BACKGROUND
biliary atresia, one of the most common causes of neonatal cholestasis, results from a fibro-inflammatory obstruction of extrahepatic bile ducts of unknown etiology.
if diagnosed early, surgical intervention with hepatoportoenterostomy has the potential to restore bile flow, but ongoing liver injury results in rapid progression to cirrhosis and the need for liver transplantation by  <dig> years of age in ~50% of children  <cit> .
contemporary studies suggest that the pathogenesis of bile duct injury results from an interplay between environmental factors and a biological response that involves cells of the immune system, injury and proliferation of the biliary epithelium, and excessive deposition of extracellular matrix  <cit> .
insight into the activation of these processes was obtained from large-scale gene expression analyses in livers of infants at the time of diagnosis  <cit>  and from extrahepatic bile ducts and gallbladder  obtained from neonatal mice in a well-established model of rotavirus-induced experimental biliary atresia  <cit> .
in this model, rhesus rotavirus type-a  infection within 2– <dig> days of life leads to an inflammatory obstruction of extrahepatic bile ducts within a week, with age-restricted susceptibility  <cit> .
this model has been shown to recapitulate many features of the tissue injury and pathogenesis observed in humans with biliary atresia  <cit> .
more recently, an investigation of livers from this mouse model suggested a potential role for micrornas in pathogenesis of disease  <cit> .
micrornas  are short non-coding rnas that regulate gene expression at post-transcriptional levels by facilitating degradation and/or inhibiting translation of target mrnas.
mirnas may play important roles in the development of the hepatobiliary system, <cit>  and cell- and tissue-specific mirna expression profiles are altered in some diseases, such as cirrhosis and cancers  <cit> .
to obtain insight into the role of mirnas in pathogenesis of biliary atresia, we performed comprehensive expression analyses of mirnas and mrna from ehbds at the times of epithelial injury, lumenal obstruction, and atresia in the experimental mouse model.
using integrative in silico approaches for mirna and mrna expression profiles, we found a coordinated temporal expression of a selected number of mirnas and their target genes, with the activation of molecular networks regulating organogenesis and immune responses.
spatially, the expression of these mirnas was higher in ehbds , and was localized in cholangiocytes and in stromal cells of the subepithelial compartment.
RESULTS
time-dependent suppression of mirnas in ehbds
to search for mirnas with potential roles in the pathogenesis of biliary atresia, we first performed a comprehensive survey of mirna expression patterns in extrahepatic bile ducts and gallbladder  of neonatal mice challenged with rrv within  <dig> hours of birth.
all datasets are deposited in the gene expression omnibus repository , with accession code gse <dig>  we focused on the analysis of ehbds, rather than the liver, because they are the primary site of tissue injury, beginning with an epithelial insult , followed by lumenal obstruction with inflammatory cells  and later by atresia   <cit> .
in these studies, all mice developed jaundice by  <dig> days, which persisted and became associated with poor weight gain until the time of death by  <dig> days after rrv challenge, as described previously  <cit> .
among a total of  <dig> mirnas,  <dig> changed their expression levels in ehbds after rrv challenge by at least  <dig> fold when compared to saline-injected control mice .
among these mirnas, only mir-let-7e* was differentially expressed in the rrv group at early phases of epithelial injury, with a  <dig>  fold decrease below controls , while a greater number of mirnas was downregulated at  <dig> days  and  <dig> days .
interestingly, only  <dig> mirnas were upregulated by more than 2-fold, which were all at day  <dig> after rrv challenge and included mir-29b, which was reported to be elevated in the liver of newborn mice subjected to the same experimental model   <cit> .
to identify candidate mirnas with a potential role in the pathogenesis of biliary injury, we selected mirnas that showed the same pattern of expression at the times of bile duct obstruction and atresia .
we found  <dig> mirnas that were consistently suppressed at both time points ; there was no mirna that was overexpressed at both time points.
most of these mirnas have been implicated in mechanisms of hepatocellular carcinoma , hepatitis c virus infection of hepatocytes , and nonalcoholic fatty liver disease and steatohepatitis .
most notably, mir-30b/c has been reported to influence transforming growth factor beta 1-induced epithelial to mesenchymal transition and biliary development and infection,  <cit>  while mir-200b,  <cit>  - <dig>   <cit>  and − <dig>  <cit>  have been linked to cholangiocarcinoma.
combined, the pattern of time-dependent suppression of mirnas and the previous link to pathogenesis of hepatobiliary diseases formed a rationale for new experiments to investigate the expression behavior of those genes predicted to be targets of this subset of mirnas at different stages of experimental biliary atresia.
