BACKGROUND
avian influenza remains a serious threat to poultry and human health.
from december  <dig> to april  <dig>  more than  <dig> human infections and  <dig> deaths have been reported to the world health organization
<cit> .
outbreaks of h5n <dig> in poultry swept from southeast asia to many parts of the world.
to date, there is still no sign that the epidemic is under control.
while it has been well documented that infection with h5n <dig> results in high mortality in humans
<cit> , the cellular pathway leading to such adverse outcome is unknown.
the naive host immune system cannot be the sole explanation as infection of other avian influenza viruses, e.g.
h9n <dig>  only results in mild infections
<cit> .
while the predilection of h5n <dig> towards cells in the lower respiratory tract contributes to the development of severe pneumonia
<cit> , the available clinico-pathological evidence indicates that the infected patients progress to multi-organ failure early in the course of illness, and the degree of organ failure is out of proportion to the involvement of infection
<cit> .
cytokine storm and reactive haemophagocytic syndrome are the key features that distinguish h5n <dig> infection from severe seasonal influenza.
these indirect mechanisms seem to play an even more important role than direct cell killing due to lytic viral infection.
mirnas, a new class of endogenous, 18– <dig> nucleotide long noncoding and single-stranded rnas, were recently discovered in both animals and plants.
they trigger translational repression and/or mrna degradation mostly through complementary binding to the 3′utr of target mrnas.
studies have shown that mirnas can regulate a wide array of biological processes such as cell proliferation, differentiation, and apoptosis
<cit> .
given the nature of viruses, being intracellular parasites and using the cellular machinery for their survival and replication, the success of the virus essentially depends on its ability to effectively and efficiently use the host machinery to propagate itself.
this dependence on the host also makes it susceptible to the host gene-regulatory mechanisms, i.e.
the host mirnas may also have direct or indirect regulatory role on viral mrnas expression.
recently, several reports indicated that mirnas can target influenza viruses and regulate influenza virus replication.
in one report,  <dig> pig-encoded mirnas and  <dig> human-encoded mirnas were found to have putative targets in swine influenza virus and swine-origin  <dig> a/h1n <dig> influenza virus genes, respectively
<cit> .
in another report, results showed that mir- <dig>  mir- <dig> and mir- <dig> could inhibit replication of h1n <dig> influenza a virus through binding to the conserved region of the pb <dig> gene
<cit> .
these mirnas could downregulate pb <dig> expression through mrna degradation instead of translation repression
<cit> .
besides targeting influenza virus, cellular mirnas were also implicated in the lethal infections of mice with a highly pathogenic  <dig> pandemic h1n <dig> influenza virus
<cit> .
a previous study on mirna gene expression in avian influenza virus infected chicken showed that mir- <dig>  which was previously reported to be associated with immune-related signal pathways in mammals, was found to be differentially expressed in infected tissues
<cit> .
moreover, a study of profiling cellular mirnas of lung tissue from cynomolgus macaques infected with a highly pathogenic h5n <dig> avian and a less pathogenic  <dig> h1n <dig> reassortant virus identified that  <dig> mirnas were associated with the extreme virulence of highly pathogenic h5n <dig> avian virus
<cit> .
also, the predicted gene targets of the identified mirnas were found to be associated with aberrant and uncontrolled inflammatory responses and increased cell death
<cit> .
this study aimed at elucidating how avian influenza infection perturbs the human gene regulatory pathways leading to adverse pathological events, e.g.
cytokine storm.
we hypothesized that mirnas could be involved in influenza virus infection response and began addressing this hypothesis using a microarray-based screening.
the ultimate goal of this study is to generate essential information for further studies to identify novel intervention targets to ameliorate the adverse outcome of infection.
RESULTS
differential mirna expression in h5n <dig> and h1n <dig> influenza virus infected cells
the cell line - nci-h <dig>  infected with various preparations of influenza viruses was analysed for mirna expression profiles subsequently.
a list of differentially expressed mirna was identified for subtypes h1n <dig> and h5n <dig>  respectively , and the temporal pattern of expression was delineated.
among the listed profiles of differentially up-regulated mirna, it was found that mir- <dig>  mir-181c*, mir- <dig>  mir29b, mir-324-5p, and mir- <dig> were up-regulated  at 3-hour post-infection with subtype h <dig> as compared with non-infected control cells.
at this time point, only mir- <dig> was found to be slightly induced in subtype h <dig> infected cells.
at 6-hour post-infection, it was found that mir-483-3p was up-regulated  in h5n <dig> infected cells while mir- <dig> was found to be up-regulated  in h1n <dig> infected cells.
at  <dig> and 24-hour post-infection, mir- <dig>  mir- <dig>  mir-574-3p, and mir- <dig> were up-regulated  in h5n <dig> infected cells.
