BACKGROUND
genetically improved farmed tilapia  is a commercially important farmed fish in southern china  <cit> .
data from the food and agriculture organization show that tilapia output reached  <dig> , <dig> tons in about  <dig> farming countries during  <dig>  in china, the tilapia output was estimated to be about  <dig> million tons in  <dig>  because of its intraspecific cross and relatively rapid growth rate, gift is highly susceptible to bacterial diseases triggered by streptococcus iniae and streptococcus agalactiae when reared in intensive culture systems  <cit> .
thus, a better understanding of the regulatory response of gift to infection will provide crucial information for both immunomodulation and gift breeding.
the non-specific immune system in fish plays a major role at all stages of infection  <cit> .
thus, understanding the immune regulation mechanism may contribute to better disease prevention.
micrornas  are small non-coding single-stranded rnas  that are found in plants, animals and some viruses.
mirnas function in rna silencing and post-transcriptional regulation of gene expression by binding to the 3’-untranslated regions of their target mrnas  <cit> .
mirna-mediated regulation has been reported in the innate immune response, inflammatory diseases, cell signal transduction, and targeted therapy  <cit> ; however, the functions of mirnas have been studied mainly in mammalian and plants  <cit> .
the mirbase database   contains  <dig>  mirnas in  <dig> species expressing  <dig>  mature mirnas.
many mature mirnas are highly conserved across vertebrate taxa  <cit> , but their role in the fish host immune response is known in only a few species, mainly zebrafish   <cit> , large yellow croaker   <cit> , and grass carp   <cit> .
several immune-specific mirnas that regulate innate and adaptive immune responses in fish have been identified and classified, including mir- <dig>  mir- <dig>  mir-194a, mir-146a/b, and mir- <dig>  for example, mir- <dig> and mir-194a were markedly decreased in zebrafish infected by vibrio harveyi, and mir-146a/b and mir- <dig> down-regulated toll-like receptor  <dig>  and downstream nuclear factor kb activity in zebrafish with lipopolysaccharide infection  <cit> .
up-regulated expression of mir-122-3p, mir-214-3p, and mir-181c was detected in infected large yellow croaker treated with polyriboinosinic: polyribocytidylic acid   <cit> .
further, let-7i and mir- <dig> were reported to down-regulate tlr <dig> and nuclear factor interleukin 3- <dig>  in response to an excessive inflammatory response in grass carp infected with aeromonas hydrophila  <cit> .
aberrant regulation of some of these mirnas can disrupt intracellular signaling networks and inflammatory responses, which can result in pathological conditions  <cit> .
in recent years, a growing number of genes related to immunology have been detected and identified in tilapia using various molecular biological techniques  <cit> .
following the successful completion of the o.niloticus genome project , studies on the expression and regulation mechanisms of mirna-mediated genes in the immune response will grow in importance.
however, o. niloticus mirna transcriptome data have not yet been reported, and changes in mirna expression profiles in the gift immune response to a foreign challenge have not been studied until now.
the aim of this study is to identify and analyze the mirnas and their target genes involved in the immune response of gift to s. iniae infection.
two small rna libraries from gift infected with s. iniae for 24 h and uninfected gift were sequenced by high-throughput sequencing.
mirnas were identified and rnahybrid and targetscan were used to predict mirna target genes, and the gene ontology  and kyoto encyclopedia of genes and genomes  databases were used to explore potential biological processing of downstream targets genes.
we identified mirnas and their target genes involved in the immune response and verified the expression profiles of mirnas by quantitative real-time pcr .
identification of mirnas involved in immune regulation is an essential prerequisite towards better understanding of their function in intracellular signaling networks and inflammatory responses.
the new insights from this study will pave the way for the development of disease control measures and for genetic improvement in gift.
