BACKGROUND
skeletal muscle hypertrophy is defined as an increase in muscle mass, which in the adult animal comes as a result of an increase in size of skeletal muscle fibers.
in the last years different mechanism of action have been reported to regulate muscle hypertrophy.
the major extracellular mediator of skeletal muscle hypertrophy is thought to be insulin growth factor- <dig>  which binds to its receptor igf1r to initiate a cascade of signaling pathways via phosphoinositide 3-kinase )
<cit> .
however, several lines of evidence suggest that igf- <dig> also mediates hypertrophy through calcineurin /nuclear factor of activated t-cells  signaling pathway
<cit> .
moreover, studies suggested that myocyte enhancer factor 2c  regulates the hypertrophic process
<cit> .
also glycogen synthase kinase  <dig> beta , that is a distinct substrate of akt, has been shown to modulate hypertrophy.
through akt phosphorylation, gsk3b activity is inhibited
<cit>  and its inhibition may induce hypertrophy by stimulating protein synthesis independent of the mtor pathway.
tead <dig>  regulates the expression of many skeletal muscle-specific genes
<cit> .
tead <dig> is a member of the tea domain family and is constitutively expressed in cardiac and skeletal muscles in pigs, mice and humans
<cit> .
the transcriptional regulation of tead <dig> to muscle-specific genes is implemented by co-operation with numerous co-factors, including mef2
<cit> .
myostatin  is a member of the transforming growth factor-b  superfamily of secreted growth and differentiation factors
<cit> .
in piedmontese cattle the double-muscled phenotype is an inherited condition associated to a point mutation in the mstn gene.
the piedmontese mstn missense mutation g938a is translated to c313y myostatin protein with the consequent loss of one of the disulphide bonds  involved in the characteristic tgf-b family cystine-knot structural motif
<cit> .
this mutation alters the function of mstn as a negative regulator of muscle growth, thereby inducing muscle hyperplasia and hypertrophy.
this breed has been systematically selected for double muscling to the point of fixation in many herds , but some difference in muscularity phenotype is still present.
micrornas  are small non-coding rna molecules , highly conserved, that regulate gene expression through binding with imperfect complementarities sequences of messenger rna
<cit> .
it is becoming increasingly evident that mirnas regulation of mrnas represents an effective way of interfering with function by acting at protein translation level.
this takes place when the mirna targets the 3′- untranslated region  of transcripts by imperfect base pairing and functions to inhibit translation.
in some cases, degradation of the target mrna is also a mechanism or a consequence of mirna-mediated suppression
<cit> .
mirnas have been shown to play critical roles in skeletal muscle development as well as in regulation of muscle cell proliferation and differentiation
<cit> .
by down-regulating gene expression, mirnas could play a role in skeletal muscle hypertrophy modulation.
in this study, we have analyzed mirnas engaged in post-transcriptional regulation of negative or positive modulators involved in the muscular hypertrophy pathway in piedmontese cattle.
in particular, we aimed to investigate on the specific mirnas involved in mstn regulation.
we screened the 3′-utr matching mirnas of several genes, such as igf <dig>  igf1r, ppp3ca, nfatc <dig>  mef2c, gsk3b, tead <dig> and mstn using a computational approach.
this analysis led to the identification of mir-27b, mir- <dig>  mir- <dig> and mir-199b-5p as possible candidates implicated in bovine skeletal muscle hypertrophy.
finally, we report the functional evidence that mir-27b is able to down-regulate mstn gene in vitro.
