BACKGROUND
aristolochic acid  is found in plants of the genus aristolochia and asarum  <cit> .
use of dietary supplements and other botanical products containing aa has caused severe nephrotoxicity and consequent renal replacement therapy  <cit> .
animal studies show that aa results in renal failure in rodents and induces tumors in the kidney and other tissues of rabbits, rats and mice  <cit> .
aa is among the most potent 2% of the carcinogens in the carcinogenic potency and genotoxicity databases.
as a result, the u.s. food and drug administration  issued a consumer advisory in  <dig> warning consumers against using dietary supplements and other botanical products containing aa, and the fda also requested a recall of these products and published a list of botanical products that contained aa.
however, products containing aa have not been banned in the us or many other countries.
aa-induced carcinogenesis has been attributed to the mutagenicity and dna adducts formed in the kidney and other tissues of aa nephropathy patients.
on the other hand, aa induced similar dna adduct formation in both the kidney and liver of mice, but tumors preferentially occurred in kidney  <cit> .
this suggests that in addition to the genetic alterations induced by aa, alternative mechanisms such as epigenetic remodeling and mirna  modulation might also play an important role in aa-induced cancers.
mirnas represent a class of non-coding small rna  that are ubiquitously present in different kinds of organisms from c.elegans to mammals  <cit> .
mirnas are involved in the post-transcriptional regulation of gene expression via binding to the 3’ utr region of target mrnas, resulting in mrna degradation or translation inhibition  <cit> .
each mirna usually targets multiple, even hundreds of mrnas  <cit> .
it is believed that one third of human genes are subject to mirna control.
mirnas regulate a variety of developmental and physiological processes, including control of leaf and flower development in plants  <cit>  and neuronal patterning in nematodes  <cit> .
recent studies indicate that mirnas are involved in the regulation of pathways that are associated with the initiation and progression of many types of tumors  <cit> .
mirna expression was found to accurately identify the tissue origin of cancers, including distal metastatic colonies of unknown primary origin  <cit> .
mirnas affect tumor metastasis, such as the lung and bone metastasis of human breast cancer  <cit> .
moreover, mirnas are associated with chemical carcinogenesis and regulate gene expression that is important in every stage  <cit> .
deep sequencing, also known as next generation sequencing, has undergone tremendous acceptance in the past few years.
by sequencing dna or rna in a massively parallel fashion, deep sequencing technologies dramatically reduce both the cost-per-base and time required to decode an entire genome or transcriptome  <cit> , making sequencing a cost-effective option for many experimental approaches.
moreover, it has very low, if any, background signal, and does not have an upper limit for quantification, resulting in a large dynamic range of expression levels over which transcripts can be detected  <cit> .
toxicoproteomics is a new discipline that applies proteomics concepts and approaches to toxicological studies.
it can elucidate pathological responses to a specific toxicant at the protein molecule level, including qualitative and quantitative measurements of protein expression, protein modifications, and protein-protein/toxicant interactions  <cit> .
a primary research objective of mirna biology is to understand the post-transcriptional regulation of the target genes and the effect on biological functions.
however, due to the different mechanisms of mirna regulation, the targets can be translationally repressed with a concordant decrease in mrna abundance or without significant mrna degradation, or have decreased mrna abundance with little protein change  <cit> .
moreover, mirnas can switch from repression to activation depending on different stages of the cell cycle  <cit> .
therefore, in order to understand mirna regulation in aa-induced kidney tumorigenesis, we integrated deep sequencing data for global mirna and mrna expression with proteomics data for global protein expression in conjunction with target identification of dysregulated mirnas from the mirtarbase  <cit> .
compared with using mirna target prediction tools such as miranda and targetscan  <cit> , we selected those targets that were published and validated to study their mirna transcriptional and translational regulations  <cit> , thus providing a novel approach to enhance our understanding of the biological role of mirna in aa-induced carcinogenesis in rat kidney.
the details of the proteomics data from rats treated with aa were reported separately  <cit> .
this work will contribute to our understanding of the biological processes driving aa-induced carcinogenesis at the mirna, mrna and protein levels as well as representing a generalizable strategy that can be extended to study the functional role of mirnas and their regulation of target expression in organs or tissues of interest.
RESULTS
aa treatment resulted in functionally differential mirna expressions in rat kidney
dysregulated mirna expression has been linked to cancer in experimental animal models and patients  <cit> .
to investigate the effects of aa exposure on mirna expression under conditions that resulted in kidney tumors, we treated  <dig> rats for 12 weeks with aa along with  <dig> control rats treated with vehicle, following a carcinogenetic protocol that has been demonstrated to result in kidney tumors  <cit> .
all the rats were sacrificed one day after the last treatment and the kidneys were removed for mirna deep sequencing analysis.
the short reads were mapped to sanger mirbase   <cit> .
there were  <dig> detectable mirnas in at least one of the  <dig> samples.
first, classification analyses were conducted to explore global alteration of mirnas by the aa treatment.
hierarchical clustering analysis  was performed to globally view the groups among the  <dig> samples in terms of the expression profile of all the detectable mirnas.
these samples are divided into two branches, one comprises  <dig> aa-treated samples and the other contains the  <dig> control samples.
this indicates that mirnas have similar and consistent expression patterns within each group, but are distinct from each other, demonstrating the profound impact of aa treatment on mirna expression in rat kidney .
the same phenomenon was revealed by the principal component analysis  analysis, further supporting that mirna expression is profoundly changed by aa treatment at the genome scale.figure  <dig> identification of differentially expressed mirna and mrna in kidney in rat treated with aa.
