BACKGROUND
adipogenesis is the development of fat cells from preadipocytes to mature adipocytes, and has been one of the most intense studied models of cellular differentiation  <cit> , which implicated in insulin resistance, type  <dig> diabetes, hypertension and atherosclerosis, collectively called the metabolic syndrome  <cit> .
wingless-type mmtv integration site family  is indispensable for the regulation of adipogenesis  <cit> , which is a family of secreted glycoproteins with autocrine and paracrine effects on the regulation of cell proliferation, survival, fate and behavior  <cit> .
in canonical wnt signaling, wnt members bind to frizzled  receptors and low-density lipoprotein receptor-related protein- <dig> or - <dig>  co-receptors, leading to inactivation of the degradation complex that includes glycogen synthase kinase -3β, axin and adenomatous polyposis coli , in turn blocking β-catenin phosphorylation by gsk-3β.
hypophosphorylation of β-catenin and translocation into the nucleus leads to binding with members of the lymphoid-enhancer-binding factor/t-cell-specific transcription factor  family and activation of wnt target genes  <cit> .
as a member of lef/tcf family, transcription factor  <dig> like  <dig>  is an important transcription factor triggering the downstream responsive genes of wnt signaling  <cit> .
previous research has shown that wnt signaling represses adipocyte differentiation by blocking the expression of pparg and cebpa, two transcription factors indispensable for adipogenesis  <cit> .
wnt10b was first identified as an inhibitor of adipogenesis, which must be suppressed for pre-adipocytes to differentiate in vitro  <cit> .
in vivo experiments also demonstrated that wnt-10b transgenic mice showed a similar decline in total body fat as well as perirenal depots  <cit> .
ectopic expression of wnt- <dig> in 3t3-l <dig> pre-adipocytes blocked adipogenesis  <cit> .
together, these results indicated that the wnt/β-catenin signaling pathway suppresses adipocyte differentiation and is therefore a molecular switch of adipogenesis; once it is activated, pre-adipocytes are maintained in an undifferentiated state  <cit> .
recently, it has been found that rnas of a new class of noncoding rnas, micrornas , regulate gene expression in metazoans from c. elegans to humans  <cit> .
mirnas are endogenous non-coding rnas, 22- <dig> nucleotides in length, which are demonstrated to modulate diverse biological processes through negatively regulating gene expressions posttranscriptionally by controlling mrna stability or translation <cit> .
many studies have shown that mirnas participate in multiple metabolic processes including energy homeostasis, sugar/lipid metabolism and cell differentiation.
in the development of lipogenesis, mir- <dig> was first reported to be up-regulated in human pre-adipocytes  <cit>  and in adult mice in vivo  <cit> , suggesting that it was involved in adipocyte differentiation; mir- <dig> has also been reported to promote 3t3-l <dig> preadipocytes differentiation  <cit> .
to sum up, these findings indicated that wnt signaling and mirnas have regulatory roles in adipogenesis.
to investigate which mirnas might modulate wnt signaling during adipogenesis, we screened out the pupative mirnas using microarray assay.
here we used 3t3-l <dig> cells to establish two models by different treatments.
one treatment was to induce differentiation with an mdi cocktail , suppressing wnt signaling.
the other was to treat the cells with lithium , which is an inhibitor of gsk-3β that further blocks the phosphorylation of β-catenin, resulting in activation of wnt signaling  <cit> .
the different mirna expression patterns of the two models were investigated by a mouse mirna microarray, enabling us to screen out a number of mirnas that might regulate adipogenesis by activating or repressing the wnt signaling pathway.
RESULTS
cell models construction
in model  <dig>  pre-adipocytes  were induced to differentiate into mature adipocytes by mdi induction assay; enlarged cells with distinct lipid droplets were observed under the light microscope .
in model  <dig>  the pre-adipocytes treating with lithium before mdi induction  failed to differentiate into mature adipocytes , as confirmed by oil red o staining .
as a control, nacl was used in place of licl before mdi induction, the pre-adipocytes differentiated normally into mature adipocytes .
the following molecular data were fully consistent with these morphological findings.
target genes of the wnt signaling pathway such as cyclin d <dig> and c-myc were markedly more highly expressed in model  <dig> than model  <dig> cells .
in time course experiments with model  <dig>  several transcription factors such as pparg, cebpa and add <dig> and differentiation-specific genes such as ap <dig> and adiponectin  <cit>  increased significantly during adipogenesis.