RESULTS
expression of igf <dig>  igf1r, ppp3ca, nfatc <dig>  mef2c, gsk3b, tead <dig> and mstn mrna in bovine skeletal muscle specimens
skeletal muscle samples were collected from cattle from two breeds with opposite carcass conformation: piedmontese and friesian bovines classified respectively as “e” and “o” muscle profile .
in order to identify possible gene expression differences linked to skeletal muscle phenotype between the two breeds, we have analyzed igf <dig>  igf1r, ppp3ca, nfatc <dig>  mef2c, gsk3b, tead <dig> and mstn genes using real-time quantitative pcr.
as shown in figure
<dig>  only mstn mrna expression displayed a strong difference between the two breeds.
in particular, in piedmontese samples, mstn mrna expression was downregulated  <dig> -fold compared with friesian muscle .
on the other hand, the expression of igf <dig>  igf1r, ppp3ca, nfatc <dig>  mef2c, gsk3b and tead <dig> were not significantly different.
mstn protein quantification
total proteins were extracted from the muscle samples and measured by western blot analysis.
as shown in figure
<dig>  mstn specific antibody recognized a ~29-kda mature mstn in both friesian and piedmontese cattle muscle extracts.
alpha-tubulin was used as loading control.
densitometric analysis of western blot indicates that in piedmontese breed there was less abundance mstn than in friesian one  .
targeting site of mirnas in igf <dig>  igf1r, ppp3ca, nfatc <dig>  mef2c, gsk3b, tead <dig> and mstn 3′-utr
to identify mirnas which could modulate bovine skeletal muscle hypertrophy, we have screened mirnas which potentially suppress expression of genes involved as negative or positive modulators in the muscular hypertrophy pathway.
for this purpose, mirnas target site prediction for bovine igf <dig>  igf1r, ppp3ca, nfatc <dig>  mef2c, gsk3b, tead <dig> and mstn were performed with the use of targetscan  <dig>  algorithms .
this analysis combined with bibliographic data research induced us to investigate mir-27b, mir- <dig>  mir- <dig> and mir-199b-5p as potential candidates implicated in bovine skeletal muscle hypertrophy.
the number of gene targets resulted higher for mir-27b than for other mirnas .
targetscan predicts biological targets of mirnas by searching for the presence of conserved 8mer and 7mer sites that match the seed region of each mirna.
in mammals, predictions are ranked based on the predicted efficacy of targeting as calculated using the context scores of the sites.
the context score is the sum of the contribution of these four features: site-type contribution; 3′ pairing contribution; local au contribution; position contribution.
expression of mir-27b, mir- <dig>  mir- <dig> and mir-199b-5p in bovine skeletal muscle
to examine which, if any, of the mirnas identified from the targetscan algorithm were expressed in bovine muscle, we used microrna taqman assay.
sample of both breeds mir-27b, mir- <dig>  mir- <dig> and mir-199b-5p were detectable.
no significant difference were observed for mir- <dig>  mir- <dig> and mir199b-5p expression.
on the contrary, mir-27b was shown significantly upregulated with a  <dig> -fold increase in piedmontese cattle  .
as a consequence of the data shown above, in piedmontese breed samples, we observed an inverse relationship between mstn transcript and mir-27b expression.
this observation was confirmed by spearman correlation test .
a correlation was also carried out between mstn abundance protein and mir-27b expression.
also in this case results showed a significant correlation .
the data of mstn expression downregulation in mrna and protein analysis, and the upregulation of mir-27b in piedmontese bovine samples prompted us to verify if mstn was a direct target of mir-27b in a functional assay.
the 3′-utr of bovine myostatin mrna is a direct target of mir-27b
through a target prediction algorithm , we identified mstn as the potential target of mir-27b.
targetscan predicts  <dig> conserved targets for mir-27a/b in the cow, of which mstn is ranked 82th on the basis of total context score.
to determine whether mstn is a direct target of mir-27b, we performed luciferase reporter assays in 293 t cells.
co-transfection of pre-mir-27b with the luciferase reporter gene linked to the wild-type 3′-utr of mstn strongly inhibited the luciferase activity  .
moreover, no effect was observed with the mutant construct of mstn 3′-utr in which mir-27b seed sequence was mutated.
co-transfection of pre-mir- <dig>  with the wild-type mstn 3′-utr construct did not alter the luciferase activity, indicating that mir-27b specifically targeted mstn .
discussion
considerable progress has been made in elucidating signalling mechanisms of skeletal muscle hypertrophy at the molecular level, but fine regulatory processes are still unknown.