a. classification of samples according to expression intensity of the  <dig> detectable mirnas.
hierarchical clustering analysis separates the  <dig> samples into two groups that are consistent with control and the treatment groups.
principal component analysis groups samples into the aa- treated and control clusters.
b. classification of  <dig> samples according to expression level of the  <dig> detectable mrnas.
hierarchical clustering analysis separates the  <dig> samples into two groups that are consistent with the control and treatment groups.
principal component analysis groups samples into the aa- treated and control clusters.
next, we investigated which mirnas were significantly dysregulated by aa treatments.
all the  <dig> detectable mirnas were used to find differentially expressed mirnas .
read count normalization, statistical analysis and p value adjustment were performed using the deseq methods.
sixty-three mirnas showed significant changes in their expression level by aa treatment using a cutoff with adjusted p values of <  <dig>  and fold change of >  <dig>  .
more up-regulated mirnas  were observed than down-regulated mirnas .rno-mir- <dig>  rno-mir- <dig>  mir-741-3p, mir-511*, mir- <dig>  mir-449a, as well as  <dig> members of mir- <dig> family, mir-34a, mir-34a*, mir-34b, mir-34b*, mir-34c, and mir-34c*, showed over 10-fold up-regulation.
in contrast, only one mirna, mir- <dig>  showed more than 10-fold down-regulation.
moreover, the oncogenic mirna, mir- <dig> was increased  <dig>  fold, whereas the tumor suppressor mirnas, let-7e, mir-135a, and mir- <dig> were decreased by  <dig> ,  <dig>  and  <dig>  fold, respectively.
in order to validate the changes in mirna expression detected by a deep-sequencing, we conducted quantitative real-time pcr analysis to examine expression levels of six mirnas including rno-mir- <dig>  rno-mir- <dig>  rno-mir- <dig>  rno-mir-34a, rno-mir-34b, and rno-mir34c.
the results from the real-time pcr analysis confirmed the sequencing data .
finally,  <dig> mirna target proteins that were dysregulated in rat kidney were inputted into the ingenuity database to explore the underlying biological and toxicological functions.
the biological function analysis showed that cancer was the top functional category significantly related to these mirnas, along with organismal injury and abnormalities, which also could result from aa treatment.
in total,  <dig> of the  <dig> mirnas were cancer-related .table  <dig>
ipa analysis of  <dig> dysregulated mirnas in rat kidney treated with aa
top networks
top diseases and functions
differentially expressed genes  from mrna sequencing data and their functions
mirnas regulate the homeostatic level of their target mrnas.
to investigate whether mrna expression was also changed by aa treatments, the  <dig> rat kidney samples were analyzed by mrna deep sequencing analysis in parallel.
the short reads were mapped to ensembl transcripts , and a data processing procedure similar to that used for the mirnas was applied to normalize the raw counts, and to calculate adjusted p values.
there were  <dig> mrnas that had detectable expression in these  <dig> samples.
as with mirna expression in rat kidney, the expression patterns of mrna were similar within the treatment or control group alone, but distinctly between the two groups in both hca and pca analysis .
a total of  <dig> richness mrnas were found to be differentially expressed based on a cutoff of p <  <dig>  and fold change >  <dig>  with 4051upregulated and  <dig> down-regulated.
ingenuity pathway analysis  of both up-regulated and down-regulated genes identified cancer as the top disease and biological function with  <dig> up-regulated and  <dig> down-regulated molecules included .
the top canonical pathways included wnt/β-actin, mitochondrial dysfunction and valine degradation.
.
an integrated multiple profiling reveals a comprehensive regulation of mirnas on their target expression
in order to search for the targets of its differentiately regulated mirnas and understand effects of the post-transcriptional regulation of mirna on gene expression in aa-induced carcinogenesis, we combined proteomic and transcriptomic data to study expression of the mirna targets that are validated and reported in mirtarbase .
this database provides the most current and comprehensive information on experimentally validated mirna-target interactions and data resources with published studies on the identification of mirna targets, molecular networks of mirna targets and systems biology.
specifically, it contains  <dig> mirna targets that are validated by one or more solid experimental methods including luciferase reporter assay, western blot, northern blot, and quantitative real-time analysis.
if a gene is targeted by multiple mirnas, the log <dig> transformed fold changes of these mirnas were summed together as the fold changes of the mirnas.
we initially conducted correlation analysis between the mirnas vs. mrnas, mirnas vs. proteins, and mrnas vs. proteins in rat kidney treated with aa in relation to the control.
there was a very weak correlation between expression of mrnas versus proteins , and mirnas vs. proteins , but there was no correlation between mirna vs. mrna expression  , indicating that regulation by mirnas on their target genes can be attributed to multiple mechanisms.
next, we divided our study into two groups including group a, the up-regulated mirnas and group b, the down-regulated mirnas, and used an adjusted p <  <dig>  and fold changes of < - <dig>  or >  <dig>  as a cutoff, indicating the difference for all these three variables.
according to the response of gene expression in group a, we separated our study into three subgroups: 1) mrnas and proteins expressed in the same direction including up-regulated , down-regulated , not changed .