interestingly, these genes were blocked completely in model  <dig> .
both the morphological and molecular data showed that wnt signaling was dramatically suppressed and adipogenesis promoted in model  <dig>  while in model  <dig>  wnt signaling was activated and adipogenesis totally repressed.
this suggested that the cell models had been constructed successfully.
mirna expression profile revealed by microarray data
expression profile of differentiation-specific mirnas
to identify the mirna related to adipocyte differentiation, mirna expression in pre-adipocytes and adipocytes was analyzed by microarray.
the results showed that  <dig> mirnas underwent at least twofold changes, significant at p <  <dig>  .
comparison between the two sets screened out most mirnas; nine were down-regulated and  <dig> up-regulated in the pre-adipocytes.
remarkably, mir-582-5p and mir-99a were up-regulated 6-fold and  <dig> -fold respectively in mature adipocytes.
conversely, mir-196a and mir-130b were down-regulated 7- and  <dig> -fold respectively in mature adipocytes.
these were all significant differences .
up-regulation means the expression of the mirnas are higher in mature adipocytes than pre-adipocytes; down-regulation is the reverse.
expression profile of wnt pathway-specific mirnas
to investigate mirna profiles associated with the wnt signaling activation, mirna expression between the preadipocytes treating with or without lithium was analyzed by microarray.
the results showed  <dig> mirnas differ-expression for twofold, significant at p <  <dig>  .
seven of these were up-regulated and eight down-regulated in lithium-treated pre-adipocytes.
mir-322* was down-regulated  <dig> -fold and mir-183* was up-regulated significantly  changing 4-fold, in lithium-treated pre-adipocytes.
notably, mir- <dig> was down-regulated dramatically, nearly 8-fold , in these cells.
up-regulation means the expression of the mirnas are higher in lithium-treated pre-adipocytes than pre-adipocytes; down-regulation means the expression of the mirnas are lower in lithium-treated pre-adipocytes than pre-adipocytes.
expression profile of wnt pathway-specific mirnas by further induction in lithium-treated cells
to clarify further the pattern of mirnas specific to wnt signaling, we reinvestigated the mirnas that were differentially expressed between the mdi  and li+mdi  groups.
as observed by light microscopy, li+mdi-treated cells did not differentiate into mature adipocytes , while the cells of the mdi group differentiated fully .
microarray analysis showed that  <dig> mirnas differed at least two-fold between these treatments : ten were down-regulated and  <dig> up-regulated in the li+mdi group.
in li+mdi-treated cells, mir-24-1* and mir-301a were up-regulated  <dig> - and  <dig> -fold respectively, whereas mir- <dig> was down-regulated  <dig> -fold; all those differences were significant .
remarkably, mir- <dig> was up-regulated about 10-fold and mir- <dig> down-regulated about 6-fold in li+mdi-treated cells.
up-regulation means the expression of the mirnas are higher in li+mdi cells than mdi cells; down-regulation means the expression of the mirnas are lower in li+mdi cells than mdi cells.
validation of microarray by real time qrt-pcr
to confirm the accuracy of the mirna microarray, stem-loop polymerase chain reaction  assay was performed on several mirnas chosen at random  <cit> .
to determine whether there was a similar trend between the microarray and real time qpcr results, the latter were normalized by fold amplification.
as shown in figure  <dig>  the qrt-pcr data  mapped the microarray results closely.
for example, the microarray data showed that mir- <dig> expression was higher in lithium-treated preadipocytes than in untreated preadipocytes, and the same trend was also observed in the qrt-pcr results.
these indicated the precision and accuracy of the mirna microarray used in the present study.
mir- <dig> promotes adipogenesis by repressing wnt signaling through targeting tcf7l2
to validate the predicted targets and functions of these identified mirnas, a mimic of mir- <dig> was transfected into 3t3-l <dig> cells for 24h before mdi induction, three days later, 100% of cell differentiated into mature adipocytes , while only less than 10% of the control cells differentiated .
meanwhile, qpcr data  showed the differentiated specific factors such as pparg and ap <dig> increased in transcription level after transfection with the mimics of mir- <dig>  while both of the two factors decreased markedly after transfection with the inhibitor of mir- <dig> .