piedmontese breed has been systematically selected for double-muscled phenotype and the peculiar muscular hypertrophy have been analyzed for a long time.
until now, this hypertrophy was traced back to mstn point mutation
<cit> .
here we demonstrate that mstn is further regulated at post-transcriptional level by mir-27b.
in fact, despite the mstn homozygosis mutation is present in more than 96% of piedmontese cattle in piedmonte area, in the north-western part of italy, some differences in muscle phenotype conformation still exist.
it has been previously observed that these differences are visible in animals submitted to homogeneous management conditions .
therefore, observed hypertrophy variations among animals are related to genetics and they are hereditable, and not linked to the environment
<cit> .
to understand witch other mechanisms, other than mstn mutation, could be involved in the piedmontese muscle phenotype we have analyzed several negative and positive regulator genes involved in the skeletal muscle hypertrophy pathway  and relative mirnas with conserved site on their 3′utr in this breed and in friesian breed used as control, for opposed carcass conformation.
according to our analysis, mstn, in addition to mutation showed a strong downregulation at mrna and protein levels.
we also observed a significant, high upregulation for mir-27b.
recently, much attention has been focused on the potential role of mirnas in gene expression regulation.
mirnas may contribute to phenotypic variability in cattle selected for the mstn gene mutation.
in texel sheep, the mstn allele is characterized by a g vs a transition in the 3′utr that creates a target site for mir- <dig> and mir- <dig>  which are highly expressed in skeletal muscle.
this causes inhibition of the mstn gene and contributes to muscular hypertrophy
<cit> .
analysis of human and murine single nucleotide polymorphism demonstrates that mutations creating or destroying putative mirna target sites are abundant and therefore might be important sources of phenotypic variation
<cit> .
current data on the role of mirnas in myogenesis has been obtained largely from studies on muscle-specific mir- <dig>  mir- <dig> and mir- <dig>  we have previously demonstrated that piedmontese and friesian female, but not male cattle showed differential expression of mir- <dig> in skeletal muscle leading to hypothesize a relationship between mirna expression and sex
<cit> .
here, we screened in males that exhibit a greater carcass hypertrophy conformation than females, the possible involvement of non-muscle-specific mirnas in myogenesis that had not been yet thoroughly examined, and we report the pattern of expression of four non-muscle-specific mirnas potentially involved in muscle hypertrophic pathway.
in particular, we selected mir-27b because recently it has been recognized to have functional roles in cardiomyocytes
<cit>  and skeletal muscle cells
<cit> .
it has been reported that nuclear receptor peroxisome proliferator-activated receptor-γ  is targeted by mir-27b in the adult mouse heart and that mir-27b overexpression in primary cultured cardiomyocytes promoted hypertrophic cell growth through this way
<cit> .
in muscle cells, mir-27b down-regulates pax <dig> expression during myogenic differentiation
<cit>  and mstn in murine myoblast promoting proliferation
<cit> .
moreover, mef2c, a muscle specific transcription factor, which plays essential roles during cardiac and skeletal myogenesis, has been demonstrated as another important target of mir-27b during heart development
<cit> .
although the specific functions and targets of mir- <dig> in hypertrophic pathway are largely unexplored, in this work we suggest that in piedmontese cattle, mstn is further regulated at post-transcriptional level by this mirna.
mature mir- <dig> sequence, as predicted by targetscan, is well conserved among a wide range of mammalian species, and our data are comparable to those obtained recently in mouse, where mstn 3′utr was demonstrated to be targeted by mir-27a/b
<cit> .
muscle samples were evaluated from male bovines based on s.e.u.r.o.p.
grid classification.
the s.e.u.r.o.p.
system is currently in use for carcass classification in european union.
the carcass classification is done after slaughter, when the cleaned carcasses are halved and a trained classifier weighs and evaluates them with respect to fatness and fleshiness.
we have shown the strong different expression of mir-27b when we compared animals from two opposite conformation such as “e” and “o”.
on the contrary, the collected samples with carcass conformation e and u did not show a specific relationship with mir-27b .
we may argue that to observe differences in muscle hypertrophy we need to extend the analysis in a larger group of animals.
the high ability of mir-27b to suppress mstn 3′utr lead us to hypothesize that different levels of expression of mir-27b may elucidate differences in muscle conformation.
a 79% of 3′utr mstn knock down of luciferase activity is an exceptional down regulation compared to the 48% that huang and colleagues have shown for mir-27a on murine mstn 3′utr.
we suppose that in piedmonte region the discrepancies among “e” and “u” subject are so small that these conformation differences are difficult to perceive at molecular level with a good statistical significance.
further studies are currently carrying out to elucidate this question.