2) mrnas and proteins expressed in the opposite direction including  <dig> down-regulated mrnas vs. up-regulated proteins and  <dig> up-regulated mrnas vs. down-regulated proteins, and 3) mirna targeted gene translation indicated by protein up-regulated  or down-regulated  without a change in mrna expression, or targeted transcription indicated by mrna up-regulated  or down-regulated  without a change in protein expression .figure  <dig> mirnas regulated their target gene expression.
a. genome-wide mrna and protein expression associated with down-regulated mirnas in rat kidney.
we identified  <dig> dysregulated mirnas and selected their targets that are validated and reported from mirtarbase and correlated with their mrna and protein expression.
b. genome-wide mrna and protein expression associated with up-regulated mirnas in rat kidney.
similarly, for group b, we separated our study into three subgroups: 1) mrnas and proteins expressed in the same direction including both up-regulated , down-regulated , and not changed ; 2) mrnas and proteins expressed in the opposite direction including  <dig> down-regulated mrnas vs. up-regulated proteins and  <dig> up-regulated mrnas vs down-regulated proteins, and 3) mirna targeted translation indicated by protein up-regulated  or down-regulated  without a change in mrna expression, or targeted transcription indicated by mrna up-regulated  or down-regulated  without a change in protein expression .
in summary, mirnas exhibited a more complex regulation on their target expression, indicated by both repression and activation at levels of transcription and translation.
also, mirnas affected gene translation without a change in mrna abundance and vice versa.
the correlation of expression between mrnas and proteins was very weak, indicating that post-transcriptional regulation by mirnas or other factors are involved in this regulation.
mirna-target interaction networks associated with cancer in rat kidney treated with aa
we examined the interactions of mirna with cancer specific targets that were expressed at the protein level and more related to their functional regulation.
we used ipa analysis to pull out all the cancer-related mirnas' targets from mirarbase and matched with  <dig> dysregulated mirnas associated with cancer in rat kidney treated with aa.
therefore, we created mirna regulation networks that link mirnas and their targets to aa-induced carcinogenesis in rat kidney.
for example, we identified  <dig> cancer-related targets associated with down-regulated mirnas.
among these,  <dig> targets were down-regulated for protein expression and only  <dig> were up-regulated .
also,  <dig> cancer related targets were associated with  <dig> up-regulated oncogenic mirnas.
among these,  <dig> targets were up-regulated for protein expression, and only  <dig> were down-regulated .
in other words, mirnas exhibited a bi-directional regulation of their targets and most cancer related target expression had the same direction as the mirna expression, an unexpected finding.figure  <dig> interaction of mirnas with their cancer-related protein targets.
mirna targets were selected from ipa analysis and matched to mirnas that were down-regulation.
mirna targets matched to mirnas that were up-regulated.
mirna targets – protein expression that has the same direction indicated by the red color and opposite direction indicated by the green color.
highlighted mirna-target interactions of mir- <dig>  mir-34a, mir- <dig>  and mir-135a.
we next selected several few specific oncogenic mirnas and examined their targets' expression.
for example, mir- <dig> was up-regulated in rat kidney.
the expression of its target genes ppp3cb and kras were down-regulated, but eight target genes including ywhah, txnrd <dig>  ldha, ace, eif5b, sparc, itprc, and pcna were up-regulated, and these targeted genes are related with several cancer signaling pathways including erk/mapk, pi3k/akt, and 14-3- <dig> mediated signaling.
mir-34a was up-regulated.
the expression of its target genes lcam <dig> and amacr were down-regulated, but  <dig> out of  <dig> mir-34a's targets were up-regulated including tubbs, ldha, nampt, eno <dig>  ggh, txnrd <dig>  and lmna which are associated with mitochondrial dysfunction, dna damage and repair, and apoptosis .
discussion
the international agency for research on cancer  has classified herbal remedies containing plant species of the genus aristolochia as human carcinogens .
in the present study, we profiled the expression of mirna, mrna and protein in the kidney of rats treated with a carcinogenetic dose of aa.
we also employed an integrative methodology to study the correlations among mirna, mrna, and protein expression during aa-induced kidney carcinogenesis.
we found that mirna expression was significantly changed by aa treatment, with  <dig> altered mirnas, accounting for 15% of  <dig> detectable mirnas.
we performed real-time pcr to validate the mirna expression changes detected by deep-sequencing analysis.
moreover, in a genome-wide analysis, we found that altered mirna expression had different effects on their target at the levels of both transcription and translation.
we further determined that mirna regulation is associated with both gene repression and activation, which could help us better understand mirna biology in aa-induced rat kidney tumorigenesis.
mirna dysregulation is recognized as a driving force in human cancer.
alteration of mirna expression is commonly observed following exposure to different carcinogens  <cit> , but the role of mirnas in aa-induced carcinogenesis was not studied previously.
genetic changes are frequently observed in experimental animal models treated with aa.
for example, we have previously shown that aa induces a high frequency of dna mutations, with a:t → t:a transversion as the predominant mutation type  <cit> .
in addition to the genetic changes induced by aa, we have found that aa treatment result in dysregulation of mirnas and that half of them are associated with cancer according to ipa analysis.
we selected up-regulation of oncogenic mirnas such as mir- <dig> as well as down-regulation of the tumor suppressor mirnas let-7e, mir-135a, and mir- <dig> for our analysis.
in patients, mir-135a inhibits cancer cell proliferation and exhibits the properties of a tumor suppressor in renal-cell carcinoma  <cit> , while its target oncoprotein bag- <dig> increases more than  <dig> fold as determined by proteomics.
a recent study demonstrates that aa treatment altered a number of mirnas in proximal tubular epithelial cells such as up-regulation of mir- <dig>  mir- <dig>  mir-450a, and mir- <dig>  mir- <dig> mediated dna damage response and recapitulated g2/m arrest via repression of murine double-minute  <dig>  a negative regulator of p <dig>  <cit> .
similarly, we found that aa treatment increased expression of mir-450a and mir- <dig>  and that aa induced a dna damage response indicated by up-regulation of mir-34a.
