BACKGROUND
insulitis is an inflammation of pancreatic langerhans islets, which is known to precede the onset of diabetes mellitus   <cit> .
post-insulitis decrease of pancreatic β cells is a major hallmark of both type  <dig>  and type  <dig> diabetes mellitus   <cit> .
it is associated to α cells dysfunctions and high glucagon secretion, which contribute to chronic hyperglycemia and ensuing clinical outcomes in dm  <cit> .
accordingly, efforts to increase our knowledge on the biomolecular mechanisms regulating proliferation, physiopathological functions, and apoptosis of α cells will likely result in improved medical approaches to the disease.
the pathways leading to mammalian β cell apoptosis, induced by proinflammatory cytokines, are complex but sufficiently known  <cit> .
on the contrary, the molecular bases of α cells higher resistance than β to the same cues are still largely uncharacterized  <cit> .
pancreatic α and β cells share common endocrine precursors , but upon differentiation they respond differently to external stimuli as proinflammatory cytokines, glucagon-like peptide  <dig> , and inhibitors of dipeptidyl peptidase- <dig>   <cit> .
micrornas  are small  noncoding rnas, which control gene expression mainly at the post-transcriptional level: mirnas have been shown to be master regulators of cell networks  <cit> .
many mirnas are involved in dm physiopathology: for instance, mir- <dig> controls β cell mass and insulin secretion  <cit> ; mir- <dig> and mir-146a are involved in β-cell apoptosis  <cit> ; mir-30d promotes insulin synthesis and protects β cells from damage by proinflammatory cytokines  <cit> ; mir- <dig> is responsible for impaired angiogenetic signaling in dm patients  <cit> .
however, there are no high-throughput  published data on α cells mirna transcriptome at steady state, its comparison with β cells steady state mirna transcriptome, and its alterations following treatment with cytokines.
to discover new physiopathologic regulatory mechanisms, specific of cytokine-treated pancreatic cells, we profiled global mirnas expression in two mouse pancreatic cell lines  at steady state and in αtc1- <dig> after treatment with proinflammatory cytokines ifn-γ, il-1β, tnf-α.
this analysis brought to the identification of:  differentially expressed  mirnas between αtc1- <dig> and βtc <dig> at steady state;  de mirnas between αtc1- <dig> treated with cytokines and matched untreated controls.
to insert our data in the appropriate biological context, we reconstructed the molecular networks regulated at steady state by de mirnas in α and β cells.
our results demonstrate that these networks are remarkably different: very likely, they increase α cells resistance to apoptosis and negatively interfere with pancreatic β cells viability after exposure to cytokines.
RESULTS
apoptosis induction by cytokines in αtc1- <dig> and βtc <dig> cells
to verify the differential response of αtc1- <dig> and βtc <dig> cells to treatment with cytokines, we assayed their propensity to undergo apoptosis during a time course experiment .
the number of apoptotic αtc1- <dig> cells did not significantly vary with respect to controls for the entire time course , demonstrating that in our system α cells appear to withstand apoptosis induction by cytokines; on the contrary, βtc <dig> cells are clearly more susceptible to treatment .
steady state mirna transcriptome profiles of αtc1- <dig> and βtc <dig> cells
to identify mirnas potentially responsible for αtc1- <dig> resistance to cytokines, we analyzed the steady state mirna transcriptome of αtc1- <dig> and βtc <dig> cells.
at steady state,  <dig> mirnas were exclusively expressed in αtc1- <dig> , while  <dig> were expressed only in βtc <dig> ;  <dig> mirnas resulted significantly more expressed in αtc1- <dig> than in βtc <dig>  whereas  <dig> were significantly more expressed in βtc <dig> compared to αtc1- <dig>  .
their assignment to specific families is shown in additional files  <dig> and  <dig>
treatment with cytokines alters αtc1- <dig> mirna transcriptome profiles
after treatment with cytokines for  <dig> h,  <dig> mirnas  were significantly differentially expressed in αtc1- <dig> as compared to matched untreated controls  .
their assignment to specific families is shown in additional file  <dig>
mirnas 296-3p and 298-5p are reliable candidates for involvement in αtc1- <dig> higher resistance than β cells to apoptosis induction by cytokines
to identify mirnas whose functions could explain the differential response to cytokines of pancreatic α and β cells, we specifically focused our attention on mir-296-3p and mir-298-5p.
both mirnas are expressed at steady state only in αtc1- <dig>  but are not synthesized in βtc <dig> ; both are part of the imprinted gnas/gnas clusters in mice and humans and share more targets than expected by chance , even though their seed regions are different: this suggests common regulatory functions.
single taqman gene expression assays  during a time course analysis  showed a highly significant downregulation of mir-296-3p in αtc1- <dig> cells at  <dig> and  <dig> h pt, compared to matched untreated controls .
mir-298-5p resulted significantly downregulated starting at  <dig> h pt and reached a highly significant downregulation at  <dig> h pt, compared to matched untreated controls: student’s t-test, bonferroni adjusted p-values, were < <dig>  and < <dig> , respectively .