CONCLUSIONS
in summary, the important novel findings of our study are that exist a relationship between mir-27b and hypertrophic phenotype in bovine with a correlation between abundance of mstn transcript/protein and mir-27b expression, and that mstn gene is a target of mir-27b.
thus, our results provide the first evidence showing that mstn-regulated mir-27b could play an important role in bovine skeletal muscle growth and hypertrophy.
to our knowledge, this is the first study to report a link between double-muscled phenotype in piedmontese bovine and mirnas.
these data indicate a specific role for mir-27b in the regulation of mstn, which might be of great interest as selection marker to improve muscular hypertrophy.

 skeletal muscle hypertrophy is defined as an increase in muscle mass, which in the adult animal comes as a result of an increase in size of skeletal muscle fibers. in the last years different mechanism of action have been reported to regulate muscle hypertrophy. the major extracellular mediator of skeletal muscle hypertrophy is thought to be insulin growth factor- which binds to its receptor igf1r to initiate a cascade of signaling pathways via phosphoinositide 3-kinase ). however, several lines of evidence suggest that igf- also mediates hypertrophy through calcineurin /nuclear factor of activated t-cells signaling pathway. moreover, studies suggested that myocyte enhancer factor 2c regulates the hypertrophic process. also glycogen synthase kinase beta, that is a distinct substrate of akt, has been shown to modulate hypertrophy. through akt phosphorylation, gsk3b activity is inhibited and its inhibition may induce hypertrophy by stimulating protein synthesis independent of the mtor pathway. tead regulates the expression of many skeletal muscle-specific genes. tead is a member of the tea domain family and is constitutively expressed in cardiac and skeletal muscles in pigs, mice and humans. the transcriptional regulation of tead to muscle-specific genes is implemented by co-operation with numerous co-factors, including mef2. myostatin is a member of the transforming growth factor-b superfamily of secreted growth and differentiation factors. in piedmontese cattle the double-muscled phenotype is an inherited condition associated to a point mutation in the mstn gene. the piedmontese mstn missense mutation g938a is translated to c313y myostatin protein with the consequent loss of one of the disulphide bonds involved in the characteristic tgf-b family cystine-knot structural motif. this mutation alters the function of mstn as a negative regulator of muscle growth, thereby inducing muscle hyperplasia and hypertrophy. this breed has been systematically selected for double muscling to the point of fixation in many herds, but some difference in muscularity phenotype is still present. micrornas are small non-coding rna molecules, highly conserved, that regulate gene expression through binding with imperfect complementarities sequences of messenger rna. it is becoming increasingly evident that mirnas regulation of mrnas represents an effective way of interfering with function by acting at protein translation level. this takes place when the mirna targets the 3′- untranslated region of transcripts by imperfect base pairing and functions to inhibit translation. in some cases, degradation of the target mrna is also a mechanism or a consequence of mirna-mediated suppression. mirnas have been shown to play critical roles in skeletal muscle development as well as in regulation of muscle cell proliferation and differentiation. by down-regulating gene expression, mirnas could play a role in skeletal muscle hypertrophy modulation. in this study, we have analyzed mirnas engaged in post-transcriptional regulation of negative or positive modulators involved in the muscular hypertrophy pathway in piedmontese cattle. in particular, we aimed to investigate on the specific mirnas involved in mstn regulation. we screened the 3′-utr matching mirnas of several genes, such as igf igf1r, ppp3ca, nfatc mef2c, gsk3b, tead and mstn using a computational approach. this analysis led to the identification of mir-27b, mir- mir- and mir-199b-5p as possible candidates implicated in bovine skeletal muscle hypertrophy. finally, we report the functional evidence that mir-27b is able to down-regulate mstn gene in vitro.  