* minus sign implies fold change below controls.
fold changes are expressed in rrv-challenged mice relative to saline-injected controls.
tgf: transforming growth factor, lps: lipopolysaccharide, hcc: hepatocellular carcinoma, hcv: hepatitis c virus, emt: epithelial-to-mesenchymal transition, nafld: nonalcoholic fatty liver disease, nash: nonalcoholic steatohepatitis, hsc: hepatic stellate cells.
increased expression of target genes in ehbds
because mirnas execute their biological roles by regulating expression levels of target genes, we developed a whole-genome expression platform using rna from ehbds at the same time points after rrv challenge .
to integrate the  <dig> mirnas with the expression levels of the related genes, we first built a list of putative target genes based on targetscan and miranda prediction algorithms, and identified  <dig>  genes shared by both algorithms.
second, we mined the gene expression platform to select genes that changed by ≥ <dig> fold either at  <dig>   <dig> and  <dig> days after rrv challenge when compared to saline controls, and found  <dig>  genes fulfilling these criteria at p <  <dig>  and 5% fdr .
a comparison of these two lists revealed that  <dig> genes were shared by both lists.
third, given that mirnas are thought to function by inhibiting the level of gene expression and that the  <dig> mirnas were suppressed after rrv challenge, we examined the list of  <dig> genes to select genes as potential mirna targets based on an increase in the expression by ≥ <dig> fold in ehbds, and got  <dig> genes at  <dig> days and  <dig> genes at  <dig> days after rrv challenge .
analyzing the association among the  <dig> mirnas and their potential target genes , we found that  <dig> mirnas were associated with increased expression of at least  <dig> target genes at the time of lumenal obstruction : a) mir-30b and -30c were associated with overexpression of mrna for pik3cd, runx <dig>  cecam <dig>  cysltr <dig>  gda, serpine <dig>  soc <dig> and cmpk2; b) mir- <dig> with hmga <dig>  il10ra, pim <dig>  il7r and has2; and c) mir-200a with runx <dig>  gbp <dig> and has <dig> .
notably, although their selection was based on an overexpression at  <dig> days, cluster analysis showed that the increase in some of these genes began as early as  <dig> days and generally persisted through the time of duct atresia .
from the genes selected based on the increased expression at the time of atresia ,  <dig> genes were increased, of which  <dig> were targets of mir-30b/c .
the linkage of coordinately overexpressed genes with a small group of mirnas  formed the rationale for informatics-based analyses to explore the relationship of these genes and mirnas in terms of biological pathways and function.
functional enrichment analysis of target genes
using toppcluster as a platform to integrate informatics, streams of gene and protein function, regulation and animal models of disease,  <cit>  we found 1st-to-3rd levels of association  that were significantly enriched for  <dig> genes predicted to be targets of  <dig> mirnas when compared to the entire genome .
among these, mir-30b/c, - <dig> and − <dig> had 1st tier links to biological processes of hematology and inflammation by influencing the expression of pim <dig>  il10ra, il7r , arrb <dig> , pik3cd, cmpk <dig>  socs <dig>  cysltr <dig>  serpine <dig> and ceacam <dig> .
the remaining mir-200a, - <dig> and -133a/b were linked to organ and tissue development via ereg , ctgf, col8a  and runx <dig> .
thus, using integrative bioinformatics we could predict a prominent position for mir-30b/c and secondary positions for mir-133a/b, - <dig>  -200a, - <dig> and − <dig> in a network based on the number of target genes and 1st tier links with biological processes and pathways that involved the regulation of immunity and organogenesis, two classes of processes previously linked to pathogenesis of biliary atresia  <cit> .