RESULTS
streptococcal disease caused by s. iniae is unquestionably one of the main bacterial diseases in tilapia.
it has been reported to cause huge mortality and economic loss in tilapia culture zones in more than four provinces in china, including guangdong, guangxi, hainan, and fujian.
however, very few studies have described the molecular mechanisms underlying the regulation of the mirna-mediated immune response in tilapia.
the pre-experiment determined that the 96 h semi-lethal s. iniae dose  in gift was 107cfuml- <dig> .
gift infected with a concentration of 107cfuml- <dig> could stimulate their own immune systems to deal with bacterial invasion 24 h post- s. iniae infection  <cit> , the survival rate was 90%.
in a previous study, we found that c-type lysozyme, heat-shock protein  <dig> , interleukin-1β , interferon-γ , and tumor necrosis factor-α  expression levels in the head kidney tissue of s. iniae-infected gift for 96 h reached peak values 24 h post-infection  <cit> .
however, prolonging the infection time weakened the fish’s own immune response, survival decreased rapidly at 36 h and 48 h post-infection in the pre-experimental group injected with  <dig> cfuml- <dig>  the following symptoms occurred after 24 h in gift infected with the 96 h ld <dig>  diseased fish began to rotate intermittently in the water, unbalanced swimming motions, corneal opacity in the white of the eye, enlarged and reddened spleen, liver congestion, hemorrhaging kidneys, enlarged gallbladder, and dark green bile.
hemorrhaging kidneys may have resulted from lymphatic cell reduction and replacement by a large number of red blood cells, and a high rate of macrophage cell infiltration  <cit> .
macrophages can reduce bacterial invasion by phagocytosis.
head kidney was found to be an important immune-regulated tissue in adult fish during the early stage of bacterial infections  <cit> , and is considered to play a similar role as bone marrow in mammals  <cit> .
head kidney is composed mainly of granulocytes, macrophages, and t and b lymphocytes in the presence of any acquired specific or non-specific immunoregulation  <cit> .
therefore, in this study, we aimed to identify mirnas involved in resistance to pathogens infection and mirna-mediated regulatory pathways, to better understand the immune-regulated response in tilapia head kidney triggered by a s. iniae challenge.
two small rna libraries from the head kidney of gift infected with s. iniae for 24 h  and from uninfected gift  were built and sequenced.
a total of  <dig> , <dig> and  <dig> , <dig> raw reads were obtained from the co and in libraries, respectively .
after removing reads that contained adaptor sequences and low-quality reads,  <dig> , <dig> and  <dig> , <dig> clean reads remained in the co and in libraries, respectively.
we found that  <dig> %  and  <dig> %  of the reads had no matches in the searched databases; these unannotated sequences were considered to be unique reads.
the total clean reads contained  <dig> %  and  <dig> %  of the conserved mirnas, and the unique reads contained  <dig> %  and  <dig> %  of the conserved mirnas .table  <dig> preliminary analysis of solexa high-throughput sequencing data
we found that  <dig>   and  <dig>   of the unique reads and  <dig> and  <dig> of the candidate novel mirnas could be mapped to the o. niloticus  genome sequence.
the lengths of the read sequences in the two libraries ranged from 10 nt to 44 nt, with the 22-nt, 21-nt, and 23-nt long reads being the most abundant .
li et al.
<cit>  and hong et al.
<cit>  also reported that 22-nt long mirnas were enriched in mirna transcriptome libraries of both apostichopus japonicus and gobiocypris rarus.fig.
<dig> length size distribution small rna sequences both the control group  and infected group
the numbers of reads in a library have been employed to estimate the abundance of the reads in a sequencing sample.
the ten most abundant mirnas in the co library were mir-6585-5p, mir-181a, mir-181a-5p, mir- <dig>  mir-92a-3p, mir-26-5p, mir-26a-5p, mir-10b-5p, mir-10b, and mir-26a , and they accounted for  <dig> % of the reads that mapped to sequences in mirbase.
eight of these mirnas were among the ten most abundant mirnas in the in library  , which accounted for  <dig> % of the reads that were mapped to sequences in mirbase.
among these mirnas, mir-181a, mir-92a-3p, mir-26a-5p, mir-10b, and mir-26a have been reported to play fundamental roles in immune-regulated signaling and inflammatory responses in human .fig.
<dig> top  <dig> lists of most abundant mirnas both the control group  and infected group
we selected and analyzed  <dig> differentially expressed mirnas with more than  <dig> reads between the co and in libraries .
among the  <dig> mirnas, eight  were significantly up-regulated and  <dig>  were significantly down-regulated in the in library compared with the co library .
we screened for putative mirna target genes using rnahybrid and targetscan with the o. niloticus genome sequence.
the candidate target genes were annotated with go terms, and  <dig> biological processes were assigned to the predicted targets .
the heat map indicates that these differentially expressed mirnas may play important roles in immune-regulation, signaling transmission, and cellular processes .fig.