expression of igf igf1r, ppp3ca, nfatc mef2c, gsk3b, tead and mstn mrna in bovine skeletal muscle specimens skeletal muscle samples were collected from cattle from two breeds with opposite carcass conformation: piedmontese and friesian bovines classified respectively as “e” and “o” muscle profile. in order to identify possible gene expression differences linked to skeletal muscle phenotype between the two breeds, we have analyzed igf igf1r, ppp3ca, nfatc mef2c, gsk3b, tead and mstn genes using real-time quantitative pcr. as shown in figure  only mstn mrna expression displayed a strong difference between the two breeds. in particular, in piedmontese samples, mstn mrna expression was downregulated -fold compared with friesian muscle. on the other hand, the expression of igf igf1r, ppp3ca, nfatc mef2c, gsk3b and tead were not significantly different. mstn protein quantification total proteins were extracted from the muscle samples and measured by western blot analysis. as shown in figure  mstn specific antibody recognized a ~29-kda mature mstn in both friesian and piedmontese cattle muscle extracts. alpha-tubulin was used as loading control. densitometric analysis of western blot indicates that in piedmontese breed there was less abundance mstn than in friesian one. targeting site of mirnas in igf igf1r, ppp3ca, nfatc mef2c, gsk3b, tead and mstn 3′-utr to identify mirnas which could modulate bovine skeletal muscle hypertrophy, we have screened mirnas which potentially suppress expression of genes involved as negative or positive modulators in the muscular hypertrophy pathway. for this purpose, mirnas target site prediction for bovine igf igf1r, ppp3ca, nfatc mef2c, gsk3b, tead and mstn were performed with the use of targetscan algorithms. this analysis combined with bibliographic data research induced us to investigate mir-27b, mir- mir- and mir-199b-5p as potential candidates implicated in bovine skeletal muscle hypertrophy. the number of gene targets resulted higher for mir-27b than for other mirnas. targetscan predicts biological targets of mirnas by searching for the presence of conserved 8mer and 7mer sites that match the seed region of each mirna. in mammals, predictions are ranked based on the predicted efficacy of targeting as calculated using the context scores of the sites. the context score is the sum of the contribution of these four features: site-type contribution; 3′ pairing contribution; local au contribution; position contribution. expression of mir-27b, mir- mir- and mir-199b-5p in bovine skeletal muscle to examine which, if any, of the mirnas identified from the targetscan algorithm were expressed in bovine muscle, we used microrna taqman assay. sample of both breeds mir-27b, mir- mir- and mir-199b-5p were detectable. no significant difference were observed for mir- mir- and mir199b-5p expression. on the contrary, mir-27b was shown significantly upregulated with a -fold increase in piedmontese cattle. as a consequence of the data shown above, in piedmontese breed samples, we observed an inverse relationship between mstn transcript and mir-27b expression. this observation was confirmed by spearman correlation test. a correlation was also carried out between mstn abundance protein and mir-27b expression. also in this case results showed a significant correlation. the data of mstn expression downregulation in mrna and protein analysis, and the upregulation of mir-27b in piedmontese bovine samples prompted us to verify if mstn was a direct target of mir-27b in a functional assay. the 3′-utr of bovine myostatin mrna is a direct target of mir-27b through a target prediction algorithm, we identified mstn as the potential target of mir-27b. targetscan predicts conserved targets for mir-27a/b in the cow, of which mstn is ranked 82th on the basis of total context score. to determine whether mstn is a direct target of mir-27b, we performed luciferase reporter assays in 293 t cells. co-transfection of pre-mir-27b with the luciferase reporter gene linked to the wild-type 3′-utr of mstn strongly inhibited the luciferase activity. moreover, no effect was observed with the mutant construct of mstn 3′-utr in which mir-27b seed sequence was mutated. co-transfection of pre-mir- with the wild-type mstn 3′-utr construct did not alter the luciferase activity, indicating that mir-27b specifically targeted mstn. discussion considerable progress has been made in elucidating signalling mechanisms of skeletal muscle hypertrophy at the molecular level, but fine regulatory processes are still unknown. piedmontese breed has been systematically selected for double-muscled phenotype