tissue and cellular expression of mirnas
mirnas are known to work in a cell- and tissue-specific manner and to influence hepatobiliary organogenesis  <cit> .
based on the period of susceptibility of balb/c mice to rrv restricted to the first 2– <dig> days of life,  <cit>  we determined the expression level of this select group of mirnas in livers and ehbds at  <dig>   <dig>  and  <dig> days of postnatal age and 6– <dig> weeks of life .
focusing first on the pattern of postnatal development in the liver, mir-30c and -200a increased in suckling mice at all time points by 2– <dig> fold above adult levels , while mir- <dig> tended to have lower levels but did not reach statistical significance.
in contrast, the developmental expression in ehbds of mir-133b, - <dig> and -200a was lowest at  <dig> days of age and increased progressively to adult levels, while mir- <dig>  - <dig> and -30c was highest during the suckling period .
by directly comparing the expression levels in the liver and ehbds, we found that mir-133a/b, - <dig>  -200a, - <dig> and − <dig> were consistently higher in ehbds above livers at most time points, whereas mir-30c expression was lower in ehbds when normalized to the expression level of u <dig> mirna as an endogenous control .
to obtain insight into the normal expression of mirnas at the cellular level, we performed in situ hybridization of mirnas in ehbds from mice at  <dig> days of life and found that mir-200a was expressed largely in cholangiocytes of the duct epithelium and peribiliary glands, and the remaining  <dig> mirnas were expressed in cholangiocytes and subepithelial stromal cells .
furthermore, to investigate whether the decreased expression of the mirnas in ehbd after rrv challenge was not a consequence of a decrease in the number of cholangiocytes during the biliary injury, we quantified the expression levels of the  <dig> mirnas in a cholangiocyte cell line  cultured in the presence or absence of rrv.
this in vitro system has been shown to recapitulate the cell-specific pattern of rrv infection and shared mechanism of virus-cholangiocyte interaction  <cit> .
after  <dig> hours of rrv infection , the expression levels were downregulated by  <dig>  to  <dig> -fold for mir- <dig>  mir- <dig>  mir-30c and mir-200a  below levels of the vehicle control.
interestingly, the expression of mir- <dig> and mir-133b trended down in rrv-challenged cholangiocytes, but did not reach to significance.
discussion
from two comprehensive datasets displaying the expression of mirnas and mrnas in ehbds at early phases of epithelial injury, onset of duct obstruction, and duct atresia, we identified mir-30b/c, -133a/b, - <dig>  -200a, - <dig> and − <dig> as candidate mirnas with potential roles in pathogenesis of experimental biliary atresia.
the selection of mirnas was based on a stringent statistical approach to identify mirnas based on the highest changes in the level of expression, the simultaneous over-expression of target genes in a tissue- and time-restricted fashion, and the expression of mirnas in the epithelial lining of ehbds.
the mirnas formed groups with 1st-to-3rd tier associations with genes involved in biological processes and pathways regulating tissue organogenesis and immune response, processes that have been linked to pathogenic mechanisms of biliary atresia  <cit> .
mirnas are increasingly recognized as regulators of liver physiology and diseases  <cit> .
by regulating the levels of specific mrnas and their posttranscriptional regulation, mirnas have been linked to the pathogenesis of viral hepatitis, nonalcoholic fatty liver disease and steatohepatitis, fibrogenesis and hepatocellular carcinoma.
for biliary atresia, a recent report identified mir-29a to be increased in the livers of neonatal mice subjected to a similar rrv infection protocol, and to target the igf <dig> and il1rap genes in vivo  <cit> .
in our study, we focused primarily on ehbds because it is the primary site of the inflammatory response that injures cholangiocytes and produces the obstructive lesion typical of experimental atresia.
previously, we used a similar approach to survey the gene expression signature in bile ducts in search of genes or gene-groups with potential roles in pathogenesis of biliary atresia  <cit> .