<dig> fold changes of differentially expressed mirnas between the control group  and infected group .
fold changes were calculated as log <dig>
fig.
<dig> cluster analysis of differentially expressed mirnas.
the color indicates the log2-fold change from high  to low , as indicated by the color scale.
the names of the mirnas and the clusters to which they belong are shown in the bottom of the panel.
a heat map was constructed based on the  <dig> biological processes of the mirna target genes indentified by a gene ontology analysis
to verify the expression levels estimated from the transcriptome sequencing data, we measured the expression levels of eight mirnas  by qrt-pcr.
the eight mirnas were selected because they had relatively high fold-changes post-infection.
the pcr results showed significant up-regulation of mir-310-3p, mir- <dig>  and mir- <dig> and significant down-regulation of mir-92d-3p, mir-375-5p, mir-146-3p, and mir- <dig> in the in group compared with the co group , in agreement with the results obtained from the transcriptome data.
however, the pcr results did not show up-regulation of mir- <dig> expression in the in group , which contradicts the result obtained from the transcriptome data.
in our study, we considered transcripts with more than  <dig> reads; however, cristino et al.
<cit>  considered that transcripts with less than  <dig> reads may be unreliable and only roughly measure relative mirna abundance.
the mir- <dig> transcript in the co library was represented by  <dig> reads, which was the lowest number of reads among the eight selected mirnas .
this might explain the difference between the mir- <dig> expression levels estimated from the transcriptome sequencing data and by qrt-pcr.fig.
<dig> quantitative real-time pcr validation of eight mirna expression levels both the control group  and infected group .
the levels of the indicated mirnas expression were showed by box and whisker plots .
the values are expressed as the relative ratio with u <dig> as an internal control.
*p <  <dig>  and **p <  <dig>  by unpaired student’s t-tests.
ns, no significant difference
previous observations have suggested that mir- <dig>  mir- <dig>  mir-92a, mir- <dig>  mir- <dig>  and mir- <dig> play vital immune-regulated functions in fish and mammals.
for example, a recently study by zhang et al.
<cit>  found that mir-92a was up-regulated in sea cucumber exposed to vibrio splendidus and lipopolysaccharides and its target gene was predicted as e <dig> ubiquitin-protein ligase , which might act as a tumor suppressor by modulating the signaling pathway involved in transforming growth factor-beta and bone morphogenetic protein  <cit> .
in breast cancer patients, down-regulated mir-92a levels were found in tumor tissues and serum  <cit> .
dysregulated mir- <dig> in transgenic cloned sheep mediated the expression of retrotransposon-like gene  <dig>  a key placental development gene that may cause developmental retardation  <cit> .
mir- <dig> was shown to be down-regulated in the liver of lass ii knockout mice and may be involved in the association between liver dysregulation and metabolism dysfunction  <cit> .
mir- <dig> over-expression was found to inhibit lung surfactant secretion, possibly by influencing actin dynamicand and injury pancreas in patients  <cit> .
as tumor suppressors or oncogenes, the mir-310/ <dig> cluster might regulate evolutionarily conserved immune signaling networks in cancer  <cit> .
seven genes involved in immune-related responses were predicted to be the targets of seven mirnas .
complement c <dig> and cytidine deaminase were predicted as potential targets for mir-92d-3p and mir- <dig>  respectively.
the levels of complement c <dig> mrna were reported to increase during the 24-h acute phase response of large yellow croaker   <cit>  and miiuy croaker   <cit>  triggered by experimental infections.
up-regulation of complement c <dig> has been linked to innate immunity, which plays an important role in mediating tissue injury after stress.
in this study, we found markedly up-regulated levels of complement c <dig> in the in library , suggesting its role as a potential modulator of the anti-inflammatory response defense against invading pathogens.
cytidine deaminase is a mutagenic activation-induced dna/rna-editing enzyme that has been implicated in human tumorigenesis.
abnormal cytidine deaminase levels were found in gastric epithelial cells infected by helicobacter pylori, and it was suggested that this might be a mechanism responsible for the accumulation of submicroscopic deletions and somatic mutations  <cit> .
cytidine deaminase levels in aquatic animals have been little researched.
our results suggest that the down-regulated expression of cytidine deaminase mrna after s.iniae infection for 24 h, may be associated with infectious diseases and immune-mediated disorders.fig.