both mirnas were co-expressed in αtc1- <dig> cells throughout the entire experimental time course  .
stas confirmed that they are not expressed either in βtc <dig> or ins- <dig> cells.
genomics of genes encoding mir-296-3p, mir-298-5p, nespas and identification of upstream cpg islands
genes encoding mirnas 296-3p and 298-5p are clustered in a genomic region, which also comprises the gene for the noncoding transcript nespas and is imprinted in mice and humans  <cit> .
sequences of mature mir-296-3p are 100% conserved between rodents and humans, whereas those of mir-298-5p are 74% identical.
analysis of this region through ucsc browser revealed the presence of two clusters of cpg islands:  one comprises two cpg islands, from  <dig>  to  <dig>  kb upstream the first nucleotide of pre-mir- <dig>  and is located  <dig>  and  <dig> kb downstream nespas transcription start site ;  the other is made of three cpg islands from  <dig>  to  <dig>  kb upstream the first nucleotide of pre-mir- <dig> and is located  <dig> - <dig> kb upstream nespas tss .
matinspector revealed a putative nespas promoter located  <dig> bp upstream- <dig> bp downstream its tss.
αtc1- <dig> transfection with mimics of mir-296-3p and mir-298-5p increases apoptosis levels induced by cytokines
to precisely define mir-296-3p and mir-298-5p biological functions, we compared apoptosis levels of αtc1- <dig> cells, transfected with either one or both mirna mimics and treated with cytokines for  <dig>   <dig>   <dig> h after transfection , with those of scramble-transfected αtc1- <dig> cells treated with cytokines by following a similar protocol.
the percentage of apoptotic αtc1- <dig> cells after transfection with mimics of mir-296-3p was comparable to scramble-transfected controls during the entire time course treatment.
on the other hand, transfection with mimics of mir-298-5p or of both mir-296-3p and mir-298-5p increased in a highly significant manner the number of αtc1- <dig> apoptotic cells, compared to matched scramble-transfected controls:  <dig>  and more than  <dig>  folds at  <dig> h pt, respectively  .
identification of mir-296-3p and mir-298-5p targets
to characterize the networks regulated by mirnas 296-3p and 298-5p, we computationally searched their validated and predicted targets.
we identified  <dig> validated target of mir-296-3p;  <dig> validated targets of mir-298-5p;  <dig> predicted targets of mir-296-3p;  <dig> predicted targets of mir-298-5p.
we focused our attention on  <dig> targets of mir-296-3p,  <dig> of mir-298-5p,  <dig> common to both mirnas: they were chosen according to their involvement in apoptosis, cell cycle progression, cell differentiation and hormone secretion .
among them, bcl <dig>  ccna <dig>  irs <dig>  nr4a <dig> are transcriptionally regulated by creb <dig>  which is a validated target of mir-296-3p  <cit> ; tnf and vdr are validated targets of mir-298-5p  <cit> .
all other targets were computationally predicted, including igf1rβ that we validated through western analysis .
modulation of mir-296-3p and mir-298-5p also alters expression of their targets
to verify whether in vitro modulation of mir-296-3p and mir-298-5p affected the expression of their targets, we performed transient transfection experiments of αtc1- <dig> cells with their mimics.
transfection efficiency at  <dig> h at was in all cases higher than 90%.
in αtc <dig> transfected with mimics of mir-296-3p or mir-298-5p, real-time pcr showed altered expression of different genes with respect to scramble-transfected cells, including igf1r, tnf, vdr .
expression of igf1rβ and tnfα is controlled by mir-296-3p and mir-298-5p in αtc1- <dig> cells
in αtc1- <dig> treated with cytokines for  <dig> h, levels of proteins igf1rβ and tnfα increased  <dig>  and  <dig>  folds with respect to untreated controls, respectively , while mirnas 296-3p and 298-5p decreased of  <dig>  and  <dig>  folds with respect to untreated controls, respectively .
we further demonstrated through western analysis that protein igf1rβ decreased about  <dig>  folds in steady-state αtc1- <dig> after transfection with mimics of mir-296-3p or mir-298-5p, as compared to scramble-transfected controls; this decrease was higher than  <dig> folds when cells were transfected with both mimics .
the decrease of igf1rβ was not detectable in αtc1- <dig> transfected with either mimic of mir-296-3p or mir-298-5p and then treated with cytokines for  <dig> h. in αtc1- <dig> treated with cytokines for  <dig> h, after transfection with mimics of both mirnas 296-3p and 298-5p, it was instead lower than at steady state .
this could also be due to the decrease of mirnas 296-3p and 298-5p following treatment with cytokines .
following transfection of αtc1- <dig> with either mimics of mir-296-3p or mir-298-5p, tnfα protein was about  <dig>  folds less expressed with respect to scramble-transfected controls; this decrease was more pronounced  when cells were transfected with both mimics .