it was followed by activation of p <dig> and the binding p <dig> to the promoter of mir-34a resulted in mir- <dig> activation  <cit> .
on the other hand, we did not see any consistency of  <dig> down-regulated mirnas as identified in that study.
this discrepancy could be explained by the fact that we used an in vivo model to study aa-induced carcinogenesis, which might be affected by many factors such as microenvironment, immune factors, and so on.
all of these are not modeled in an in vitro study.
it is likely that the carcinogenic effects of aa depend upon the balance between the oncogenic mirnas and tumor-suppressor mirnas.
thus, the alterations in these mirnas might play an important role in promoting tumor development in rat kidney as well as inducing kidney toxicity, suggesting that identifying the mirna targets involved in these processes are important.
we combined multiple levels of profiling to study mirna target expression and identified those targets of dysregulated mirnas that are validated and reported from mirtarbase.
currently, mirna target identification is primarily based on computational predications that could result in a vast number of targets with errors, raising the problem of identifying functional targets for validation  <cit> .
from our knowledge, this is the first time that this novel approach has been used to study the relationship among three levels of expression of mirna, mrna and protein on a genome-wide scale.
even though there might be some missing targets for mirnas identified in this study in the mirtarbase, we have identified several categories of mirna-target relationship that might represent general mirna regulation patterns and the biological operating during aa-induced carcinogenesis.
in general, there are two possible post-transcriptional mechanisms accounting for mirna regulation of its target genes, mrna cleavage or translational repression, both of which negatively regulate target expression.
if an mrna target is perfectly or near-perfectly complementary to the mirna, the mrna will be cleaved and degraded.
mrna translation also can be repressed.
consequently, we identified a number of mirna targets that show translational repression or mrna destabilization or both.
on the other hand, a number of mirna targets identified in this study pair with proteins show expression in opposite direction to the change in mirna expression, but the mrna level remains unchanged.
this is consistent with previous reports that most animal mirnas complement their targets imperfectly so they function primarily via translational repression rather than mrna cleavage and degradation  <cit> .
however, we identified a number of mirna targets with expression of mirnas and proteins in the same direction.
moreover, we have discovered a network of mirnas and their functional target interactions associated with aa-induced tumors that show both directions of mirna - target regulation and most of them are actually expressed in the same direction.
in certain cases mirnas and their potential targets were observed to have similar expression patterns  <cit> .
such a phenomenon might be explained by counter-regulation of different posttranscriptional mechanisms or possibly the down-regulation of protein production being offset by a feedback mechanism  <cit> .
in this study, it remains intriguing that up-regulation of mirnas such as mir- <dig> and mir-34a is associated with activation of their target oncoprotein expression after aa treatment for 12 weeks.
it remains unknown how mirnas could maintain the target overexpression that could be a driving force for aa-induced carcinogenesis.
so far, mirna biology studies mainly have focused on mirna targets with an opposite regulation or a similar expression with the anti-targets.
our research is based on a genome-wide approach and highlights the importance of considering both sides of mirna regulation, including translational repression resulting in activation in biological functions and diseases.
finally, we found that there was only a weak correlation between mrna and protein expression or even opposite expression for a number of genes.
one possibility for this lack of association is the different stability of proteins in the rat  <cit> .
for example, the half-life of different proteins varies from minutes to days, but the variability for mrnas is quite a bit less.
also, post-transcriptional and post-translational regulation might affect protein expression.
mirnas are also likely involved in this process and might regulate gene expression through feedback mechanisms, but the mechanisms for these pathways are largely unknown.
however, we do not preclude the possibility that mechanisms may be important including transcriptional factors, translational or posttranslational regulation that would affect mirna target expression.
in summary, we treated rats with a carcinogenic dose of aa and identified dysregulated expression of  <dig> mirnas in rat kidney by performing a deep sequencing analysis.
ipa analysis indicated that half of them are associated with cancer.
we next selected mirna targets based on those dysregulated mirnas and combined mirna data with the rna microarray and proteomics data in order to understand mirna regulation of their targets regulation.
the ability to compare mirna, mrna, and protein expression in the identical samples defined a comprehensive mirna regulatory network that indicated rna destabilization, translational repression and activation.
CONCLUSIONS
we employed global profiling of mirna, transcriptome, and proteome expression in parallel in order to integrate three biological system levels to better understand aa-induced carcinogenesis in rat kidney.
aa induced a number of dysregulated mirnas that are closely associated with cancer initiation and progression.
moreover, the use of integrated approaches helps us better understand the relationships among expression of mirnas, mrnas and proteins with a focus on mirna regulation on genome-wide repression and activation.
we identified a cancer-related network that is associated with mirna target regulation, primarily regulating translational activation.
this novel systematic study provides us information on mirna target regulation on a genome-wide scale.
the study supports application of mirna expression as a biomarker for identifying the genotoxicity and carcinogenicity of aa and other of unknown exposures.