<dig> quantitative real-time pcr validation of seven gene mrna expression levels both the control group  and infected group .
the expression levels of the indicated immune-regulated or signaling transmission genes in the co and in groups  were normalized to that of 18 s rrna.
statistical analyses were performed using unpaired student’s t-tests.
*p <  <dig>  and **p <  <dig>  by unpaired student’s t-tests
regulators of g protein signaling  are multi-functional gtpase-accelerating proteins that have become a focus of attention in recent years  <cit> .
down-regulated rgs <dig> was reported in androgen-independent prostate tumor cells, which were inhibited by over-expression of rgs <dig> in vivo and in vitro  <cit> .
rgs <dig> was a predicted target gene of mir-310-3p in our study.
mir-310-3p was down-regulated in the in library , suggesting that the reduction of rgs <dig> may be associated with an increased inflammation response against infection.
signaling pathways of mitogen-activated protein kinases  include extracellular signal-regulated kinase1/ <dig> , p <dig>  cmyc, and c-jun n-terminal kinase , which mediate the activities of several transcription factors  <cit> .
modulation of the mapk signaling pathway may regulate the transcription of cell cycle genes, which was shown to be essential for mediating many cellular processes, including inflammatory response, cell stress, cell differentiation, and cell proliferation  <cit> .
tnf-α is a major pro-inflammatory cytokine in cancer cells  <cit> .
meili et al.
<cit>  and zhu et al.
<cit>  reported that tnf-α expression affected the downstream mapk effector pathway.
a significant increase in the expression of tnf-α mrna was found in s. iniae-infected gift at 24 h post-infection  <cit> , which might lead to activation of mapk  <dig> and the immune response.
metabotropic glutamate receptors  are widely known for their roles in synaptic signaling.
aberrant glutamate signaling has been shown to be involved in the transformation and maintenance for human cancer, including glioma, melanoma skin cancer, breast cancer, and prostate cancer  <cit> .
in this study, mglur <dig> was identified as a potential target of mir-375-5p, and mglur <dig> mrna was found to be up-regulated in the in library, suggesting that mglur <dig> may promote inflammatory cell infiltration via activation of the mapk signaling pathways  <cit> .
the calcium-sensing receptor  is an important mediator of inflammation , and dysregulation of intestinal barrier integrity was reported to be induced in casr knockout mice  <cit> .
our findings suggest that down-regulation of casr mrna expression may alter the composition of the gut microbiota and disrupt immune homeostasis in s. iniae-infected gift at 24 h post-infection.
microtubule-associated protein 1s  was the predicted target gene of mir- <dig>  as a novel member of the microtubule-associatedprotein  <dig>  family, map1s is important in inducing microtubule stabilization, bonding the microtubule organizing center to the centrosome, and regulation of mitotic cell cycle progression  <cit> .
zou et al.
<cit>  reported that the interaction of suppressor of cytokine signaling  <dig>  and map1s, and the integrity of the microtubule cytoskeleton was an important modulator of interleukin- <dig>  signaling.
our results suggest that map1s may be crucial in the response to pathogenic infection, and the regulation of map1s by mir- <dig> may be involved in inflammatory regulating networks.
CONCLUSIONS
in this study, we used high-throughput sequencing and analysis to screen for different mirnas between normal and infected tilapia.
we confirmed the expression levels of seven differentially expressed mirnas and their predicted target genes, which may play essential roles in triggering the immune response of head kidney in s. iniae-infected gift at 24 h post-infection.
these mirnas and their putative target genes were associated mainly with autoimmune inflammation and regulation of cytokine signaling.
our findings provide insights into the role of mirnas in the immune system of gift, and will help to form the basis for understanding mirna-mediated control of gene expression against infection in gift.
further in vivo and invitro studies are needed to gain more insight into the association and the regulatory response between mirnas and their predicted target genes, biological pathways, and molecular pathogenesis in tilapia.