and the peculiar muscular hypertrophy have been analyzed for a long time. until now, this hypertrophy was traced back to mstn point mutation. here we demonstrate that mstn is further regulated at post-transcriptional level by mir-27b. in fact, despite the mstn homozygosis mutation is present in more than 96% of piedmontese cattle in piedmonte area, in the north-western part of italy, some differences in muscle phenotype conformation still exist. it has been previously observed that these differences are visible in animals submitted to homogeneous management conditions. therefore, observed hypertrophy variations among animals are related to genetics and they are hereditable, and not linked to the environment. to understand witch other mechanisms, other than mstn mutation, could be involved in the piedmontese muscle phenotype we have analyzed several negative and positive regulator genes involved in the skeletal muscle hypertrophy pathway and relative mirnas with conserved site on their 3′utr in this breed and in friesian breed used as control, for opposed carcass conformation. according to our analysis, mstn, in addition to mutation showed a strong downregulation at mrna and protein levels. we also observed a significant, high upregulation for mir-27b. recently, much attention has been focused on the potential role of mirnas in gene expression regulation. mirnas may contribute to phenotypic variability in cattle selected for the mstn gene mutation. in texel sheep, the mstn allele is characterized by a g vs a transition in the 3′utr that creates a target site for mir- and mir- which are highly expressed in skeletal muscle. this causes inhibition of the mstn gene and contributes to muscular hypertrophy. analysis of human and murine single nucleotide polymorphism demonstrates that mutations creating or destroying putative mirna target sites are abundant and therefore might be important sources of phenotypic variation. current data on the role of mirnas in myogenesis has been obtained largely from studies on muscle-specific mir- mir- and mir- we have previously demonstrated that piedmontese and friesian female, but not male cattle showed differential expression of mir- in skeletal muscle leading to hypothesize a relationship between mirna expression and sex. here, we screened in males that exhibit a greater carcass hypertrophy conformation than females, the possible involvement of non-muscle-specific mirnas in myogenesis that had not been yet thoroughly examined, and we report the pattern of expression of four non-muscle-specific mirnas potentially involved in muscle hypertrophic pathway. in particular, we selected mir-27b because recently it has been recognized to have functional roles in cardiomyocytes and skeletal muscle cells. it has been reported that nuclear receptor peroxisome proliferator-activated receptor-γ is targeted by mir-27b in the adult mouse heart and that mir-27b overexpression in primary cultured cardiomyocytes promoted hypertrophic cell growth through this way. in muscle cells, mir-27b down-regulates pax expression during myogenic differentiation and mstn in murine myoblast promoting proliferation. moreover, mef2c, a muscle specific transcription factor, which plays essential roles during cardiac and skeletal myogenesis, has been demonstrated as another important target of mir-27b during heart development. although the specific functions and targets of mir- in hypertrophic pathway are largely unexplored, in this work we suggest that in piedmontese cattle, mstn is further regulated at post-transcriptional level by this mirna. mature mir- sequence, as predicted by targetscan, is well conserved among a wide range of mammalian species, and our data are comparable to those obtained recently in mouse, where mstn 3′utr was demonstrated to be targeted by mir-27a/b. muscle samples were evaluated from male bovines based on s.e.u.r.o.p. grid classification. the s.e.u.r.o.p. system is currently in use for carcass classification in european union. the carcass classification is done after slaughter, when the cleaned carcasses are halved and a trained classifier weighs and evaluates them with respect to fatness and fleshiness. we have shown the strong different expression of mir-27b when we compared animals from two opposite conformation such as “e” and “o”. on the contrary, the collected samples with carcass conformation e and u did not show a specific relationship with mir-27b. we may argue that to observe differences in