 aristolochic acid is found in plants of the genus aristolochia and asarum. use of dietary supplements and other botanical products containing aa has caused severe nephrotoxicity and consequent renal replacement therapy. animal studies show that aa results in renal failure in rodents and induces tumors in the kidney and other tissues of rabbits, rats and mice. aa is among the most potent 2% of the carcinogens in the carcinogenic potency and genotoxicity databases. as a result, the u.s. food and drug administration issued a consumer advisory in warning consumers against using dietary supplements and other botanical products containing aa, and the fda also requested a recall of these products and published a list of botanical products that contained aa. however, products containing aa have not been banned in the us or many other countries. aa-induced carcinogenesis has been attributed to the mutagenicity and dna adducts formed in the kidney and other tissues of aa nephropathy patients. on the other hand, aa induced similar dna adduct formation in both the kidney and liver of mice, but tumors preferentially occurred in kidney. this suggests that in addition to the genetic alterations induced by aa, alternative mechanisms such as epigenetic remodeling and mirna modulation might also play an important role in aa-induced cancers. mirnas represent a class of non-coding small rna that are ubiquitously present in different kinds of organisms from c.elegans to mammals. mirnas are involved in the post-transcriptional regulation of gene expression via binding to the 3’ utr region of target mrnas, resulting in mrna degradation or translation inhibition. each mirna usually targets multiple, even hundreds of mrnas. it is believed that one third of human genes are subject to mirna control. mirnas regulate a variety of developmental and physiological processes, including control of leaf and flower development in plants and neuronal patterning in nematodes. recent studies indicate that mirnas are involved in the regulation of pathways that are associated with the initiation and progression of many types of tumors. mirna expression was found to accurately identify the tissue origin of cancers, including distal metastatic colonies of unknown primary origin. mirnas affect tumor metastasis, such as the lung and bone metastasis of human breast cancer. moreover, mirnas are associated with chemical carcinogenesis and regulate gene expression that is important in every stage. deep sequencing, also known as next generation sequencing, has undergone tremendous acceptance in the past few years. by sequencing dna or rna in a massively parallel fashion, deep sequencing technologies dramatically reduce both the cost-per-base and time required to decode an entire genome or transcriptome, making sequencing a cost-effective option for many experimental approaches. moreover, it has very low, if any, background signal, and does not have an upper limit for quantification, resulting in a large dynamic range of expression levels over which transcripts can be detected. toxicoproteomics is a new discipline that applies proteomics concepts and approaches to toxicological studies. it can elucidate pathological responses to a specific toxicant at the protein molecule level, including qualitative and quantitative measurements of protein expression, protein modifications, and protein-protein/toxicant interactions. a primary research objective of mirna biology is to understand the post-transcriptional regulation of the target genes and the effect on biological functions. however, due to the different mechanisms of mirna regulation, the targets can be translationally repressed with a concordant decrease in mrna abundance or without significant mrna degradation, or have decreased mrna abundance with little protein change. moreover, mirnas can switch from repression to activation depending on different stages of the cell cycle. therefore, in order to understand mirna regulation in aa-induced kidney tumorigenesis, we integrated deep sequencing data for global mirna and mrna expression with proteomics data for global protein expression in conjunction with target identification of dysregulated mirnas from the mirtarbase. compared with using mirna target prediction tools such as miranda and targetscan, we selected those targets that were published and validated to study their mirna transcriptional and translational regulations, thus providing a novel approach to enhance our understanding of the biological role of mirna in aa-induced carcinogenesis in rat kidney. the details of the proteomics data from rats treated with aa were reported separately. this work will contribute to our understanding of the biological processes driving aa-induced carcinogenesis at the mirna, mrna and protein levels as well as representing a generalizable strategy that can be extended to study the functional role of mirnas and their regulation of target expression in organs or tissues of interest.  aa treatment resulted in functionally differential mirna expressions in rat kidney dysregulated mirna expression has been linked to cancer in experimental animal models and patients. to investigate the effects of aa exposure on mirna expression under conditions that resulted in kidney tumors, we treated rats for 12 weeks with aa along with control rats treated with vehicle, following a carcinogenetic protocol that has been demonstrated to result in kidney tumors. all the rats were sacrificed one day after the last treatment and the kidneys were removed for mirna deep sequencing analysis. the short reads were mapped to sanger mirbase. there were detectable mirnas in at least one of the samples. first, classification analyses were conducted to explore global alteration of mirnas by the aa treatment. hierarchical clustering analysis was performed to globally view the groups among the samples in terms of the expression profile of all the detectable mirnas. these samples are divided into two branches, one comprises aa-treated samples and the other contains the control samples. this indicates that mirnas have similar and consistent expression patterns within each group, but are distinct from each other, demonstrating the profound impact of aa treatment on mirna expression in rat kidney. the same phenomenon was revealed by the principal component analysis analysis, further supporting that mirna expression is profoundly changed by aa treatment at the genome scale.figure identification of differentially expressed mirna and mrna in kidney in rat treated with aa. a. classification of samples according to expression intensity of the detectable mirnas. hierarchical clustering analysis separates the samples into two groups that are consistent with control and the treatment groups. principal component analysis groups samples into the aa- treated and control clusters. b. classification of samples according to expression level of the detectable mrnas. hierarchical clustering analysis separates the samples into two groups that are consistent with the control and treatment groups. principal component analysis groups samples into the aa- treated and control clusters. next, we investigated which mirnas were significantly dysregulated by aa treatments. all the detectable mirnas were used to find differentially expressed mirnas. read count