 genetically improved farmed tilapia is a commercially important farmed fish in southern china. data from the food and agriculture organization show that tilapia output reached, tons in about farming countries during in china, the tilapia output was estimated to be about million tons in because of its intraspecific cross and relatively rapid growth rate, gift is highly susceptible to bacterial diseases triggered by streptococcus iniae and streptococcus agalactiae when reared in intensive culture systems. thus, a better understanding of the regulatory response of gift to infection will provide crucial information for both immunomodulation and gift breeding. the non-specific immune system in fish plays a major role at all stages of infection. thus, understanding the immune regulation mechanism may contribute to better disease prevention. micrornas are small non-coding single-stranded rnas that are found in plants, animals and some viruses. mirnas function in rna silencing and post-transcriptional regulation of gene expression by binding to the 3’-untranslated regions of their target mrnas. mirna-mediated regulation has been reported in the innate immune response, inflammatory diseases, cell signal transduction, and targeted therapy; however, the functions of mirnas have been studied mainly in mammalian and plants. the mirbase database contains mirnas in species expressing mature mirnas. many mature mirnas are highly conserved across vertebrate taxa, but their role in the fish host immune response is known in only a few species, mainly zebrafish, large yellow croaker, and grass carp. several immune-specific mirnas that regulate innate and adaptive immune responses in fish have been identified and classified, including mir- mir- mir-194a, mir-146a/b, and mir- for example, mir- and mir-194a were markedly decreased in zebrafish infected by vibrio harveyi, and mir-146a/b and mir- down-regulated toll-like receptor and downstream nuclear factor kb activity in zebrafish with lipopolysaccharide infection. up-regulated expression of mir-122-3p, mir-214-3p, and mir-181c was detected in infected large yellow croaker treated with polyriboinosinic: polyribocytidylic acid. further, let-7i and mir- were reported to down-regulate tlr and nuclear factor interleukin 3- in response to an excessive inflammatory response in grass carp infected with aeromonas hydrophila. aberrant regulation of some of these mirnas can disrupt intracellular signaling networks and inflammatory responses, which can result in pathological conditions. in recent years, a growing number of genes related to immunology have been detected and identified in tilapia using various molecular biological techniques. following the successful completion of the o.niloticus genome project, studies on the expression and regulation mechanisms of mirna-mediated genes in the immune response will grow in importance. however, o. niloticus mirna transcriptome data have not yet been reported, and changes in mirna expression profiles in the gift immune response to a foreign challenge have not been studied until now. the aim of this study is to identify and analyze the mirnas and their target genes involved in the immune response of gift to s. iniae infection. two small rna libraries from gift infected with s. iniae for 24 h and uninfected gift were sequenced by high-throughput sequencing. mirnas were identified and rnahybrid and targetscan were used to predict mirna target genes, and the gene ontology and kyoto encyclopedia of genes and genomes databases were used to explore potential biological processing of downstream targets genes. we identified mirnas and their target genes involved in the immune response and verified the expression profiles of mirnas by quantitative real-time pcr. identification of mirnas involved in immune regulation is an essential prerequisite towards better understanding of their function in intracellular signaling networks and inflammatory responses. the new insights from this study will pave the way for the development of disease control measures and for genetic improvement in gift.  streptococcal disease caused by s. iniae is unquestionably one of the main bacterial diseases in tilapia. it has been reported to cause huge mortality and economic loss in tilapia culture zones in more than four provinces in china, including guangdong, guangxi, hainan, and fujian. however, very few studies have described the molecular mechanisms underlying the regulation of the mirna-mediated immune response in tilapia. the pre-experiment determined that the 96 h semi-lethal s. iniae dose in gift was 107cfuml-. gift infected with a concentration of 107cfuml- could stimulate their own immune systems to deal with bacterial invasion 24 h post- s. iniae infection, the survival rate was 90%. in a previous study, we found that c-type lysozyme, heat-shock protein, interleukin-1β, interferon-γ, and tumor necrosis factor-α expression levels in the head kidney tissue of s. iniae-infected gift for 96 h reached peak values 24 h post-infection. however, prolonging the infection time weakened the fish’s own immune response, survival decreased rapidly at 36 h and 48 h post-infection in the pre-experimental group injected with cfuml- the following symptoms occurred after 24 h in gift infected with the 96 h ld diseased fish began to rotate intermittently in the water, unbalanced swimming motions, corneal opacity in the white of the eye, enlarged and reddened spleen, liver congestion, hemorrhaging kidneys, enlarged gallbladder, and dark green bile. hemorrhaging kidneys may have resulted from lymphatic cell reduction and replacement by a large number of red blood cells, and a high rate of macrophage cell infiltration. macrophages can reduce bacterial invasion by phagocytosis. head kidney was found to be an important immune-regulated tissue in adult fish during the early stage of bacterial infections, and is considered to