muscle hypertrophy we need to extend the analysis in a larger group of animals. the high ability of mir-27b to suppress mstn 3′utr lead us to hypothesize that different levels of expression of mir-27b may elucidate differences in muscle conformation. a 79% of 3′utr mstn knock down of luciferase activity is an exceptional down regulation compared to the 48% that huang and colleagues have shown for mir-27a on murine mstn 3′utr. we suppose that in piedmonte region the discrepancies among “e” and “u” subject are so small that these conformation differences are difficult to perceive at molecular level with a good statistical significance. further studies are currently carrying out to elucidate this question.  in summary, the important novel findings of our study are that exist a relationship between mir-27b and hypertrophic phenotype in bovine with a correlation between abundance of mstn transcript/protein and mir-27b expression, and that mstn gene is a target of mir-27b. thus, our results provide the first evidence showing that mstn-regulated mir-27b could play an important role in bovine skeletal muscle growth and hypertrophy. to our knowledge, this is the first study to report a link between double-muscled phenotype in piedmontese bovine and mirnas. these data indicate a specific role for mir-27b in the regulation of mstn, which might be of great interest as selection marker to improve muscular hypertrophy. 
this analysis led to the identification of mir-27b, mir- mir- and mir-199b-5p as possible candidates. cotransfection of mir-27b and psi-check vector with the luciferase reporter gene linked to the bovine wild-type 3′-utr of mstn strongly inhibited the luciferase activity. these data demonstrate that bovine mstn is a specific target of mir-27b and that mirnas contribute to explain additive phenotypic hypertrophy in piedmontese cattle selected for the mstn gene mutation, possibly outlining a more precise genetic signature able to elucidate differences in muscle conformation. the piedmontese mstn missense mutation g938a is translated to c313y myostatin protein. this mutation alters mstn function as a negative regulator of muscle growth, thereby inducing muscle hypertrophy. mirnas could play a role in skeletal muscle hypertrophy modulation by down-regulating gene expression. after identifying a 3′-utr consensus sequence of several negative and positive modulator genes involved in the skeletal muscle hypertrophy pathway, such as igf igf1r, ppp3ca, nfatc mef2c, gsk3b, tead and mstn, we screened mirnas matching to it. further, we report that the level of mstn mrna was about 5-fold lower in piedmontese cattle vs friesian cattle and that less mature mstn protein was detected in the piedmontese one. in piedmontese cattle the double-muscled phenotype is an inherited condition associated to a point mutation in the myostatin gene.
 in piedmontese cattle the double-muscled phenotype is an inherited condition associated to a point mutation in the myostatin gene. the piedmontese mstn missense mutation g938a is translated to c313y myostatin protein. this mutation alters mstn function as a negative regulator of muscle growth, thereby inducing muscle hypertrophy. mirnas could play a role in skeletal muscle hypertrophy modulation by down-regulating gene expression.  after identifying a 3′-utr consensus sequence of several negative and positive modulator genes involved in the skeletal muscle hypertrophy pathway, such as igf igf1r, ppp3ca, nfatc mef2c, gsk3b, tead and mstn, we screened mirnas matching to it. this analysis led to the identification of mir-27b, mir- mir- and mir-199b-5p as possible candidates. we collected samples of longissimus thoracis from twenty piedmontese and twenty friesian male bovines. in piedmontese group mir-27b was up-regulated -fold. further, we report that the level of mstn mrna was about 5-fold lower in piedmontese cattle vs friesian cattle and that less mature mstn protein was detected in the piedmontese one. cotransfection of mir-27b and psi-check vector with the luciferase reporter gene linked to the bovine wild-type 3′-utr of mstn strongly inhibited the luciferase activity.  these data demonstrate that bovine mstn is a specific target of mir-27b and that mirnas contribute to explain additive phenotypic hypertrophy in piedmontese cattle selected for the mstn gene mutation, possibly outlining a more precise genetic signature able to elucidate differences in muscle conformation. micrornabovineskeletal musclehypertrophy 