the predicted target of mir- <dig>  tcf7l <dig> was repressed at the transcriptional level in a dose-dependent manner , and the activity of luciferase reporter containing the mir- <dig> binding site in the 3'utr of tcf7l <dig> mrna  decreased for nearly 60% in hek293ft cells .
in lithium-treated 3t3-l <dig> cells, β-catenin protien was increased not only in the cytoplasm but also in the nucleus after transfection with mir- <dig> .
these findings indicated that mir- <dig> promotes the lipids formation by repressing wnt signaling through targeting tcf7l <dig>
discussion
for years, the mechanism and development of lipid formation have been actively investigated in the study of adipogenesis and obesity  <cit> .
many studies about the switch of adipogenesis, wnt signaling pathway, just focused on protein-coding genes such as wnt <dig>  wnt <dig> and wnt10b  <cit> .
recently, mirnas have been found to have wide-ranging regulatory functions in cell proliferation, differentiation and many other processes  <cit> , but only a few studies have focused on the interaction with wnt signaling during adipogenesis.
strikingly, one report showed that mirnas have interaction with wnt signaling during the adipogenesis and mir- <dig> family members including mir-200a/b/c, mir- <dig> and mir- <dig> promoted adipogenesis  <cit> .
thus, mirnas are believed to elucidate the mechanism of adipogenesis by regulating the wnt/β-catenin signaling pathway  <cit> .
in the current study, wnt-suppressed cells , i.e.
pre-adipocytes treated with a wnt-antagonist such as insulin, differentiated into mature adipocytes .
meanwhile, the expression of adipocyte differentiation-specific genes such as pparg, cebpa etc.
increased significantly  after induction .
these data indicated that the wnt-suppressed cell model was successfully established.
in contrast, in the wnt-activated cell model , i.e.
preadipocytes treated with the wnt signaling activator lithium, adipogenesis was blocked completely  and the expression of adipogenesis-specific genes was totally suppressed  , which suggests that the wnt-activated cell model was successfully established and the following experiments were reliable.
besides, combined investigation of wnt signaling pathway and mirnas could offer a novel sight into the mechanism of adipogenesis.
according to the predicted potential function in adipogenesis by regulating wnt signaling, these mirnas can be divided into two sets; one that suppresses wnt signaling and one that activates it.
the former set should be characterized by up-regulation in mature adipocytes and down-regulation in both lithium-treated pre-adipocytes and cells of the li+mdi group.
these mirnas also target the key factors in the wnt pathway, including members of wnt family such as wnt10b, wnt <dig> and axin <dig>  and molecules in the wnt pathway such as lrp5/ <dig>  tcf and dvl .
the aforementioned screened pattern indicated that some mirnas such as mir- <dig>  mir- <dig>  mir-148a, mir- <dig> and mir- <dig> accorded with this prediction.
a previous report has shown that mir- <dig> was required for adipocyte differentiation, as confirmed by northern blotting  <cit> .
in the present study, mir- <dig>  which was markedly up-regulated in differentiating adipocytes , was predicted to target wnt3a and consequently to promote adipogenesis; thus, our prediction was consistent with the previous study.
the second set of mirna  is characterized by down-regulation in mature adipocytes and up-regulation in lithium-treated pre-adipocytes and adipocytes in the li+mdi group.
importantly, these mirnas mostly target important transcriptional factors or differentiation-specific marker genes during adipogenesis, such as pparg, cebpa etc.
.
they included mir-301a, mir-130a/b, mir-27a/b, mir-18a, mir- <dig> and mir-181a/b/c/d, and were predicted to activate wnt signaling, resulting in the suppression of adipogenesis.
a previous study showed that the mir- <dig> gene family was down-regulated during adipocytes differentiation.
over-expression of mir- <dig> specifically inhibited adipocytes formation and blocked the expression of pparg and cebpa as well as fat tissue in obese mice  <cit> .
a similar result in the present study validated this previous report.
as shown in table  <dig>  mir-27b was up-regulated significantly by about  <dig> -fold  in li+mdi group cells, indicating that it was less necessary for adipogenesis; mir-27a/b was also predicted to target a master regulator in adipogenesis, i.e.
pparg, indicating that they might be antagonistic to adipogenesis, which was consistent with the previous report.
to sum up: using microarray data, we screened out two sets of mirnas that might play a role in suppressing or activating wnt signaling during adipogenesis.
this predicted pattern was shown in figure  <dig>  which offers a model for the study of adipogenesis related to mirnas and wnt signaling.