the strength of the approach was validated in subsequent studies showing that the suppression of gene groups induced by the loss of cd8+ lymphocytes  <cit>  and natural killer  <cit>  and dendritic cells  <cit>  substantially decreased epithelial injury, prevented bile duct obstruction, and improved cholestasis.
reasoning that the highest fold changes in mirna expression in the microarray are more likely to be validated by other techniques  and represent activation or suppression of their gene products, we primarily selected those mirnas with ≥ <dig> fold increase or decrease relative to age-matched saline-controls.
this stringent approach selected  <dig> mirnas, from which we focused on  <dig> mirnas based on their shared expression behavior at  <dig> and  <dig> days, times when ehbds undergo inflammatory obstruction and atresia, respectively.
among the  <dig> mirnas, mir-30b/c, -133a/b, - <dig>  -200a, - <dig> and − <dig> emerged as chief candidates of pathogenesis based on the inverse relationship with the increased expression level of their target genes in ehbd, high mirna expression in ehbds relative to livers , expression in cholangiocytes of the biliary epithelium, and a decreased expression of mir- <dig>  - <dig>  - <dig> and -200a in a cholangiocyte line infected with rrv.
this list of mirnas is much smaller than the broad mirna profile of livers from rrv-challenged mice reported by hand et.al.
<cit>  notably, our findings of suppressed expression of mir-30b/c, - <dig>  -200a and − <dig> in ehbds are similar to the suppression reported previously;  <cit>  however, the finding of high level of hepatic mir-29a was not strictly reproduced in ehbds, which in fact had an increased expression of the family member mir-29b to  <dig>  fold above saline controls at the time of duct obstruction.
it is possible that different members of the mir- <dig> family may produce differential biological effects in a tissue-specific fashion , although we cannot rule out the possibility that the lower expression in our model may relate to a progressive loss of cholangiocytes populating the bile duct mucosa.
the main biological processes and diseases previously assigned to most of the  <dig> selected mirnas included hepatocellular and cholangiocarcinoma, nonalcoholic fatty liver disease and steatohepatitis, and viral hepatitis infection.
we also found genes associated with morphogenesis/development despite the use of control newborn mice that were age-matched to rrv-infected mice, thus avoiding the confounding variable of post-natal age.
this suggests a biological scenario in which the selected genes have a role in the cellular response to rrv in addition to their traditional role in development.
perhaps more relevant to our experimental model of a biliary disease , only mir-30b/c, -200b, - <dig> and − <dig> have been reported to change their expression levels in cholangiocarcinoma tissues or cell lines,  <cit>  with mir- <dig> family members increasing in lipopolysaccharide-induced nfκb activation in cholangiocytes and after cryptosporidium parvum infection, and being required for hepatobiliary development  <cit> .
consistent with an involvement in our model, mir-30b/c had the largest number of target genes with increased expression at the times of duct obstruction and atresia, and the largest number of 1st tier links with genes regulating inflammation and immunity, which are thought to be chief biological processes involved in the pathogenesis of biliary atresia.
CONCLUSIONS
in summary, using integrative bioinformatics to screen the biliary mirna and mrna expression profiles in a complementary fashion, the expression of mirnas in a tissue and cell-specific fashion, and the predicted interaction with genes and gene-groups, we identified mir-30b/c, -133a/b, - <dig>  -200a, - <dig> and − <dig> as candidate mirnas with potential roles in pathogenesis of experimental biliary atresia.
among these, our data point to mir-30b/c as an attractive mirna for future mechanistic studies to define how its expression in cholangiocytes and subepithelial cells modulate pathogenic mechanisms of disease by regulation of their target genes.