normalization, statistical analysis and p value adjustment were performed using the deseq methods. sixty-three mirnas showed significant changes in their expression level by aa treatment using a cutoff with adjusted p values of <  and fold change of > . more up-regulated mirnas were observed than down-regulated mirnas.rno-mir- rno-mir- mir-741-3p, mir-511*, mir- mir-449a, as well as members of mir- family, mir-34a, mir-34a*, mir-34b, mir-34b*, mir-34c, and mir-34c*, showed over 10-fold up-regulation. in contrast, only one mirna, mir- showed more than 10-fold down-regulation. moreover, the oncogenic mirna, mir- was increased fold, whereas the tumor suppressor mirnas, let-7e, mir-135a, and mir- were decreased by, and fold, respectively. in order to validate the changes in mirna expression detected by a deep-sequencing, we conducted quantitative real-time pcr analysis to examine expression levels of six mirnas including rno-mir- rno-mir- rno-mir- rno-mir-34a, rno-mir-34b, and rno-mir34c. the results from the real-time pcr analysis confirmed the sequencing data. finally, mirna target proteins that were dysregulated in rat kidney were inputted into the ingenuity database to explore the underlying biological and toxicological functions. the biological function analysis showed that cancer was the top functional category significantly related to these mirnas, along with organismal injury and abnormalities, which also could result from aa treatment. in total, of the mirnas were cancer-related.table ipa analysis of dysregulated mirnas in rat kidney treated with aa top networks top diseases and functions differentially expressed genes from mrna sequencing data and their functions mirnas regulate the homeostatic level of their target mrnas. to investigate whether mrna expression was also changed by aa treatments, the rat kidney samples were analyzed by mrna deep sequencing analysis in parallel. the short reads were mapped to ensembl transcripts, and a data processing procedure similar to that used for the mirnas was applied to normalize the raw counts, and to calculate adjusted p values. there were mrnas that had detectable expression in these samples. as with mirna expression in rat kidney, the expression patterns of mrna were similar within the treatment or control group alone, but distinctly between the two groups in both hca and pca analysis. a total of richness mrnas were found to be differentially expressed based on a cutoff of p <  and fold change >  with 4051upregulated and down-regulated. ingenuity pathway analysis of both up-regulated and down-regulated genes identified cancer as the top disease and biological function with up-regulated and down-regulated molecules included. the top canonical pathways included wnt/β-actin, mitochondrial dysfunction and valine degradation.. an integrated multiple profiling reveals a comprehensive regulation of mirnas on their target expression in order to search for the targets of its differentiately regulated mirnas and understand effects of the post-transcriptional regulation of mirna on gene expression in aa-induced carcinogenesis, we combined proteomic and transcriptomic data to study expression of the mirna targets that are validated and reported in mirtarbase. this database provides the most current and comprehensive information on experimentally validated mirna-target interactions and data resources with published studies on the identification of mirna targets, molecular networks of mirna targets and systems biology. specifically, it contains mirna targets that are validated by one or more solid experimental methods including luciferase reporter assay, western blot, northern blot, and quantitative real-time analysis. if a gene is targeted by multiple mirnas, the log transformed fold changes of these mirnas were summed together as the fold changes of the mirnas. we initially conducted correlation analysis between the mirnas vs. mrnas, mirnas vs. proteins, and mrnas vs. proteins in rat kidney treated with aa in relation to the control. there was a very weak correlation between expression of mrnas versus proteins, and mirnas vs. proteins, but there was no correlation between mirna vs. mrna expression, indicating that regulation by mirnas on their target genes can be attributed to multiple mechanisms. next, we divided our study into two groups including group a, the up-regulated mirnas and group b, the down-regulated mirnas, and used an adjusted p <  and fold changes of < - or > as a cutoff, indicating the difference for all these three variables. according to the response of gene expression in group a, we separated our study into three subgroups: 1) mrnas and proteins expressed in the same direction including up-regulated, down-regulated, not changed. 2) mrnas and proteins expressed in the opposite direction including down-regulated mrnas vs. up-regulated proteins and up-regulated mrnas vs. down-regulated proteins, and 3) mirna targeted gene translation indicated by protein up-regulated or down-regulated without a change in mrna expression, or targeted transcription indicated by mrna up-regulated or down-regulated without a change in protein expression.figure mirnas regulated their target gene expression. a. genome-wide mrna and protein expression associated with down-regulated mirnas in rat kidney. we identified dysregulated mirnas and selected their targets that are validated and reported from mirtarbase and correlated with their mrna and protein expression. b. genome-wide mrna and protein expression associated with up-regulated mirnas in rat kidney. similarly, for group b, we separated our study into three subgroups: 1) mrnas and proteins expressed in the same direction including both up-regulated, down-regulated, and not changed; 2) mrnas and proteins expressed in the opposite direction including down-regulated mrnas vs. up-regulated proteins and up-regulated mrnas vs down-regulated proteins, and 3) mirna targeted translation indicated by protein up-regulated or down-regulated without a change in mrna expression, or targeted transcription indicated by mrna up-regulated or down-regulated without a change in protein expression. in summary, mirnas exhibited a more complex regulation on their target expression, indicated by both repression and activation at levels of transcription and translation. also, mirnas affected gene translation without a change in mrna abundance and vice versa. the correlation of expression between mrnas and proteins was very weak, indicating that post-transcriptional regulation by mirnas or other factors are involved in this regulation. mirna-target interaction networks associated with cancer in rat kidney treated with aa we examined the interactions of mirna with cancer specific targets that were expressed at the protein level and more related to their functional regulation. we used ipa analysis to pull out all the cancer-related mirnas' targets from mirarbase and matched with dysregulated mirnas associated with cancer in rat kidney treated with aa. therefore, we created mirna regulation networks that link mirnas and their targets to aa-induced carcinogenesis in rat kidney. for example, we identified cancer-related targets associated with down-regulated mirnas. among these, targets were down-regulated for protein expression and only were up-regulated. also, cancer related targets were