for h1n <dig> infected cells, at  <dig> and 24-hour post-infection, mir-188-5p, mir- <dig>  mir-1274a, mir-1274b, mir <dig>  mir183*, mir-18b, mir-19a, mir21*, mir-301a, mir- <dig>  mir- <dig>  and mir- <dig> were found to be up-regulated  .
among the listed profiles of differentially down-regulated mirna as compared with non-infected control cells, it was found that mir-574-5p was down regulated  in h5n <dig> infected cells at 3-hour post-infection.
for h1n <dig> infected cells, mir-23a, mir-574-3p and mir-574-5p were down-regulated  at this time point.
at 6-hour post-infection, mir- <dig>  mir-20a*, mir-362-5p, mir- <dig>  mir- <dig>  and mir574-5p were found to be down-regulated  in h5n <dig> infected cells.
at the same time point , mir-15a*, mir- <dig>  mir-183*, mir-34b, mir- <dig>  and mir-574-5p were found to be down-regulated  in h1n <dig> infected cells.
furthermore, at  <dig>  and 24-hour post-infection, mir- <dig>  mir-1274a, mir-1274b, mir- <dig>  mir-18b, mir-21*, mir- <dig>  mir-100*, mir- <dig>  mir <dig>  and mir21* were found to be down-regulated  in h5n <dig> infected cells.
at these time points, only mir- <dig>  and mir-17* were found to be down-regulated  in h1n <dig> infected cells .
from the results, we found that similar changes in mirna profiles were observed in both h1n <dig> and h5n <dig> infection.
however, the magnitude of fold-changes which occurred in h1n <dig> infection were much lower than that in h5n <dig> infection.
confirmation of mirna expression profile by real-time pcr
the microarray data were further confirmed using taqman quantitative rt-pcr  assays.
there were general consistency between taqman qrt-pcr assays and microarray results.
it was found that six mirnas  were initially up-regulated at  <dig> hours post-infection.
the degree of up-regulation was more prominent in h5n <dig> infection  than in h1n <dig> infection .
it was also found that these mirnas became down-regulated during 6-to- <dig> hours post-infection.
the degree of down-regulation was also higher in h5n <dig> infection than in h1n <dig> infection .
target prediction of the mirna expression profile
we then examined the list of targets predicted by targetscan computer software  for the mirna species that had the most consistent and significant changes in expression following influenza a virus infection
<cit> .
the targetscan results showed that many of the target genes were involved in the inflammatory response and cell death pathways.
interestingly, one of the target prediction results showed that there was a 3′ untranslated region  binding site on tgf-β <dig> for mir- <dig>  the mir- <dig> sequence is: 3′- gguagaaauggucugucacaau - 5′, while that of tgf-β <dig> 3′utr is: 5′-agagccuugguucaucaguguua-3′.
we had previously reported that tgf-β <dig> was an important cytokine involved in the inflammatory response of avian influenza a virus infection
<cit>  and, together with the results showing that the expression of mir- <dig> was altered during the time course of influenza a virus infection, we selected mir- <dig> for further functional analysis in this study.
mir- <dig> represses the expression of tgf-β <dig> mrna
in addition to the mirna target prediction results, by using ecoptic expression of mir- <dig>  the level of tgf-β <dig> mrna was found to be significantly decreased in mir- <dig> transfected cells but not in negative-control mirna mimic transfected cells .
in this over-expression system we could determine that the 3′utr was the mir- <dig> target and the decreased tgf-β <dig> mrna level might be due to the binding of mir- <dig> to the 3′utr of tgf-β <dig> mrna which reduced the half-lives of tgf-β <dig> mrna.
effect of inhibition of mir- <dig> in influenza a virus infection
the functional relevance of changes in mir- <dig> expression during influenza a virus infection was assessed using mirna inhibitors.
chemically modified, single stranded nucleic acids anti-mir mir- <dig> inhibitor and negative control were transfected into h <dig> cells for  <dig> hours.
we had previously shown that this was sufficient time to obtain oligonucleotide delivery in h <dig> cells when examining the inhibition of tgf-β <dig> mrna expression.
after the cells were pre-treated with anti-mir mir- <dig> for  <dig> hours, they were then infected with h1n <dig> or h5n <dig>  respectively.
after the infection processes, anti-mir mir- <dig> was transfected again into the virus infected cells and incubated for another  <dig> hours.
the results of this experiment showed that the anti-mir mir- <dig> inhibitor could cause an increase in tgf-β <dig> protein expression in h1n <dig> or h5n <dig> infected cells, as compared to cells only infected with h1n <dig> or h5n <dig> but without anti-mir mir- <dig> inhibitor treatment .