play a similar role as bone marrow in mammals. head kidney is composed mainly of granulocytes, macrophages, and t and b lymphocytes in the presence of any acquired specific or non-specific immunoregulation. therefore, in this study, we aimed to identify mirnas involved in resistance to pathogens infection and mirna-mediated regulatory pathways, to better understand the immune-regulated response in tilapia head kidney triggered by a s. iniae challenge. two small rna libraries from the head kidney of gift infected with s. iniae for 24 h and from uninfected gift were built and sequenced. a total of, and, raw reads were obtained from the co and in libraries, respectively. after removing reads that contained adaptor sequences and low-quality reads,, and, clean reads remained in the co and in libraries, respectively. we found that % and % of the reads had no matches in the searched databases; these unannotated sequences were considered to be unique reads. the total clean reads contained % and % of the conserved mirnas, and the unique reads contained % and % of the conserved mirnas.table preliminary analysis of solexa high-throughput sequencing data we found that and of the unique reads and and of the candidate novel mirnas could be mapped to the o. niloticus genome sequence. the lengths of the read sequences in the two libraries ranged from 10 nt to 44 nt, with the 22-nt, 21-nt, and 23-nt long reads being the most abundant. li et al. and hong et al. also reported that 22-nt long mirnas were enriched in mirna transcriptome libraries of both apostichopus japonicus and gobiocypris rarus.fig. length size distribution small rna sequences both the control group and infected group the numbers of reads in a library have been employed to estimate the abundance of the reads in a sequencing sample. the ten most abundant mirnas in the co library were mir-6585-5p, mir-181a, mir-181a-5p, mir- mir-92a-3p, mir-26-5p, mir-26a-5p, mir-10b-5p, mir-10b, and mir-26a, and they accounted for % of the reads that mapped to sequences in mirbase. eight of these mirnas were among the ten most abundant mirnas in the in library, which accounted for % of the reads that were mapped to sequences in mirbase. among these mirnas, mir-181a, mir-92a-3p, mir-26a-5p, mir-10b, and mir-26a have been reported to play fundamental roles in immune-regulated signaling and inflammatory responses in human.fig. top lists of most abundant mirnas both the control group and infected group we selected and analyzed differentially expressed mirnas with more than reads between the co and in libraries. among the mirnas, eight were significantly up-regulated and were significantly down-regulated in the in library compared with the co library. we screened for putative mirna target genes using rnahybrid and targetscan with the o. niloticus genome sequence. the candidate target genes were annotated with go terms, and biological processes were assigned to the predicted targets. the heat map indicates that these differentially expressed mirnas may play important roles in immune-regulation, signaling transmission, and cellular processes.fig. fold changes of differentially expressed mirnas between the control group and infected group. fold changes were calculated as log fig. cluster analysis of differentially expressed mirnas. the color indicates the log2-fold change from high to low, as indicated by the color scale. the names of the mirnas and the clusters to which they belong are shown in the bottom of the panel. a heat map was constructed based on the biological processes of the mirna target genes indentified by a gene ontology analysis to verify the expression levels estimated from the transcriptome sequencing data, we measured the expression levels of eight mirnas by qrt-pcr. the eight mirnas were selected because they had relatively high fold-changes post-infection. the pcr results showed significant up-regulation of mir-310-3p, mir- and mir- and significant down-regulation of mir-92d-3p, mir-375-5p, mir-146-3p, and mir- in the in group compared with the co group, in agreement with the results obtained from the transcriptome data. however, the pcr results did not show up-regulation of mir- expression in the in group, which contradicts the result obtained from the transcriptome data. in our study, we considered transcripts with more than reads; however, cristino et al. considered that transcripts with less than reads may be unreliable and only roughly measure relative mirna abundance. the mir- transcript in the co library was represented by reads, which was the lowest number of reads among the eight selected mirnas. this might explain the difference between the mir- expression levels estimated from the transcriptome sequencing data and by qrt-pcr.fig. quantitative real-time pcr validation of eight mirna expression levels both the control group and infected group. the levels of the indicated mirnas expression were showed by box and whisker plots. the values are expressed as the relative ratio with u as an internal control. *p <  and **p <  by unpaired student’s t-tests. ns, no significant difference previous observations have suggested that mir- mir- mir-92a, mir- mir- and mir- play vital immune-regulated functions in fish and mammals. for example, a recently study by zhang et al. found that mir-92a was up-regulated in sea cucumber exposed to vibrio splendidus and lipopolysaccharides and its target gene was predicted as e ubiquitin-protein ligase, which might act as a tumor suppressor by modulating the signaling pathway involved in transforming growth factor-beta and bone morphogenetic protein. in breast cancer patients, down-regulated mir-92a levels were found in tumor tissues and serum. dysregulated mir- in transgenic cloned sheep mediated the expression of retrotransposon-like gene a key placental development gene that may cause developmental retardation. mir- was shown to be down-regulated in the liver of