to date, only a small number of predicted targets have been experimentally validated, so further investigation is needed to clarify the function of these mirnas  <cit> .
microarray data showed that mir- <dig> was up-regulated in adipocytes, which suggested the potential role of mir- <dig> in promoting lipogenesis.
after transfection of mir- <dig>  the lipogenesis was dramatically promoted , but when antagonizing mir- <dig>  the differentiation specific genes such as pparg and ap <dig> were makedly suppressed, which suggested the specific role of mir- <dig> in promoting adipogenesis .
tcf7l <dig>  a key transcription factor in activation by wnt signaling  <cit> , is essential for the β-catenin/tcf complex downstream of wnt signaling.
as predicted, mir- <dig> targets tcf7l <dig> with hybrid free energy - <dig>  kcal/mol.
after transfection of mir- <dig>  tcf7l <dig> was markedly repressed at the transcriptional level  and the activity of luciferase reporter decreased markedly , which indicated that mir- <dig> promoted the lipid formation and tcf7l <dig> should be targeted by mir- <dig>  when wnt signaling was activated by lithium, β-catenin protein decreased dramatically.
interestingly, after transfection with mir- <dig>  the β-catenin protein increased not only in the cytoplasm but also in the nucleus .
these lines of evidence suggested that mir- <dig> probably plays a role in promoting the adipogenesis and repressing wnt signaling through targeting the transcription factor tcf7l <dig>  these experiments not only validated our hypothesis but also confirmed the accuracy and reliability of the predicted target of these mirnas.
mir- <dig>  which was reported to be required during adipocyte differentiation  <cit> , was not listed in our microarray data.
in the present study, micrornas that changed at least twofold and differed markedly  between the two cell models were identified as potentially interesting.
qrt-pcr showed that mir- <dig> expression in adipocytes after seven days' differentiation was  <dig>  fold higher than in pre-adipocytes .
in a previous study, northern blotting indicated that mir- <dig> changed significantly on the 9th day after mdi induction, while the sample in the current work was harvested on the 7th day.
as for the strict screening criterion, the expression of mir- <dig> does not change dramatically before the 9th day of differentiation and therefore was not shown in table  <dig> <cit> .
the list of hybrids represents the free energies needed for micrrnas to bind their targets: the lower the value, the more stable the binding, which offers an alternative way of assessing the reliability of predictions.
to evaluate this screening criterion, we carried out experiments to find a reliable hybrid score by transfection of mirna in vitro and conducting semi-quantitative pcr to detect the target genes.
the results showed a good correlation in the mirnas and predicted targets when the hybrid free energy was less than - <dig> kcal/mol .
consistent with our observation, this criterion was validated by other reports using a similar approach  <cit> .
for example, the hybrid free energy of mir-146a and its predicted target wnt5a was - <dig>  kcal/mol; our prediction was confirmed by previous study.
therefore, hybrid free energy is recommended for consideration in the prediction of mirna targets; the prediction is more reliable when hybrid score is less than - <dig> kcal/mol.
CONCLUSIONS
in summary, the current work used microarray experiments to examine global mirna expression in two 3t3-l <dig> cell models, i.e.
activation or suppression of wnt signaling by mdi induction or lithium treatment.
mirna target prediction and further functional characterization of mir- <dig> will supplement the results of this study and help to elucidate the specific roles in adipogenesis.
the findings demonstrated crosstalk between wnt signaling and mirnas, highlighting the role of mirnas during adipogenesis, i.e.
repression and promotion of adipogenesis by regulating wnt signaling.
the global changes in mirna expression discovered in the present study may for the first time elucidate the preliminary mechanisms underlying wnt signaling-mediated changes in lipid formation during adipogenesis.