the effect was also more prominent in h5n <dig> infection than that of h1n <dig>
discussion
in this study we examined the connection between influenza a virus infection and the global patterns of cellular mirna expression.
the major observations from this work were that influenza a virus infection resulted in the altered regulation of cellular mirnas.
avian influenza a virus can alter cellular mirnas to a greater extent than that of seasonal human influenza a virus.
influenza a virus affects the regulation of many cellular processes.
in some cases, these changes are directed by the virus for its advantage and others are cellular defense responses to infection.
here, we found that influenza a virus infection led to altered regulation of cellular mirnas.
given the number of genes that can be regulated by individual mirnas and the number of mirnas expressed in cells, this greatly expands the range of possible virus-host regulatory interactions.
the complexity is underscored by there being no uniform global pattern of regulation; rather, it appears that individual  mirna are independently regulated, some positively and some negatively.
persistent and transient effects were seen, and changes in mirna expression profiles were linked to the time course of infection.
as a summary, mir- <dig>  mir- <dig> and mir-574-3p were up-regulated  at 24-hour post-infection with subtype h <dig> as compared with non-infected control cells.
moreover, mir-100*, mir-21*, mir- <dig>  mir-1274a and mir1274b were found to be down-regulated  in infection with subtype h <dig>  particularly at  <dig> or  <dig> hours post-infection as compared with non-infected control cells.
interestingly, many of the virally regulated mirnas were predicted by targetscan to target important biological pathways, immune-related signal pathways and have altered regulation in some cellular defense and some states of cellular differentiation
<cit> .
in our study, we found that the expression of mir- <dig> was affected by influenza a virus infection.
to validate the in silico findings empirically on the target of mir- <dig>  we checked whether transient-transfection of anti- and pre-mir- <dig> into nci-h <dig> cells resulted in tgf-β <dig> regulation.
in our experiment, the transfection efficiency was an important factor affecting the degree of regulation on the target gene.
in the case of higher transfection efficiency, as more mirna would be transfected into the cells, the effect of gene regulation by mirna transfected would be greater.
in our study, the transfection efficiency was about  <dig>  ±  <dig> % , which was considered to be adequate for further functional analyses.
during transfection, some oligonucleotide molecules were sequestered in internal vesicles and physically separated from their targets in the cytoplasm; and then released during cell lysis.
therefore monitoring mirnas by qpcr after transfection would not be valuable.
previous researchers of this procedure had highly recommended investigating the target mrnas and proteins instead of mirna quantification.
the time point of 24-hour post-transfection or post-infection was chosen for evaluation because mir- <dig> induction was observed at the early stage of virus infection, and sufficient time might be required for the mir- <dig> to have effect on its target, so we had chosen 24-hour post-transfection or post-infection for evaluation of the effect of this mirna.
indeed, upon detecting the tgf-β <dig> expression at mrna and protein levels, we found that the altered mir- <dig> expression would affect the expression of the cytokine- tgf-β <dig>  literature search on the background of mir- <dig> confirmed that mir- <dig> is a member of the mir- <dig> family .
previous studies of mir- <dig> were mainly on its role in cancer.
it has been reported that mir- <dig> were markedly downregulated in cells that had undergone epithelial to mesenchymal in response to tgf-β.
mir- <dig> was also found to be overexpressed in ovarian and colorectal cancers
<cit>  and down-regulated in prostate, hepatocellular, renal cell carcinoma and in gastric cancer tissues
<cit>  raising a controversial issue about the role of mir- <dig> in cancer progression.
furthermore, the mir- <dig> family members play roles in maintaining the epithelial phenotype of cancer cells
<cit> .
a member of this family - mir-200a was also found to be differentially expressed in response to influenza virus infection in another study
<cit> .
the targets of mir-200a are associated with viral gene replication and the jak-stat signaling pathway, which is closely related to type i interferon-mediated innate immune response
<cit> .
however, the effect of mir- <dig> on virus infection was not known, except one recent report showing that enterovirus can induce mir- <dig> and contribute to the shutoff of host protein translation by targeting the translation initiation factor eif4e
<cit> .
in addition, evidence suggests that influenza a virus infection reduces or promotes the expression of the host mir- <dig> in a time dependent manner.
we found that tgf-β <dig> mrna was suppressed in mir- <dig> overexpressed cells.
our observation is in line with another study showing that the 3′utr of tgf-β <dig> mrna contained a target site for mir-141/200a and the expression of tgf-β <dig> was significantly decreased in mir-141/200a transfected cells
<cit> .
furthermore, mir- <dig> may not only work as translational repressors of target mrnas, because it was observed that they also caused a decrease in tgf-β <dig> mrna levels.