 biliary atresia, one of the most common causes of neonatal cholestasis, results from a fibro-inflammatory obstruction of extrahepatic bile ducts of unknown etiology. if diagnosed early, surgical intervention with hepatoportoenterostomy has the potential to restore bile flow, but ongoing liver injury results in rapid progression to cirrhosis and the need for liver transplantation by years of age in ~50% of children. contemporary studies suggest that the pathogenesis of bile duct injury results from an interplay between environmental factors and a biological response that involves cells of the immune system, injury and proliferation of the biliary epithelium, and excessive deposition of extracellular matrix. insight into the activation of these processes was obtained from large-scale gene expression analyses in livers of infants at the time of diagnosis and from extrahepatic bile ducts and gallbladder obtained from neonatal mice in a well-established model of rotavirus-induced experimental biliary atresia. in this model, rhesus rotavirus type-a infection within 2– days of life leads to an inflammatory obstruction of extrahepatic bile ducts within a week, with age-restricted susceptibility. this model has been shown to recapitulate many features of the tissue injury and pathogenesis observed in humans with biliary atresia. more recently, an investigation of livers from this mouse model suggested a potential role for micrornas in pathogenesis of disease. micrornas are short non-coding rnas that regulate gene expression at post-transcriptional levels by facilitating degradation and/or inhibiting translation of target mrnas. mirnas may play important roles in the development of the hepatobiliary system, and cell- and tissue-specific mirna expression profiles are altered in some diseases, such as cirrhosis and cancers. to obtain insight into the role of mirnas in pathogenesis of biliary atresia, we performed comprehensive expression analyses of mirnas and mrna from ehbds at the times of epithelial injury, lumenal obstruction, and atresia in the experimental mouse model. using integrative in silico approaches for mirna and mrna expression profiles, we found a coordinated temporal expression of a selected number of mirnas and their target genes, with the activation of molecular networks regulating organogenesis and immune responses. spatially, the expression of these mirnas was higher in ehbds, and was localized in cholangiocytes and in stromal cells of the subepithelial compartment.  time-dependent suppression of mirnas in ehbds to search for mirnas with potential roles in the pathogenesis of biliary atresia, we first performed a comprehensive survey of mirna expression patterns in extrahepatic bile ducts and gallbladder of neonatal mice challenged with rrv within hours of birth. all datasets are deposited in the gene expression omnibus repository, with accession code gse we focused on the analysis of ehbds, rather than the liver, because they are the primary site of tissue injury, beginning with an epithelial insult, followed by lumenal obstruction with inflammatory cells and later by atresia. in these studies, all mice developed jaundice by days, which persisted and became associated with poor weight gain until the time of death by days after rrv challenge, as described previously. among a total of mirnas, changed their expression levels in ehbds after rrv challenge by at least fold when compared to saline-injected control mice. among these mirnas, only mir-let-7e* was differentially expressed in the rrv group at early phases of epithelial injury, with a fold decrease below controls, while a greater number of mirnas was downregulated at days and days. interestingly, only mirnas were upregulated by more than 2-fold, which were all at day after rrv challenge and included mir-29b, which was reported to be elevated in the liver of newborn mice subjected to the same experimental model. to identify candidate mirnas with a potential role in the pathogenesis of biliary injury, we selected mirnas that showed the same pattern of expression at the times of bile duct obstruction and atresia. we found mirnas that were consistently suppressed at both time points; there was no mirna that was overexpressed at both time points. most of these mirnas have been implicated in mechanisms of hepatocellular carcinoma, hepatitis c virus infection of hepatocytes, and nonalcoholic fatty liver disease and steatohepatitis. most notably, mir-30b/c has been reported to influence transforming growth factor beta 1-induced epithelial to mesenchymal transition and biliary development and infection, while mir-200b, - and − have been linked to cholangiocarcinoma. combined, the pattern of time-dependent suppression of mirnas and the previous link to pathogenesis of hepatobiliary diseases formed a rationale for new experiments to investigate the expression behavior of those genes predicted to be targets of this subset of mirnas at different stages of experimental biliary atresia. * minus sign implies fold change below controls. fold changes are expressed in rrv-challenged mice relative to saline-injected controls. tgf: transforming growth factor, lps: lipopolysaccharide, hcc: hepatocellular carcinoma, hcv: hepatitis c virus, emt: epithelial-to-mesenchymal transition, nafld: nonalcoholic fatty liver disease, nash: nonalcoholic steatohepatitis, hsc: hepatic stellate cells. increased expression of target genes in ehbds because mirnas execute their biological roles by regulating