 adipogenesis is the development of fat cells from preadipocytes to mature adipocytes, and has been one of the most intense studied models of cellular differentiation, which implicated in insulin resistance, type diabetes, hypertension and atherosclerosis, collectively called the metabolic syndrome. wingless-type mmtv integration site family is indispensable for the regulation of adipogenesis, which is a family of secreted glycoproteins with autocrine and paracrine effects on the regulation of cell proliferation, survival, fate and behavior. in canonical wnt signaling, wnt members bind to frizzled receptors and low-density lipoprotein receptor-related protein- or - co-receptors, leading to inactivation of the degradation complex that includes glycogen synthase kinase -3β, axin and adenomatous polyposis coli, in turn blocking β-catenin phosphorylation by gsk-3β. hypophosphorylation of β-catenin and translocation into the nucleus leads to binding with members of the lymphoid-enhancer-binding factor/t-cell-specific transcription factor family and activation of wnt target genes. as a member of lef/tcf family, transcription factor like is an important transcription factor triggering the downstream responsive genes of wnt signaling. previous research has shown that wnt signaling represses adipocyte differentiation by blocking the expression of pparg and cebpa, two transcription factors indispensable for adipogenesis. wnt10b was first identified as an inhibitor of adipogenesis, which must be suppressed for pre-adipocytes to differentiate in vitro. in vivo experiments also demonstrated that wnt-10b transgenic mice showed a similar decline in total body fat as well as perirenal depots. ectopic expression of wnt- in 3t3-l pre-adipocytes blocked adipogenesis. together, these results indicated that the wnt/β-catenin signaling pathway suppresses adipocyte differentiation and is therefore a molecular switch of adipogenesis; once it is activated, pre-adipocytes are maintained in an undifferentiated state. recently, it has been found that rnas of a new class of noncoding rnas, micrornas, regulate gene expression in metazoans from c. elegans to humans. mirnas are endogenous non-coding rnas, 22- nucleotides in length, which are demonstrated to modulate diverse biological processes through negatively regulating gene expressions posttranscriptionally by controlling mrna stability or translation. many studies have shown that mirnas participate in multiple metabolic processes including energy homeostasis, sugar/lipid metabolism and cell differentiation. in the development of lipogenesis, mir- was first reported to be up-regulated in human pre-adipocytes and in adult mice in vivo, suggesting that it was involved in adipocyte differentiation; mir- has also been reported to promote 3t3-l preadipocytes differentiation. to sum up, these findings indicated that wnt signaling and mirnas have regulatory roles in adipogenesis. to investigate which mirnas might modulate wnt signaling during adipogenesis, we screened out the pupative mirnas using microarray assay. here we used 3t3-l cells to establish two models by different treatments. one treatment was to induce differentiation with an mdi cocktail, suppressing wnt signaling. the other was to treat the cells with lithium, which is an inhibitor of gsk-3β that further blocks the phosphorylation of β-catenin, resulting in activation of wnt signaling. the different mirna expression patterns of the two models were investigated by a mouse mirna microarray, enabling us to screen out a number of mirnas that might regulate adipogenesis by activating or repressing the wnt signaling pathway.  cell models construction in model pre-adipocytes were induced to differentiate into mature adipocytes by mdi induction assay; enlarged cells with distinct lipid droplets were observed under the light microscope. in model the pre-adipocytes treating with lithium before mdi induction failed to differentiate into mature adipocytes, as confirmed by oil red o staining. as a control, nacl was used in place of licl before mdi induction, the pre-adipocytes differentiated normally into mature adipocytes. the following molecular data were fully consistent with these morphological findings. target genes of the wnt signaling pathway such as cyclin d and c-myc were markedly more highly expressed in model than model cells. in time course experiments with model several transcription factors such as pparg, cebpa and add and differentiation-specific genes such as ap and adiponectin increased significantly during adipogenesis. interestingly, these genes were blocked completely in model. both the morphological and molecular data showed that wnt signaling was dramatically suppressed and adipogenesis promoted in model while in model wnt signaling was activated and adipogenesis totally repressed. this suggested that the cell models had been constructed successfully. mirna expression profile revealed by microarray data expression profile of differentiation-specific mirnas to identify the mirna related to adipocyte differentiation, mirna expression in pre-adipocytes and adipocytes was analyzed by microarray. the results showed that mirnas underwent at least twofold changes, significant at p <. comparison between the two sets screened out most mirnas; nine were down-regulated and up-regulated in the pre-adipocytes. remarkably, mir-582-5p and mir-99a were up-regulated 6-fold and -fold respectively in mature adipocytes. conversely, mir-196a and mir-130b were down-regulated 7- and -fold respectively in mature adipocytes. these were all significant differences. up-regulation means the expression of the mirnas are higher in mature adipocytes than pre-adipocytes; down-regulation is the reverse. expression profile of wnt pathway-specific mirnas to investigate mirna profiles associated with the wnt signaling