lass ii knockout mice and may be involved in the association between liver dysregulation and metabolism dysfunction. mir- over-expression was found to inhibit lung surfactant secretion, possibly by influencing actin dynamicand and injury pancreas in patients. as tumor suppressors or oncogenes, the mir-310/ cluster might regulate evolutionarily conserved immune signaling networks in cancer. seven genes involved in immune-related responses were predicted to be the targets of seven mirnas. complement c and cytidine deaminase were predicted as potential targets for mir-92d-3p and mir- respectively. the levels of complement c mrna were reported to increase during the 24-h acute phase response of large yellow croaker and miiuy croaker triggered by experimental infections. up-regulation of complement c has been linked to innate immunity, which plays an important role in mediating tissue injury after stress. in this study, we found markedly up-regulated levels of complement c in the in library, suggesting its role as a potential modulator of the anti-inflammatory response defense against invading pathogens. cytidine deaminase is a mutagenic activation-induced dna/rna-editing enzyme that has been implicated in human tumorigenesis. abnormal cytidine deaminase levels were found in gastric epithelial cells infected by helicobacter pylori, and it was suggested that this might be a mechanism responsible for the accumulation of submicroscopic deletions and somatic mutations. cytidine deaminase levels in aquatic animals have been little researched. our results suggest that the down-regulated expression of cytidine deaminase mrna after s.iniae infection for 24 h, may be associated with infectious diseases and immune-mediated disorders.fig. quantitative real-time pcr validation of seven gene mrna expression levels both the control group and infected group. the expression levels of the indicated immune-regulated or signaling transmission genes in the co and in groups were normalized to that of 18 s rrna. statistical analyses were performed using unpaired student’s t-tests. *p <  and **p <  by unpaired student’s t-tests regulators of g protein signaling are multi-functional gtpase-accelerating proteins that have become a focus of attention in recent years. down-regulated rgs was reported in androgen-independent prostate tumor cells, which were inhibited by over-expression of rgs in vivo and in vitro. rgs was a predicted target gene of mir-310-3p in our study. mir-310-3p was down-regulated in the in library, suggesting that the reduction of rgs may be associated with an increased inflammation response against infection. signaling pathways of mitogen-activated protein kinases include extracellular signal-regulated kinase1/, p cmyc, and c-jun n-terminal kinase, which mediate the activities of several transcription factors. modulation of the mapk signaling pathway may regulate the transcription of cell cycle genes, which was shown to be essential for mediating many cellular processes, including inflammatory response, cell stress, cell differentiation, and cell proliferation. tnf-α is a major pro-inflammatory cytokine in cancer cells. meili et al. and zhu et al. reported that tnf-α expression affected the downstream mapk effector pathway. a significant increase in the expression of tnf-α mrna was found in s. iniae-infected gift at 24 h post-infection, which might lead to activation of mapk and the immune response. metabotropic glutamate receptors are widely known for their roles in synaptic signaling. aberrant glutamate signaling has been shown to be involved in the transformation and maintenance for human cancer, including glioma, melanoma skin cancer, breast cancer, and prostate cancer. in this study, mglur was identified as a potential target of mir-375-5p, and mglur mrna was found to be up-regulated in the in library, suggesting that mglur may promote inflammatory cell infiltration via activation of the mapk signaling pathways. the calcium-sensing receptor is an important mediator of inflammation, and dysregulation of intestinal barrier integrity was reported to be induced in casr knockout mice. our findings suggest that down-regulation of casr mrna expression may alter the composition of the gut microbiota and disrupt immune homeostasis in s. iniae-infected gift at 24 h post-infection. microtubule-associated protein 1s was the predicted target gene of mir- as a novel member of the microtubule-associatedprotein family, map1s is important in inducing microtubule stabilization, bonding the microtubule organizing center to the centrosome, and regulation of mitotic cell cycle progression. zou et al. reported that the interaction of suppressor of cytokine signaling and map1s, and the integrity of the microtubule cytoskeleton was an important modulator of interleukin- signaling. our results suggest that map1s may be crucial in the response to pathogenic infection, and the regulation of map1s by mir- may be involved in inflammatory regulating networks.  in this study, we used high-throughput sequencing and analysis to screen for different mirnas between normal and infected tilapia. we confirmed the expression levels of seven differentially expressed mirnas and their predicted target genes, which may play essential roles in triggering the immune response of head kidney in s. iniae-infected gift at 24 h post-infection. these mirnas and their putative target genes were associated mainly with autoimmune inflammation and regulation of cytokine signaling. our findings provide insights into the role of mirnas in the immune system of gift, and will help to form the basis for understanding mirna-mediated control of gene expression against infection in gift. further in vivo and invitro studies are needed to gain more insight into the association and the regulatory response between mirnas and their predicted target genes, biological pathways, and molecular pathogenesis in tilapia. 