expression levels of target genes, we developed a whole-genome expression platform using rna from ehbds at the same time points after rrv challenge. to integrate the mirnas with the expression levels of the related genes, we first built a list of putative target genes based on targetscan and miranda prediction algorithms, and identified genes shared by both algorithms. second, we mined the gene expression platform to select genes that changed by ≥ fold either at and days after rrv challenge when compared to saline controls, and found genes fulfilling these criteria at p < and 5% fdr. a comparison of these two lists revealed that genes were shared by both lists. third, given that mirnas are thought to function by inhibiting the level of gene expression and that the mirnas were suppressed after rrv challenge, we examined the list of genes to select genes as potential mirna targets based on an increase in the expression by ≥ fold in ehbds, and got genes at days and genes at days after rrv challenge. analyzing the association among the mirnas and their potential target genes, we found that mirnas were associated with increased expression of at least target genes at the time of lumenal obstruction: a) mir-30b and -30c were associated with overexpression of mrna for pik3cd, runx cecam cysltr gda, serpine soc and cmpk2; b) mir- with hmga il10ra, pim il7r and has2; and c) mir-200a with runx gbp and has. notably, although their selection was based on an overexpression at days, cluster analysis showed that the increase in some of these genes began as early as days and generally persisted through the time of duct atresia. from the genes selected based on the increased expression at the time of atresia, genes were increased, of which were targets of mir-30b/c. the linkage of coordinately overexpressed genes with a small group of mirnas formed the rationale for informatics-based analyses to explore the relationship of these genes and mirnas in terms of biological pathways and function. functional enrichment analysis of target genes using toppcluster as a platform to integrate informatics, streams of gene and protein function, regulation and animal models of disease, we found 1st-to-3rd levels of association that were significantly enriched for genes predicted to be targets of mirnas when compared to the entire genome. among these, mir-30b/c, - and − had 1st tier links to biological processes of hematology and inflammation by influencing the expression of pim il10ra, il7r, arrb, pik3cd, cmpk socs cysltr serpine and ceacam. the remaining mir-200a, - and -133a/b were linked to organ and tissue development via ereg, ctgf, col8a and runx. thus, using integrative bioinformatics we could predict a prominent position for mir-30b/c and secondary positions for mir-133a/b, - -200a, - and − in a network based on the number of target genes and 1st tier links with biological processes and pathways that involved the regulation of immunity and organogenesis, two classes of processes previously linked to pathogenesis of biliary atresia. tissue and cellular expression of mirnas mirnas are known to work in a cell- and tissue-specific manner and to influence hepatobiliary organogenesis. based on the period of susceptibility of balb/c mice to rrv restricted to the first 2– days of life, we determined the expression level of this select group of mirnas in livers and ehbds at and days of postnatal age and 6– weeks of life. focusing first on the pattern of postnatal development in the liver, mir-30c and -200a increased in suckling mice at all time points by 2– fold above adult levels, while mir- tended to have lower levels but did not reach statistical significance. in contrast, the developmental expression in ehbds of mir-133b, - and -200a was lowest at days of age and increased progressively to adult levels, while mir- - and -30c was highest during the suckling period. by directly comparing the expression levels in the liver and ehbds, we found that mir-133a/b, - -200a, - and − were consistently higher in ehbds above livers at most time points, whereas mir-30c expression was lower in ehbds when normalized to the expression level of u mirna as an endogenous control. to obtain insight into the normal expression of mirnas at the cellular level, we performed in situ hybridization of mirnas in ehbds from mice at days of life and found that mir-200a was expressed largely in cholangiocytes of the duct epithelium and peribiliary glands, and the remaining mirnas were expressed in cholangiocytes and subepithelial stromal cells. furthermore, to investigate whether the decreased expression of the mirnas in ehbd after rrv challenge was not a consequence of a decrease in the number of cholangiocytes during the biliary injury, we quantified the expression levels of the mirnas in a cholangiocyte cell line cultured in the presence or absence of rrv. this in vitro system has been shown to recapitulate the cell-specific pattern of rrv infection and shared mechanism of virus-cholangiocyte interaction. after hours of rrv infection, the expression levels were downregulated by to -fold for mir- mir- mir-30c and mir-200a below levels of the vehicle control. interestingly, the expression of mir- and mir-133b trended down in rrv-challenged cholangiocytes, but did not reach to significance. discussion from two comprehensive datasets displaying the expression of mirnas and mrnas in ehbds at early phases of epithelial injury, onset of duct obstruction, and duct atresia, we identified