these findings are similar to recent data demonstrating that some mirnas can alter the mrna levels of target genes
<cit> .
this ability is probably independent of the ability of these mirnas to regulate the translation of target mrnas
<cit> .
we also noted that antagomir- <dig> moderately increased the accumulation of tgf-β <dig> protein during influenza virus infection.
this might be because, by the use of anti-mir mir- <dig> inhibitor, which decreases the cellular pool of mir- <dig>  the translation control of the tgf-β <dig> mrna was subsequently released and caused the tgf-β <dig> protein to express and accumulate during virus infection.
however, it was also observed that when there was an increase in tgf-β <dig> mrna level, the corresponding tgf-β <dig> protein expression level would be increased, except in the case of non-mir-141-inhibitor treated h5n <dig> infected cells.
in this case, there was a decrease in tgf-β <dig> mrna level, while the tgf-β <dig> protein was increased.
this might be explained by the fact that tgf-β <dig> mrna degradation induced by mir- <dig> might be much faster than that of the corresponding protein degradation.
recently, we had also reported that h1n <dig> was the only subtype that could induce a sustained increase in tgf-β <dig> at protein level
<cit> .
that observation coincides with our results in this study, showing that h1n <dig> infection induced a little amount of mir- <dig> expression, while h5n <dig> infection induced a higher amount of mir- <dig> expression at the early phase of infection.
as a consequence of the higher amount of mir- <dig> in h5n <dig> infection, tgf-β <dig> expression might be more greatly reduced than that in h1n <dig> infection.
since tgf-β <dig> can act as both an immunosuppressive agent and a potent proinflammatory molecule through its ability to attract and regulate inflammatory molecules, it plays a vital role in t-cell inhibition.
furthermore, it has been reported that tgf-β <dig> inhibits th <dig> cytokine-mediated induction of ccl-2/mcp- <dig>  ccl-3/mip-1α, ccl-4/mip-1β, ccl-5/rantes, ccl-9/mip-1γ, cxcl-2/mip- <dig>  and cxcl-10/ip-10
<cit> .
moreover, the pro-inflammatory responses during influenza a virus infection are tightly controlled by anti-inflammatory mediators, such as tgf-β <dig>  to protect the easily damageable lung tissue from destructive side effects associated with virus induced inflammation.
therefore, the downregulation of tgf-β <dig> protein by mir- <dig> may be an important step in the excessive inflammation progression during influenza a virus infection, particularly in h5n <dig> infection.
however, whether the recovery of tgf-β <dig> expression by anti-mir mir- <dig> inhibitor could resolve the hypercytokinemia stage of h5n <dig> infection needs to be further studied.
although our findings were obtained from an in vitro model, we could apply these to the real situation of an in vivo model or tissue comprised of different cell types.
in real bronchial environments, lung epithelial cells are the key target of influenza viruses
<cit> .
after these cells are infected, they will activate an inflammatory cascade which launches a quick antimicrobial reaction and directs adaptive immunity to mount a protective response.
bronchial epithelial cells therefore modulate the activation of monocytes, macrophages, dendritic cells , and t lymphocytes through cytokines and chemokines.
cytokines and chemokines generally function in an autocrine  or paracrine  manner.
these mediators will contribute to the generation of a specific bronchial homeostatic microenvironment that affects the way in which the body copes with the viruses.
this homeostatic “circuit” can inhibit excessive inflammatory response in lung tissues
<cit> .
for example, tgf-β had been reported to mediate a cross-talk between alveolar macrophages and epithelial cells
<cit> .
however, our findings show that, during highly pathogenic h5n <dig> avian virus infection, mir- <dig> would be induced shortly after infection.
with high level of mir- <dig>  the expression of tgf-β would be suppressed from the lung epithelial cells.
without sufficient tgf- β, the pro-inflammatory response might not be tightly controlled in cases of highly pathogenic h5n <dig> avian virus infection.
this might explain the mechanism concerning bronchial infiltration of inflammatory cells, particularly lymphocytes and eosinophils, and the subsequent hyperresponsiveness of the bronchial wall induced by viral infection.
our study has some limitations that will need to be addressed in future studies.
firstly, we did not assess the roles of other mirnas whose expression were also altered after infection.
the mirna microarrays that we used did not contain probes for every known mirna; thus it is possible that influenza a virus infection affects the expression of some other mirnas not yet covered by the kit used in the current study.
secondly, the virus may interact with mirna regulatory pathways differently in other cell or tissue types, or in other physiological status.