the role of micrornas in the regulatory response of gift to s. iniae infection has been underestimated and has not yet been well studied. seven target genes, which encode immune-related proteins were predicted to play crucial roles in the gift response to s. iniae infection. understanding the mirna transcriptome of s. iniae-infected gift is important for exploring the immune responses regulated by mirnas as well as for studying novel regulated networks to prevent and treat s. iniae infections in the future. rnahybrid and targetscan were used to identify complementary mirna and mrna target sites, and the gene ontology and kyoto encyclopedia of genes and genomes databases were used to annotate and predict potential downstream regulation of biological pathways. the expressions of these mirnas were verified by quantitative real-time pcr. bioinformatics analysis identified and conserved mirnas and and novel mirnas in the co and in libraries, respectively. head kidney has an important immune function in teleost fishes. the main aim of this study was to determine the possible function of mirnas in head kidney of s. iniae-infected gift. high-throughput deep sequencing of two libraries of rna extracted from gift head kidney tissues generated, and, clean reads. genetically improved farmed tilapia are susceptible to infection by streptococcus iniae when maintained in modern intensive culture systems. giftmicrornadeep sequencingtarget predictionstreptococcus iniaehttp://dx.doi.org/ /501100001809national natural science foundation of china 31502143qiang jun national science and technology program 2012bad26b03-1he jie special fund for agro-scientific research in the public interest project 2017jbfm08qiang jun issue-copyright-statement© the author 2017 mirnas are small, non-coding rnas that regulate gene expression by binding to the 3’-untranslated regions of their target mrnas.
 genetically improved farmed tilapia are susceptible to infection by streptococcus iniae when maintained in modern intensive culture systems. gift are commercially important fishes that are cultured widely in southern china. the role of micrornas in the regulatory response of gift to s. iniae infection has been underestimated and has not yet been well studied. head kidney has an important immune function in teleost fishes. the main aim of this study was to determine the possible function of mirnas in head kidney of s. iniae-infected gift. mirnas are small, non-coding rnas that regulate gene expression by binding to the 3’-untranslated regions of their target mrnas. mirnas are known to regulate immune-regulated signaling and inflammatory response pathways.  high-throughput deep sequencing of two libraries of rna extracted from gift head kidney tissues generated, and, clean reads. bioinformatics analysis identified and conserved mirnas and and novel mirnas in the co and in libraries, respectively. three mirnas were found to be up-regulated and four mirnas were found to be down-regulated in the s. iniae-infected gift. the expressions of these mirnas were verified by quantitative real-time pcr. rnahybrid and targetscan were used to identify complementary mirna and mrna target sites, and the gene ontology and kyoto encyclopedia of genes and genomes databases were used to annotate and predict potential downstream regulation of biological pathways. seven target genes, which encode immune-related proteins were predicted to play crucial roles in the gift response to s. iniae infection.  s. iniae outbreaks have hindered the development of the tilapia industry in china. understanding the mirna transcriptome of s. iniae-infected gift is important for exploring the immune responses regulated by mirnas as well as for studying novel regulated networks to prevent and treat s. iniae infections in the future. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords giftmicrornadeep sequencingtarget predictionstreptococcus iniaehttp://dx.doi.org/ /501100001809national natural science foundation of china 31502143qiang jun national science and technology program 2012bad26b03-1he jie special fund for agro-scientific research in the public interest project 2017jbfm08qiang jun issue-copyright-statement© the author 2017 