mir-30b/c, -133a/b, - -200a, - and − as candidate mirnas with potential roles in pathogenesis of experimental biliary atresia. the selection of mirnas was based on a stringent statistical approach to identify mirnas based on the highest changes in the level of expression, the simultaneous over-expression of target genes in a tissue- and time-restricted fashion, and the expression of mirnas in the epithelial lining of ehbds. the mirnas formed groups with 1st-to-3rd tier associations with genes involved in biological processes and pathways regulating tissue organogenesis and immune response, processes that have been linked to pathogenic mechanisms of biliary atresia. mirnas are increasingly recognized as regulators of liver physiology and diseases. by regulating the levels of specific mrnas and their posttranscriptional regulation, mirnas have been linked to the pathogenesis of viral hepatitis, nonalcoholic fatty liver disease and steatohepatitis, fibrogenesis and hepatocellular carcinoma. for biliary atresia, a recent report identified mir-29a to be increased in the livers of neonatal mice subjected to a similar rrv infection protocol, and to target the igf and il1rap genes in vivo. in our study, we focused primarily on ehbds because it is the primary site of the inflammatory response that injures cholangiocytes and produces the obstructive lesion typical of experimental atresia. previously, we used a similar approach to survey the gene expression signature in bile ducts in search of genes or gene-groups with potential roles in pathogenesis of biliary atresia. the strength of the approach was validated in subsequent studies showing that the suppression of gene groups induced by the loss of cd8+ lymphocytes and natural killer and dendritic cells substantially decreased epithelial injury, prevented bile duct obstruction, and improved cholestasis. reasoning that the highest fold changes in mirna expression in the microarray are more likely to be validated by other techniques and represent activation or suppression of their gene products, we primarily selected those mirnas with ≥ fold increase or decrease relative to age-matched saline-controls. this stringent approach selected mirnas, from which we focused on mirnas based on their shared expression behavior at and days, times when ehbds undergo inflammatory obstruction and atresia, respectively. among the mirnas, mir-30b/c, -133a/b, - -200a, - and − emerged as chief candidates of pathogenesis based on the inverse relationship with the increased expression level of their target genes in ehbd, high mirna expression in ehbds relative to livers, expression in cholangiocytes of the biliary epithelium, and a decreased expression of mir- - - and -200a in a cholangiocyte line infected with rrv. this list of mirnas is much smaller than the broad mirna profile of livers from rrv-challenged mice reported by hand et.al. notably, our findings of suppressed expression of mir-30b/c, - -200a and − in ehbds are similar to the suppression reported previously; however, the finding of high level of hepatic mir-29a was not strictly reproduced in ehbds, which in fact had an increased expression of the family member mir-29b to fold above saline controls at the time of duct obstruction. it is possible that different members of the mir- family may produce differential biological effects in a tissue-specific fashion, although we cannot rule out the possibility that the lower expression in our model may relate to a progressive loss of cholangiocytes populating the bile duct mucosa. the main biological processes and diseases previously assigned to most of the selected mirnas included hepatocellular and cholangiocarcinoma, nonalcoholic fatty liver disease and steatohepatitis, and viral hepatitis infection. we also found genes associated with morphogenesis/development despite the use of control newborn mice that were age-matched to rrv-infected mice, thus avoiding the confounding variable of post-natal age. this suggests a biological scenario in which the selected genes have a role in the cellular response to rrv in addition to their traditional role in development. perhaps more relevant to our experimental model of a biliary disease, only mir-30b/c, -200b, - and − have been reported to change their expression levels in cholangiocarcinoma tissues or cell lines, with mir- family members increasing in lipopolysaccharide-induced nfκb activation in cholangiocytes and after cryptosporidium parvum infection, and being required for hepatobiliary development. consistent with an involvement in our model, mir-30b/c had the largest number of target genes with increased expression at the times of duct obstruction and atresia, and the largest number of 1st tier links with genes regulating inflammation and immunity, which are thought to be chief biological processes involved in the pathogenesis of biliary atresia.  in summary, using integrative bioinformatics to screen the biliary mirna and mrna expression profiles in a complementary fashion, the expression of mirnas in a tissue and cell-specific fashion, and the predicted interaction with genes and gene-groups, we identified mir-30b/c, -133a/b, - -200a, - and − as candidate mirnas with potential roles in pathogenesis of experimental biliary atresia. among these, our data point to mir-30b/c as an attractive mirna for future mechanistic studies to define how its expression in cholangiocytes and subepithelial cells modulate pathogenic mechanisms of disease by regulation of their target genes. 