activation, mirna expression between the preadipocytes treating with or without lithium was analyzed by microarray. the results showed mirnas differ-expression for twofold, significant at p <. seven of these were up-regulated and eight down-regulated in lithium-treated pre-adipocytes. mir-322* was down-regulated -fold and mir-183* was up-regulated significantly changing 4-fold, in lithium-treated pre-adipocytes. notably, mir- was down-regulated dramatically, nearly 8-fold, in these cells. up-regulation means the expression of the mirnas are higher in lithium-treated pre-adipocytes than pre-adipocytes; down-regulation means the expression of the mirnas are lower in lithium-treated pre-adipocytes than pre-adipocytes. expression profile of wnt pathway-specific mirnas by further induction in lithium-treated cells to clarify further the pattern of mirnas specific to wnt signaling, we reinvestigated the mirnas that were differentially expressed between the mdi and li+mdi groups. as observed by light microscopy, li+mdi-treated cells did not differentiate into mature adipocytes, while the cells of the mdi group differentiated fully. microarray analysis showed that mirnas differed at least two-fold between these treatments: ten were down-regulated and up-regulated in the li+mdi group. in li+mdi-treated cells, mir-24-1* and mir-301a were up-regulated - and -fold respectively, whereas mir- was down-regulated -fold; all those differences were significant. remarkably, mir- was up-regulated about 10-fold and mir- down-regulated about 6-fold in li+mdi-treated cells. up-regulation means the expression of the mirnas are higher in li+mdi cells than mdi cells; down-regulation means the expression of the mirnas are lower in li+mdi cells than mdi cells. validation of microarray by real time qrt-pcr to confirm the accuracy of the mirna microarray, stem-loop polymerase chain reaction assay was performed on several mirnas chosen at random. to determine whether there was a similar trend between the microarray and real time qpcr results, the latter were normalized by fold amplification. as shown in figure the qrt-pcr data mapped the microarray results closely. for example, the microarray data showed that mir- expression was higher in lithium-treated preadipocytes than in untreated preadipocytes, and the same trend was also observed in the qrt-pcr results. these indicated the precision and accuracy of the mirna microarray used in the present study. mir- promotes adipogenesis by repressing wnt signaling through targeting tcf7l2 to validate the predicted targets and functions of these identified mirnas, a mimic of mir- was transfected into 3t3-l cells for 24h before mdi induction, three days later, 100% of cell differentiated into mature adipocytes, while only less than 10% of the control cells differentiated. meanwhile, qpcr data showed the differentiated specific factors such as pparg and ap increased in transcription level after transfection with the mimics of mir- while both of the two factors decreased markedly after transfection with the inhibitor of mir-. the predicted target of mir- tcf7l was repressed at the transcriptional level in a dose-dependent manner, and the activity of luciferase reporter containing the mir- binding site in the 3'utr of tcf7l mrna decreased for nearly 60% in hek293ft cells. in lithium-treated 3t3-l cells, β-catenin protien was increased not only in the cytoplasm but also in the nucleus after transfection with mir-. these findings indicated that mir- promotes the lipids formation by repressing wnt signaling through targeting tcf7l discussion for years, the mechanism and development of lipid formation have been actively investigated in the study of adipogenesis and obesity. many studies about the switch of adipogenesis, wnt signaling pathway, just focused on protein-coding genes such as wnt wnt and wnt10b. recently, mirnas have been found to have wide-ranging regulatory functions in cell proliferation, differentiation and many other processes, but only a few studies have focused on the interaction with wnt signaling during adipogenesis. strikingly, one report showed that mirnas have interaction with wnt signaling during the adipogenesis and mir- family members including mir-200a/b/c, mir- and mir- promoted adipogenesis. thus, mirnas are believed to elucidate the mechanism of adipogenesis by regulating the wnt/β-catenin signaling pathway. in the current study, wnt-suppressed cells, i.e. pre-adipocytes treated with a wnt-antagonist such as insulin, differentiated into mature adipocytes. meanwhile, the expression of adipocyte differentiation-specific genes such as pparg, cebpa etc. increased significantly after induction. these data indicated that the wnt-suppressed cell model was successfully established. in contrast, in the wnt-activated cell model, i.e. preadipocytes treated with the wnt signaling activator lithium, adipogenesis was blocked completely and the expression of adipogenesis-specific genes was totally suppressed, which suggests that the wnt-activated cell model was successfully established and the following experiments were reliable. besides, combined investigation of wnt signaling pathway and mirnas could offer a novel sight into the mechanism of adipogenesis. according to the predicted potential function in adipogenesis by regulating wnt signaling, these mirnas can be divided into two sets; one that suppresses wnt signaling and one that activates it. the former set should be characterized by up-regulation in mature adipocytes and down-regulation in both lithium-treated pre-adipocytes and cells of the li+mdi group. these mirnas also target the key factors in the wnt pathway, including members of wnt family such as wnt10b, wnt and axin and molecules in the wnt pathway such as lrp5/ tcf and dvl. the aforementioned screened pattern indicated that some mirnas such