CONCLUSIONS
in conclusion, based on the broad-catching mirna microarray approach, we found that dysregulation of mirna expression is mainly observed in highly pathogenic avian influenza infection.
we identified that mir- <dig> was induced at early time points upon influenza a virus infection.
the induction was higher in h5n <dig> infection than that of seasonal h1n <dig> infection.
moreover, tgf-β <dig>  which plays an important role in regulating inflammatory processes, was identified as a target of mir- <dig> binding.
as a result, influenza a virus infection, in particular highly pathogenic h5n <dig>  could affect the inflammatory processes via mir- <dig> induction.

 avian influenza remains a serious threat to poultry and human health. from december to april more than human infections and deaths have been reported to the world health organization. outbreaks of h5n in poultry swept from southeast asia to many parts of the world. to date, there is still no sign that the epidemic is under control. while it has been well documented that infection with h5n results in high mortality in humans, the cellular pathway leading to such adverse outcome is unknown. the naive host immune system cannot be the sole explanation as infection of other avian influenza viruses, e.g. h9n only results in mild infections. while the predilection of h5n towards cells in the lower respiratory tract contributes to the development of severe pneumonia, the available clinico-pathological evidence indicates that the infected patients progress to multi-organ failure early in the course of illness, and the degree of organ failure is out of proportion to the involvement of infection. cytokine storm and reactive haemophagocytic syndrome are the key features that distinguish h5n infection from severe seasonal influenza. these indirect mechanisms seem to play an even more important role than direct cell killing due to lytic viral infection. mirnas, a new class of endogenous, 18– nucleotide long noncoding and single-stranded rnas, were recently discovered in both animals and plants. they trigger translational repression and/or mrna degradation mostly through complementary binding to the 3′utr of target mrnas. studies have shown that mirnas can regulate a wide array of biological processes such as cell proliferation, differentiation, and apoptosis. given the nature of viruses, being intracellular parasites and using the cellular machinery for their survival and replication, the success of the virus essentially depends on its ability to effectively and efficiently use the host machinery to propagate itself. this dependence on the host also makes it susceptible to the host gene-regulatory mechanisms, i.e. the host mirnas may also have direct or indirect regulatory role on viral mrnas expression. recently, several reports indicated that mirnas can target influenza viruses and regulate influenza virus replication. in one report, pig-encoded mirnas and human-encoded mirnas were found to have putative targets in swine influenza virus and swine-origin a/h1n influenza virus genes, respectively. in another report, results showed that mir- mir- and mir- could inhibit replication of h1n influenza a virus through binding to the conserved region of the pb gene. these mirnas could downregulate pb expression through mrna degradation instead of translation repression. besides targeting influenza virus, cellular mirnas were also implicated in the lethal infections of mice with a highly pathogenic pandemic h1n influenza virus. a previous study on mirna gene expression in avian influenza virus infected chicken showed that mir- which was previously reported to be associated with immune-related signal pathways in mammals, was found to be differentially expressed in infected tissues. moreover, a study of profiling cellular mirnas of lung tissue from cynomolgus macaques infected with a highly pathogenic h5n avian and a less pathogenic h1n reassortant virus identified that mirnas were associated with the extreme virulence of highly pathogenic h5n avian virus. also, the predicted gene targets of the identified mirnas were found to be associated with aberrant and uncontrolled inflammatory responses and increased cell death. this study aimed at elucidating how avian influenza infection perturbs the human gene regulatory pathways leading to adverse pathological events, e.g. cytokine storm. we hypothesized that mirnas could be involved in influenza virus infection response and began addressing this hypothesis using a microarray-based screening. the ultimate goal of this study is to generate essential information for further studies to identify novel intervention targets to ameliorate the adverse outcome of infection.  differential mirna expression in h5n and h1n influenza virus infected cells the cell line - nci-h infected with various preparations of influenza viruses was analysed for mirna expression profiles subsequently. a list of differentially expressed mirna was identified for subtypes h1n and h5n respectively, and the temporal pattern of expression was delineated. among the listed profiles of differentially up-regulated mirna, it was found that mir- mir-181c*, mir- mir29b, mir-324-5p, and mir- were up-regulated at 3-hour post-infection with subtype h as compared with non-infected control cells. at this time point, only mir- was found to be slightly induced in subtype h infected cells. at 6-hour post-infection, it was found that mir-483-3p was up-regulated in h5n infected cells while mir- was found to be up-regulated in h1n infected cells. at and 24-hour post-infection, mir- mir- mir-574-3p, and mir- were up-regulated in h5n infected cells. for h1n infected cells, at and 24-hour post-infection, mir-188-5p, mir- mir-1274a, mir-1274b, mir mir183*, mir-18b, mir-19a, mir21*, mir-301a, mir- mir- and mir- were