified in this study, in particular mir- and based on which most correlations were built cumulatively targeted neurodegeneration related pathways, implying their future potential in diagnosis, prognosis and possible therapies for hiv-mediated and possibly other neurodegenerative diseases.
 hiv-associated dementia is the most common dementia type in young adults less than years of age. although the neurotoxins, oxidative/metabolic stress and impaired activity of neurotrophic factors are believed to be underlying reasons for the development of had, the genomic basis, which ultimately defines the virus-host interaction and leads to neurologic manifestation of hiv disease is lacking. therefore, identifying hiv fingerprints on the host gene machinery and its regulation by microrna holds a great promise and potential for improving our understanding of had pathogenesis, its diagnosis and therapy.  a parallel profiling of mrna and mirna of the frontal cortex autopsies from hiv positive patients with and without dementia was performed using illumina human- beadchip and affymetrix version mirna array, respectively. the gene ontology and pathway analysis of the two data sets showed high concordance between mirna and mrnas, revealing significant interference with the host axon guidance and its downstream signalling pathways in had brains. moreover, the differentially expressed mirnas identified in this study, in particular mir- and based on which most correlations were built cumulatively targeted neurodegeneration related pathways, implying their future potential in diagnosis, prognosis and possible therapies for hiv-mediated and possibly other neurodegenerative diseases. furthermore, this relationship between de mirnas and de mrnas was also reflected in correlation analysis using bayesian networks by splitting-averaging strategy, which revealed statistically significant correlated mirna-mrna pairs according to pearson’s correlation test.  our study provides the first evidence on unambiguous support for intrinsic functional relationship between mrna and mirna in the context of hiv-mediated neurodegeneration, which shows that neurologic manifestation in hiv patients possibly occurs through the interference with the host axon guidance and its downstream signalling pathways. these data provide an excellent avenue for the development of new generation of diagnostic/prognostic biomarkers and therapeutic intervention strategies for hiv-associated neurodegeneration. hiv-associated dementianeurodegenerationhivmicroarraymicrornaaxon guidance 