as mir- mir- mir-148a, mir- and mir- accorded with this prediction. a previous report has shown that mir- was required for adipocyte differentiation, as confirmed by northern blotting. in the present study, mir- which was markedly up-regulated in differentiating adipocytes, was predicted to target wnt3a and consequently to promote adipogenesis; thus, our prediction was consistent with the previous study. the second set of mirna is characterized by down-regulation in mature adipocytes and up-regulation in lithium-treated pre-adipocytes and adipocytes in the li+mdi group. importantly, these mirnas mostly target important transcriptional factors or differentiation-specific marker genes during adipogenesis, such as pparg, cebpa etc.. they included mir-301a, mir-130a/b, mir-27a/b, mir-18a, mir- and mir-181a/b/c/d, and were predicted to activate wnt signaling, resulting in the suppression of adipogenesis. a previous study showed that the mir- gene family was down-regulated during adipocytes differentiation. over-expression of mir- specifically inhibited adipocytes formation and blocked the expression of pparg and cebpa as well as fat tissue in obese mice. a similar result in the present study validated this previous report. as shown in table mir-27b was up-regulated significantly by about -fold in li+mdi group cells, indicating that it was less necessary for adipogenesis; mir-27a/b was also predicted to target a master regulator in adipogenesis, i.e. pparg, indicating that they might be antagonistic to adipogenesis, which was consistent with the previous report. to sum up: using microarray data, we screened out two sets of mirnas that might play a role in suppressing or activating wnt signaling during adipogenesis. this predicted pattern was shown in figure which offers a model for the study of adipogenesis related to mirnas and wnt signaling. to date, only a small number of predicted targets have been experimentally validated, so further investigation is needed to clarify the function of these mirnas. microarray data showed that mir- was up-regulated in adipocytes, which suggested the potential role of mir- in promoting lipogenesis. after transfection of mir- the lipogenesis was dramatically promoted, but when antagonizing mir- the differentiation specific genes such as pparg and ap were makedly suppressed, which suggested the specific role of mir- in promoting adipogenesis. tcf7l a key transcription factor in activation by wnt signaling, is essential for the β-catenin/tcf complex downstream of wnt signaling. as predicted, mir- targets tcf7l with hybrid free energy - kcal/mol. after transfection of mir- tcf7l was markedly repressed at the transcriptional level and the activity of luciferase reporter decreased markedly, which indicated that mir- promoted the lipid formation and tcf7l should be targeted by mir- when wnt signaling was activated by lithium, β-catenin protein decreased dramatically. interestingly, after transfection with mir- the β-catenin protein increased not only in the cytoplasm but also in the nucleus. these lines of evidence suggested that mir- probably plays a role in promoting the adipogenesis and repressing wnt signaling through targeting the transcription factor tcf7l these experiments not only validated our hypothesis but also confirmed the accuracy and reliability of the predicted target of these mirnas. mir- which was reported to be required during adipocyte differentiation, was not listed in our microarray data. in the present study, micrornas that changed at least twofold and differed markedly between the two cell models were identified as potentially interesting. qrt-pcr showed that mir- expression in adipocytes after seven days' differentiation was fold higher than in pre-adipocytes. in a previous study, northern blotting indicated that mir- changed significantly on the 9th day after mdi induction, while the sample in the current work was harvested on the 7th day. as for the strict screening criterion, the expression of mir- does not change dramatically before the 9th day of differentiation and therefore was not shown in table. the list of hybrids represents the free energies needed for micrrnas to bind their targets: the lower the value, the more stable the binding, which offers an alternative way of assessing the reliability of predictions. to evaluate this screening criterion, we carried out experiments to find a reliable hybrid score by transfection of mirna in vitro and conducting semi-quantitative pcr to detect the target genes. the results showed a good correlation in the mirnas and predicted targets when the hybrid free energy was less than - kcal/mol. consistent with our observation, this criterion was validated by other reports using a similar approach. for example, the hybrid free energy of mir-146a and its predicted target wnt5a was - kcal/mol; our prediction was confirmed by previous study. therefore, hybrid free energy is recommended for consideration in the prediction of mirna targets; the prediction is more reliable when hybrid score is less than - kcal/mol.  in summary, the current work used microarray experiments to examine global mirna expression in two 3t3-l cell models, i.e. activation or suppression of wnt signaling by mdi induction or lithium treatment. mirna target prediction and further functional characterization of mir- will supplement the results of this study and help to elucidate the specific roles in adipogenesis. the findings demonstrated crosstalk between wnt signaling and mirnas, highlighting the role of mirnas during adipogenesis, i.e. repression and promotion of adipogenesis by regulating wnt signaling. the global changes in mirna expression discovered in the present study may for the first time elucidate the preliminary mechanisms underlying wnt signaling-mediated changes in lipid formation during adipogenesis. 