found to be up-regulated. among the listed profiles of differentially down-regulated mirna as compared with non-infected control cells, it was found that mir-574-5p was down regulated in h5n infected cells at 3-hour post-infection. for h1n infected cells, mir-23a, mir-574-3p and mir-574-5p were down-regulated at this time point. at 6-hour post-infection, mir- mir-20a*, mir-362-5p, mir- mir- and mir574-5p were found to be down-regulated in h5n infected cells. at the same time point, mir-15a*, mir- mir-183*, mir-34b, mir- and mir-574-5p were found to be down-regulated in h1n infected cells. furthermore, at and 24-hour post-infection, mir- mir-1274a, mir-1274b, mir- mir-18b, mir-21*, mir- mir-100*, mir- mir and mir21* were found to be down-regulated in h5n infected cells. at these time points, only mir- and mir-17* were found to be down-regulated in h1n infected cells. from the results, we found that similar changes in mirna profiles were observed in both h1n and h5n infection. however, the magnitude of fold-changes which occurred in h1n infection were much lower than that in h5n infection. confirmation of mirna expression profile by real-time pcr the microarray data were further confirmed using taqman quantitative rt-pcr assays. there were general consistency between taqman qrt-pcr assays and microarray results. it was found that six mirnas were initially up-regulated at hours post-infection. the degree of up-regulation was more prominent in h5n infection than in h1n infection. it was also found that these mirnas became down-regulated during 6-to- hours post-infection. the degree of down-regulation was also higher in h5n infection than in h1n infection. target prediction of the mirna expression profile we then examined the list of targets predicted by targetscan computer software for the mirna species that had the most consistent and significant changes in expression following influenza a virus infection. the targetscan results showed that many of the target genes were involved in the inflammatory response and cell death pathways. interestingly, one of the target prediction results showed that there was a 3′ untranslated region binding site on tgf-β for mir- the mir- sequence is: 3′- gguagaaauggucugucacaau - 5′, while that of tgf-β 3′utr is: 5′-agagccuugguucaucaguguua-3′. we had previously reported that tgf-β was an important cytokine involved in the inflammatory response of avian influenza a virus infection and, together with the results showing that the expression of mir- was altered during the time course of influenza a virus infection, we selected mir- for further functional analysis in this study. mir- represses the expression of tgf-β mrna in addition to the mirna target prediction results, by using ecoptic expression of mir- the level of tgf-β mrna was found to be significantly decreased in mir- transfected cells but not in negative-control mirna mimic transfected cells. in this over-expression system we could determine that the 3′utr was the mir- target and the decreased tgf-β mrna level might be due to the binding of mir- to the 3′utr of tgf-β mrna which reduced the half-lives of tgf-β mrna. effect of inhibition of mir- in influenza a virus infection the functional relevance of changes in mir- expression during influenza a virus infection was assessed using mirna inhibitors. chemically modified, single stranded nucleic acids anti-mir mir- inhibitor and negative control were transfected into h cells for hours. we had previously shown that this was sufficient time to obtain oligonucleotide delivery in h cells when examining the inhibition of tgf-β mrna expression. after the cells were pre-treated with anti-mir mir- for hours, they were then infected with h1n or h5n respectively. after the infection processes, anti-mir mir- was transfected again into the virus infected cells and incubated for another hours. the results of this experiment showed that the anti-mir mir- inhibitor could cause an increase in tgf-β protein expression in h1n or h5n infected cells, as compared to cells only infected with h1n or h5n but without anti-mir mir- inhibitor treatment. the effect was also more prominent in h5n infection than that of h1n discussion in this study we examined the connection between influenza a virus infection and the global patterns of cellular mirna expression. the major observations from this work were that influenza a virus infection resulted in the altered regulation of cellular mirnas. avian influenza a virus can alter cellular mirnas to a greater extent than that of seasonal human influenza a virus. influenza a virus affects the regulation of many cellular processes. in some cases, these changes are directed by the virus for its advantage and others are cellular defense responses to infection. here, we found that influenza a virus infection led to altered regulation of cellular mirnas. given the number of genes that can be regulated by individual mirnas and the number of mirnas expressed in cells, this greatly expands the range of possible virus-host regulatory interactions. the complexity is underscored by there being no uniform global pattern of regulation; rather, it appears that individual mirna are independently regulated, some positively and some negatively. persistent and transient effects were seen, and changes in mirna expression profiles were linked to the time course of infection. as a summary, mir- mir- and mir-574-3p were up-regulated at 24-hour post-infection with subtype h as compared with non-infected control cells. moreover, mir-100*, mir-21*, mir- mir-1274a and mir1274b were found to be down-regulated in infection with subtype h particularly at or hours post-infection as compared with non-infected control cells. interestingly, many of the virally regulated mirnas were predicted by targetscan to target important biological