associated with up-regulated oncogenic mirnas. among these, targets were up-regulated for protein expression, and only were down-regulated. in other words, mirnas exhibited a bi-directional regulation of their targets and most cancer related target expression had the same direction as the mirna expression, an unexpected finding.figure interaction of mirnas with their cancer-related protein targets. mirna targets were selected from ipa analysis and matched to mirnas that were down-regulation. mirna targets matched to mirnas that were up-regulated. mirna targets – protein expression that has the same direction indicated by the red color and opposite direction indicated by the green color. highlighted mirna-target interactions of mir- mir-34a, mir- and mir-135a. we next selected several few specific oncogenic mirnas and examined their targets' expression. for example, mir- was up-regulated in rat kidney. the expression of its target genes ppp3cb and kras were down-regulated, but eight target genes including ywhah, txnrd ldha, ace, eif5b, sparc, itprc, and pcna were up-regulated, and these targeted genes are related with several cancer signaling pathways including erk/mapk, pi3k/akt, and 14-3- mediated signaling. mir-34a was up-regulated. the expression of its target genes lcam and amacr were down-regulated, but out of mir-34a's targets were up-regulated including tubbs, ldha, nampt, eno ggh, txnrd and lmna which are associated with mitochondrial dysfunction, dna damage and repair, and apoptosis. discussion the international agency for research on cancer has classified herbal remedies containing plant species of the genus aristolochia as human carcinogens. in the present study, we profiled the expression of mirna, mrna and protein in the kidney of rats treated with a carcinogenetic dose of aa. we also employed an integrative methodology to study the correlations among mirna, mrna, and protein expression during aa-induced kidney carcinogenesis. we found that mirna expression was significantly changed by aa treatment, with altered mirnas, accounting for 15% of detectable mirnas. we performed real-time pcr to validate the mirna expression changes detected by deep-sequencing analysis. moreover, in a genome-wide analysis, we found that altered mirna expression had different effects on their target at the levels of both transcription and translation. we further determined that mirna regulation is associated with both gene repression and activation, which could help us better understand mirna biology in aa-induced rat kidney tumorigenesis. mirna dysregulation is recognized as a driving force in human cancer. alteration of mirna expression is commonly observed following exposure to different carcinogens, but the role of mirnas in aa-induced carcinogenesis was not studied previously. genetic changes are frequently observed in experimental animal models treated with aa. for example, we have previously shown that aa induces a high frequency of dna mutations, with a:t → t:a transversion as the predominant mutation type. in addition to the genetic changes induced by aa, we have found that aa treatment result in dysregulation of mirnas and that half of them are associated with cancer according to ipa analysis. we selected up-regulation of oncogenic mirnas such as mir- as well as down-regulation of the tumor suppressor mirnas let-7e, mir-135a, and mir- for our analysis. in patients, mir-135a inhibits cancer cell proliferation and exhibits the properties of a tumor suppressor in renal-cell carcinoma, while its target oncoprotein bag- increases more than fold as determined by proteomics. a recent study demonstrates that aa treatment altered a number of mirnas in proximal tubular epithelial cells such as up-regulation of mir- mir- mir-450a, and mir- mir- mediated dna damage response and recapitulated g2/m arrest via repression of murine double-minute a negative regulator of p. similarly, we found that aa treatment increased expression of mir-450a and mir- and that aa induced a dna damage response indicated by up-regulation of mir-34a. it was followed by activation of p and the binding p to the promoter of mir-34a resulted in mir- activation. on the other hand, we did not see any consistency of down-regulated mirnas as identified in that study. this discrepancy could be explained by the fact that we used an in vivo model to study aa-induced carcinogenesis, which might be affected by many factors such as microenvironment, immune factors, and so on. all of these are not modeled in an in vitro study. it is likely that the carcinogenic effects of aa depend upon the balance between the oncogenic mirnas and tumor-suppressor mirnas. thus, the alterations in these mirnas might play an important role in promoting tumor development in rat kidney as well as inducing kidney toxicity, suggesting that identifying the mirna targets involved in these processes are important. we combined multiple levels of profiling to study mirna target expression and identified those targets of dysregulated mirnas that are validated and reported from mirtarbase. currently, mirna target identification is primarily based on computational predications that could result in a vast number of targets with errors, raising the problem of identifying functional targets for validation. from our knowledge, this is the first time that this novel approach has been used to study the relationship among three levels of expression of mirna, mrna and protein on a genome-wide scale. even though there might be some missing targets for mirnas identified in this study in the mirtarbase, we have identified several categories of mirna-target relationship that might represent general mirna regulation patterns and the biological operating during aa-induced carcinogenesis. in general, there are two possible post-transcriptional mechanisms accounting for mirna regulation of its target genes, mrna cleavage or translational repression, both of which negatively regulate target expression. if an mrna target is perfectly or near-perfectly complementary to the mirna, the mrna will be cleaved and degraded. mrna translation also can be repressed. consequently, we identified a number of mirna targets that show translational repression or mrna destabilization or both. on the other hand, a number of mirna targets identified in this study pair with proteins show expression in opposite direction to the change in mirna expression, but the mrna level remains unchanged. this is consistent with previous reports that most animal mirnas complement their targets imperfectly so they function primarily via translational repression rather than mrna cleavage and degradation. however, we identified a number of mirna targets with expression of mirnas and proteins in the same direction. moreover, we have discovered a network of mirnas and their functional target interactions associated with aa-induced tumors that show both directions of mirna - target regulation and most of them are actually expressed in the same direction. in certain cases mirnas and their potential targets were observed to have similar expression patterns. such a phenomenon might be explained by counter-regulation of different posttranscriptional mechanisms or possibly the down-regulation of protein production being offset by a feedback mechanism. in this study, it remains intriguing that