the results suggest the presence of mirnas in two cell models, providing insights into wnt pathway-specific mirnas that can be further characterized for their potential roles in adipogenesis. to our knowledge, present study represents the first attempt to unveil the profile of mirnas involed in adipogenesis by modulating wnt signaling pathway, which contributed to deeper investigation of the mechanism of adipogenesis. to validate the predicted targets and the potential functions of these identified mirnas, the mimics of mir- were transfected into 3t3-l cells and enlarged cells with distinct lipid droplets were observed; meanwhile, transfection with the inhibitor of mir- could markedly decrease differentiation-specific factors at the transcription level, which suggested the specific role of mir- in promoting adipogenesis. as a molecular switch, the canonical wnt/β-catenin signaling pathway should be suppressed during the adipogenesis; however, activation of this pathway leads to the inhibition of lipid depots formation. furthermore, the activity of luciferase reporter bearing tcf7l mrna 3' utr was decreased after co-transfection with mir- in hek-293ft cells. here we established two types of cell model, activation and repression of wnt signaling, and investigated the expression profile of micrornas using microarray assay. tcf7l the predicted target of mir- is a transcription factor triggering the downstream responsive genes of wnt signaling, was blocked at transcription level. micrornas are a large class of tiny non-coding rnas that regulate diverse biological processes at the posttranscriptional level by controlling mrna stability or translation.
 micrornas are a large class of tiny non-coding rnas that regulate diverse biological processes at the posttranscriptional level by controlling mrna stability or translation. as a molecular switch, the canonical wnt/β-catenin signaling pathway should be suppressed during the adipogenesis; however, activation of this pathway leads to the inhibition of lipid depots formation. the aim of our studies was to identify mirnas that might be involved in adipogenesis by modulating wnt signaling pathway. here we established two types of cell model, activation and repression of wnt signaling, and investigated the expression profile of micrornas using microarray assay.  the high throughput microarray data revealed mirnas that might promote adipogenesis by repressing wnt signaling: mir- mir-148a, mir- mir- etc. meanwhile, we also identified mirnas that might have negative effect on adipogenesis by activating wnt signaling: mir- mir- and mir- etc. the targets of these mirnas were also analysed by bioinformatics. to validate the predicted targets and the potential functions of these identified mirnas, the mimics of mir- were transfected into 3t3-l cells and enlarged cells with distinct lipid droplets were observed; meanwhile, transfection with the inhibitor of mir- could markedly decrease differentiation-specific factors at the transcription level, which suggested the specific role of mir- in promoting adipogenesis. tcf7l the predicted target of mir- is a transcription factor triggering the downstream responsive genes of wnt signaling, was blocked at transcription level. furthermore, the activity of luciferase reporter bearing tcf7l mrna 3' utr was decreased after co-transfection with mir- in hek-293ft cells. last but not least, the protein expression level of β-catenin was increased in the lithium treated 3t3-l cells after transfection with mir- these findings suggested that mir- could promote adipogenesis by repressing wnt signaling through targeting tcf7l  the results suggest the presence of mirnas in two cell models, providing insights into wnt pathway-specific mirnas that can be further characterized for their potential roles in adipogenesis. to our knowledge, present study represents the first attempt to unveil the profile of mirnas involed in adipogenesis by modulating wnt signaling pathway, which contributed to deeper investigation of the mechanism of adipogenesis. 