pathways, immune-related signal pathways and have altered regulation in some cellular defense and some states of cellular differentiation. in our study, we found that the expression of mir- was affected by influenza a virus infection. to validate the in silico findings empirically on the target of mir- we checked whether transient-transfection of anti- and pre-mir- into nci-h cells resulted in tgf-β regulation. in our experiment, the transfection efficiency was an important factor affecting the degree of regulation on the target gene. in the case of higher transfection efficiency, as more mirna would be transfected into the cells, the effect of gene regulation by mirna transfected would be greater. in our study, the transfection efficiency was about ± %, which was considered to be adequate for further functional analyses. during transfection, some oligonucleotide molecules were sequestered in internal vesicles and physically separated from their targets in the cytoplasm; and then released during cell lysis. therefore monitoring mirnas by qpcr after transfection would not be valuable. previous researchers of this procedure had highly recommended investigating the target mrnas and proteins instead of mirna quantification. the time point of 24-hour post-transfection or post-infection was chosen for evaluation because mir- induction was observed at the early stage of virus infection, and sufficient time might be required for the mir- to have effect on its target, so we had chosen 24-hour post-transfection or post-infection for evaluation of the effect of this mirna. indeed, upon detecting the tgf-β expression at mrna and protein levels, we found that the altered mir- expression would affect the expression of the cytokine- tgf-β literature search on the background of mir- confirmed that mir- is a member of the mir- family. previous studies of mir- were mainly on its role in cancer. it has been reported that mir- were markedly downregulated in cells that had undergone epithelial to mesenchymal in response to tgf-β. mir- was also found to be overexpressed in ovarian and colorectal cancers and down-regulated in prostate, hepatocellular, renal cell carcinoma and in gastric cancer tissues raising a controversial issue about the role of mir- in cancer progression. furthermore, the mir- family members play roles in maintaining the epithelial phenotype of cancer cells. a member of this family - mir-200a was also found to be differentially expressed in response to influenza virus infection in another study. the targets of mir-200a are associated with viral gene replication and the jak-stat signaling pathway, which is closely related to type i interferon-mediated innate immune response. however, the effect of mir- on virus infection was not known, except one recent report showing that enterovirus can induce mir- and contribute to the shutoff of host protein translation by targeting the translation initiation factor eif4e. in addition, evidence suggests that influenza a virus infection reduces or promotes the expression of the host mir- in a time dependent manner. we found that tgf-β mrna was suppressed in mir- overexpressed cells. our observation is in line with another study showing that the 3′utr of tgf-β mrna contained a target site for mir-141/200a and the expression of tgf-β was significantly decreased in mir-141/200a transfected cells. furthermore, mir- may not only work as translational repressors of target mrnas, because it was observed that they also caused a decrease in tgf-β mrna levels. these findings are similar to recent data demonstrating that some mirnas can alter the mrna levels of target genes. this ability is probably independent of the ability of these mirnas to regulate the translation of target mrnas. we also noted that antagomir- moderately increased the accumulation of tgf-β protein during influenza virus infection. this might be because, by the use of anti-mir mir- inhibitor, which decreases the cellular pool of mir- the translation control of the tgf-β mrna was subsequently released and caused the tgf-β protein to express and accumulate during virus infection. however, it was also observed that when there was an increase in tgf-β mrna level, the corresponding tgf-β protein expression level would be increased, except in the case of non-mir-141-inhibitor treated h5n infected cells. in this case, there was a decrease in tgf-β mrna level, while the tgf-β protein was increased. this might be explained by the fact that tgf-β mrna degradation induced by mir- might be much faster than that of the corresponding protein degradation. recently, we had also reported that h1n was the only subtype that could induce a sustained increase in tgf-β at protein level. that observation coincides with our results in this study, showing that h1n infection induced a little amount of mir- expression, while h5n infection induced a higher amount of mir- expression at the early phase of infection. as a consequence of the higher amount of mir- in h5n infection, tgf-β expression might be more greatly reduced than that in h1n infection. since tgf-β can act as both an immunosuppressive agent and a potent proinflammatory molecule through its ability to attract and regulate inflammatory molecules, it plays a vital role in t-cell inhibition. furthermore, it has been reported that tgf-β inhibits th cytokine-mediated induction of ccl-2/mcp- ccl-3/mip-1α, ccl-4/mip-1β, ccl-5/rantes, ccl-9/mip-1γ, cxcl-2/mip- and cxcl-10/ip-10. moreover, the pro-inflammatory responses during influenza a virus infection are tightly controlled by anti-inflammatory mediators, such as tgf-β to protect the easily damageable lung tissue from destructive side effects associated with virus induced inflammation. therefore, the downregulation of tgf-β protein by mir- may be an important step in the excessive