these mirnas showed higher expression in ehbds above livers, a unique expression in cholangiocytes and the subepithelial compartment, and were downregulated in a cholangiocyte cell line after rrv infection. in this study, we aimed to investigate the entire biliary transcriptome to identify mirnas with potential role in the pathogenesis of bile duct obstruction. next, we obtained putative target genes of the mirnas using in silico target prediction algorithms. by integrating this result with a genome-wide gene expression analysis of the same tissue, we identified potential target genes with coordinate expression by the mirnas. functional analysis of these target genes revealed a significant relevance of mir-30b/c, -133a/b, - -200a, - and − based on increases in expression of at least target genes in the same tissue and 1st-to-3rd tier links with genes and gene-groups regulating organogenesis and immune response. integrative genomics reveals functional relevance of mir-30b/c, -133a/b, - -200a, - and − the coordinate expression of mirnas and target genes in a temporal-spatial fashion suggests a regulatory role of these mirnas in pathogenesis of experimental biliary atresia. by profiling the expression levels of mirna in extrahepatic bile ducts and gallbladder from a murine model of biliary atresia, we identified mirnas whose expression was suppressed at the times of duct obstruction and atresia. biliary atresia is a fibroinflammatory obstruction of extrahepatic bile duct that leads to end-stage liver disease in children. despite advances in understanding the pathogenesis of biliary atresia, very little is known about the role of micrornas in onset and progression of the disease.
 biliary atresia is a fibroinflammatory obstruction of extrahepatic bile duct that leads to end-stage liver disease in children. despite advances in understanding the pathogenesis of biliary atresia, very little is known about the role of micrornas in onset and progression of the disease. in this study, we aimed to investigate the entire biliary transcriptome to identify mirnas with potential role in the pathogenesis of bile duct obstruction.  by profiling the expression levels of mirna in extrahepatic bile ducts and gallbladder from a murine model of biliary atresia, we identified mirnas whose expression was suppressed at the times of duct obstruction and atresia. next, we obtained putative target genes of the mirnas using in silico target prediction algorithms. by integrating this result with a genome-wide gene expression analysis of the same tissue, we identified potential target genes with coordinate expression by the mirnas. functional analysis of these target genes revealed a significant relevance of mir-30b/c, -133a/b, - -200a, - and − based on increases in expression of at least target genes in the same tissue and 1st-to-3rd tier links with genes and gene-groups regulating organogenesis and immune response. these mirnas showed higher expression in ehbds above livers, a unique expression in cholangiocytes and the subepithelial compartment, and were downregulated in a cholangiocyte cell line after rrv infection.  integrative genomics reveals functional relevance of mir-30b/c, -133a/b, - -200a, - and − the coordinate expression of mirnas and target genes in a temporal-spatial fashion suggests a regulatory role of these mirnas in pathogenesis of experimental biliary atresia. cholestasisextrahepatic bile ductinformaticscholangiocytes 