up-regulation of mirnas such as mir- and mir-34a is associated with activation of their target oncoprotein expression after aa treatment for 12 weeks. it remains unknown how mirnas could maintain the target overexpression that could be a driving force for aa-induced carcinogenesis. so far, mirna biology studies mainly have focused on mirna targets with an opposite regulation or a similar expression with the anti-targets. our research is based on a genome-wide approach and highlights the importance of considering both sides of mirna regulation, including translational repression resulting in activation in biological functions and diseases. finally, we found that there was only a weak correlation between mrna and protein expression or even opposite expression for a number of genes. one possibility for this lack of association is the different stability of proteins in the rat. for example, the half-life of different proteins varies from minutes to days, but the variability for mrnas is quite a bit less. also, post-transcriptional and post-translational regulation might affect protein expression. mirnas are also likely involved in this process and might regulate gene expression through feedback mechanisms, but the mechanisms for these pathways are largely unknown. however, we do not preclude the possibility that mechanisms may be important including transcriptional factors, translational or posttranslational regulation that would affect mirna target expression. in summary, we treated rats with a carcinogenic dose of aa and identified dysregulated expression of mirnas in rat kidney by performing a deep sequencing analysis. ipa analysis indicated that half of them are associated with cancer. we next selected mirna targets based on those dysregulated mirnas and combined mirna data with the rna microarray and proteomics data in order to understand mirna regulation of their targets regulation. the ability to compare mirna, mrna, and protein expression in the identical samples defined a comprehensive mirna regulatory network that indicated rna destabilization, translational repression and activation.  we employed global profiling of mirna, transcriptome, and proteome expression in parallel in order to integrate three biological system levels to better understand aa-induced carcinogenesis in rat kidney. aa induced a number of dysregulated mirnas that are closely associated with cancer initiation and progression. moreover, the use of integrated approaches helps us better understand the relationships among expression of mirnas, mrnas and proteins with a focus on mirna regulation on genome-wide repression and activation. we identified a cancer-related network that is associated with mirna target regulation, primarily regulating translational activation. this novel systematic study provides us information on mirna target regulation on a genome-wide scale. the study supports application of mirna expression as a biomarker for identifying the genotoxicity and carcinogenicity of aa and other of unknown exposures. 
our findings suggest that dysregulated mirna expression plays an important role in aa-induced carcinogenesis in rat kidney, and that the integrated approach of multiple profiling provides a new insight into a post-transcriptional regulation of mirnas on their target repression and activation in a genome-wide scale. furthermore, we identified that up-regulation of oncogenic mirnas was associated with translational activation of out of cancer-related targets. in addition to translational repression and transcriptional destabilization, we also found that mirnas and their target genes were expressed in the same direction at levels of transcription or translation. to further investigate the influence of mirnas on kidney mrna and protein expression, we combined proteomic and transcriptomic data in conjunction with mirna target selection as confirmed and reported in mirtarbase. aristolochic acid, a natural component of aristolochia plants that is found in a variety of herbal remedies and health supplements, is classified as a group carcinogen by the international agency for research on cancer. the expression of selected mirnas was validated by quantitative real-time pcr analysis. given that micrornas are involved in cancer initiation and progression and their role remains unknown in aa-induced carcinogenesis, we examined genome-wide aa-induced dysregulation of mirnas as well as the regulation of mirnas on their target gene expression in rat kidney. aa treatment resulted in significant differential expression of mirnas, mrnas and proteins as measured by both principal component analysis and hierarchical clustering analysis. specially, mirnas, mrnas, and proteins were significantly altered by aa treatment. aristolochic acidcarcinogenesiskidney tumormicrornaproteomicsdeep-sequencingrna arraytarget predictionratissue-copyright-statement© the author 2015 we treated rats with 10 mg/kg aa and vehicle control for 12 weeks and eight kidney samples were used for examining mirna and mrna expression by deep sequencing, and protein expression by proteomics.
 aristolochic acid, a natural component of aristolochia plants that is found in a variety of herbal remedies and health supplements, is classified as a group carcinogen by the international agency for research on cancer. given that micrornas are involved in cancer initiation and progression and their role remains unknown in aa-induced carcinogenesis, we examined genome-wide aa-induced dysregulation of mirnas as well as the regulation of mirnas on their target gene expression in rat kidney.  we treated rats with 10 mg/kg aa and vehicle control for 12 weeks and eight kidney samples were used for examining mirna and mrna expression by deep sequencing, and protein expression by proteomics. aa treatment resulted in significant differential expression of mirnas, mrnas and proteins as measured by both principal component analysis and hierarchical clustering analysis. specially, mirnas, mrnas, and proteins were significantly altered by aa treatment. the expression of selected mirnas was validated by quantitative real-time pcr analysis. ingenuity pathways analysis showed that cancer is the top network and disease associated with those dysregulated mirnas. to further investigate the influence of mirnas on kidney mrna and protein expression, we combined proteomic and transcriptomic data in conjunction with mirna target selection as confirmed and reported in mirtarbase. in addition to translational repression and transcriptional destabilization, we also found that mirnas and their target genes were expressed in the same direction at levels of transcription or translation. furthermore, we identified that up-regulation of oncogenic mirnas was associated with translational activation of out of cancer-related targets.  our findings suggest that dysregulated mirna expression plays an important role in aa-induced carcinogenesis in rat kidney, and that the integrated approach of multiple profiling provides a new insight into a post-transcriptional regulation of mirnas on their target repression and activation in a genome-wide scale. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords aristolochic acidcarcinogenesiskidney tumormicrornaproteomicsdeep-sequencingrna arraytarget predictionratissue-copyright-statement© the author 2015 