inflammation progression during influenza a virus infection, particularly in h5n infection. however, whether the recovery of tgf-β expression by anti-mir mir- inhibitor could resolve the hypercytokinemia stage of h5n infection needs to be further studied. although our findings were obtained from an in vitro model, we could apply these to the real situation of an in vivo model or tissue comprised of different cell types. in real bronchial environments, lung epithelial cells are the key target of influenza viruses. after these cells are infected, they will activate an inflammatory cascade which launches a quick antimicrobial reaction and directs adaptive immunity to mount a protective response. bronchial epithelial cells therefore modulate the activation of monocytes, macrophages, dendritic cells, and t lymphocytes through cytokines and chemokines. cytokines and chemokines generally function in an autocrine or paracrine manner. these mediators will contribute to the generation of a specific bronchial homeostatic microenvironment that affects the way in which the body copes with the viruses. this homeostatic “circuit” can inhibit excessive inflammatory response in lung tissues. for example, tgf-β had been reported to mediate a cross-talk between alveolar macrophages and epithelial cells. however, our findings show that, during highly pathogenic h5n avian virus infection, mir- would be induced shortly after infection. with high level of mir- the expression of tgf-β would be suppressed from the lung epithelial cells. without sufficient tgf- β, the pro-inflammatory response might not be tightly controlled in cases of highly pathogenic h5n avian virus infection. this might explain the mechanism concerning bronchial infiltration of inflammatory cells, particularly lymphocytes and eosinophils, and the subsequent hyperresponsiveness of the bronchial wall induced by viral infection. our study has some limitations that will need to be addressed in future studies. firstly, we did not assess the roles of other mirnas whose expression were also altered after infection. the mirna microarrays that we used did not contain probes for every known mirna; thus it is possible that influenza a virus infection affects the expression of some other mirnas not yet covered by the kit used in the current study. secondly, the virus may interact with mirna regulatory pathways differently in other cell or tissue types, or in other physiological status.  in conclusion, based on the broad-catching mirna microarray approach, we found that dysregulation of mirna expression is mainly observed in highly pathogenic avian influenza infection. we identified that mir- was induced at early time points upon influenza a virus infection. the induction was higher in h5n infection than that of seasonal h1n infection. moreover, tgf-β which plays an important role in regulating inflammatory processes, was identified as a target of mir- binding. as a result, influenza a virus infection, in particular highly pathogenic h5n could affect the inflammatory processes via mir- induction. 
since tgf-β is an important cytokine that can act as both an immunosuppressive agent and a potent proinflammatory molecule through its ability to attract and regulate inflammatory molecules, and previous report showed that only seasonal influenza h1n could induce a persistent expression of tgf-β we speculate that the modulation of tgf-β expression by different influenza subtypes via mir- might be a critical step for determining the outcome of either normal or excessive inflammation progression. the results showed that dysregulation of mirna expression was mainly observed in highly pathogenic avian influenza a h5n infection. we found that mir-21*, mir-100*, mir- mir-574-3p, mir-1274a and mir1274b were differentially expressed in response to influenza a virus infection. this study aims at elucidating how avian influenza infection perturbs the host’s mirna regulatory pathways that may lead to adverse pathological events, such as cytokine storm, using the mirna microarray approach. avian influenza remains a serious threat to human health. the consequence of human infection varies markedly among different subtypes of avian influenza viruses.
 avian influenza remains a serious threat to human health. the consequence of human infection varies markedly among different subtypes of avian influenza viruses. in addition to viral factors, the difference in host cellular response is likely to play a critical role. this study aims at elucidating how avian influenza infection perturbs the host’s mirna regulatory pathways that may lead to adverse pathological events, such as cytokine storm, using the mirna microarray approach.  the results showed that dysregulation of mirna expression was mainly observed in highly pathogenic avian influenza a h5n infection. we found that mir-21*, mir-100*, mir- mir-574-3p, mir-1274a and mir1274b were differentially expressed in response to influenza a virus infection. interestingly, we demonstrated that mir- which was more highly induced by h5n than by h1n, had an ability to suppress the expression of a cytokine - transforming growth factor -β this was supported by the observation that the inhibitory effect could be reversed by antagomir-  since tgf-β is an important cytokine that can act as both an immunosuppressive agent and a potent proinflammatory molecule through its ability to attract and regulate inflammatory molecules, and previous report showed that only seasonal influenza h1n could induce a persistent expression of tgf-β we speculate that the modulation of tgf-β expression by different influenza subtypes via mir- might be a critical step for determining the outcome of either normal or excessive inflammation progression. micrornainfluenza a virush1n1h5n1inflammationhypercytokinemiapathogenesis 