each of the two mirna mimics decreased the expression of tnfα more than  <dig> folds after  <dig> h of treatment with cytokines, but the silencing effect was not potentiated when the two mimics were transfected together .
activation of irs- <dig> and erk- <dig> is also under control of mir-296-3p and mir-298-5p
to verify if the igf1r signaling pathway was controlled by mir-296-3p and mir-298-5p through igf1rβ, we assayed the phosphorylation levels of irs- <dig> and erk  in αtc1- <dig> transfected with mimics of each one or both mirnas; this analysis was performed both after treatment with cytokines for  <dig> h or on untreated cells, exploiting as controls matched scramble-transfected αtc1- <dig> cells.
the expression of phospho-irs- <dig> didn’t change in untreated αtc1- <dig> transfected with mimics of each mirna alone, while it decreased about  <dig>  folds in αtc1- <dig> transfected with mimics of both mir-296-3p and mir-298-5p .
by using the same controls, we detected a slight decrease of phospho-irs- <dig>  in αtc1- <dig> transfected with mimics of both mir-296-3p and mir-298-5p and treated with cytokines .
interestingly, also the activation of erk- <dig> appears to be regulated by mir-296-3p and mir-298-5p: in the absence of treatment with cytokines, αtc1- <dig> cells transfected with mimics of mir-296-3p showed levels of phospho-erk- <dig>  similar to those found in scramble-transfected αtc1- <dig> cells; the transfection with mimics of mir-298-5p or of both mirnas led instead to a decrease of the protein  .
under cytokine treatment, the amount of phospho-erk- <dig>  did not change in αtc1- <dig> transfected with either mimics of mir-296-3p or of mir-298-5p alone, compared to scramble-transfected cells; in αtc1- <dig> simultaneously transfected with mimics of both mirnas, we detected a decrease of  <dig>  folds of phospho-erk- <dig>  levels, compared to scramble-transfected cells .
identification of tfs regulating de mirnas
in the genomic region comprising the genes encoding mir-296-3p, mir-298-5p and nespas, matinspector identified transcription factor binding sites  for sixty seven transcription factors .
four of them  are known to be expressed in the islets of langerhans, and mafb is known to be expressed only in rodent islet α cells within adult pancreas  <cit> ; the presence of binding sites for this tf within the promoter of the genes encoding mir-296-3p and mir-298-5p suggests that it may regulate the expression of both mirnas.
multi experiment matrix  showed a statistically highly significant negative correlation  between mafb and igf1r.
interestingly, microrna.org predicts two binding sites for mmu-mir-296-3p and mmu-mir-298-5p on mafb mrna 3’ utr.
differences between αtc1- <dig> and βtc <dig> steady state regulatory networks
<dig> of  <dig> mirnas, more abundantly or exclusively expressed at steady state in αtc1- <dig> with respect to βtc <dig>  have validated targets known to be expressed in mammalian pancreatic α cells .
by using these data, we generated a network comprised of  <dig> nodes and  <dig> edges : from this network, we inferred a subnetwork made of  <dig> α-mirnas for which validated targets have been identified, consisting of  <dig> nodes and  <dig> edges .
in the case of βtc <dig> cells, we found that  <dig> of  <dig> mirnas more abundantly or exclusively expressed with respect to αtc1- <dig> have validated targets known to be expressed in pancreatic β cells : a network of  <dig> nodes and  <dig> edges was generated from them .
the subnetwork made of  <dig> β-mirnas consisted of  <dig> nodes and  <dig> edges .
the enrichment in specific biological processes was calculated in pancreatic αtc1- <dig> versus βtc <dig> cells subnetworks  .
interestingly, in the network of genes regulated by α-mirnas, pancreatic α cell fate commitment is a biological process overrepresented with respect to the network regulated by β-mirnas .
within the network of genes regulated by α-mirnas, insulin-like growth factor receptor signaling pathway is a biological process significantly enriched among the genes interacting with targets of mir-296-3p and mir-298-5p, with respect to the genes linked to the targets of the other mirnas .
biological processes are ranked based on increasing adjusted p-values .
discussion
the data reported in this paper and those from the literature  <cit>  confirm that mammalian pancreatic α cells are more resistant than β cells to apoptosis induced by cytokines .
our results suggest that mir-296-3p and mir-298-5p play a pivotal role in determining this trait.
due to dm epidemic spreading  <cit> , its molecular characterization has become an important biological and translational problem in biomedicine  <cit> .
our ht and network analyses of mirna transcriptome in α and β cells highlighted very different profiles at steady state .
computational analysis of these data allowed us to identify a set of mirnas, specifically synthesized in pancreatic α cells, which appear to also regulate pancreatic α cell commitment.
specific enrichment of pathways, regulated by α-mirnas within this biological process , allowed the identification of mirnas that could be involved in the acquisition of the trait resistance to cytokines-induced cell death upon pancreatic α cells differentiation.
among them, mir-296-3p and mir-298-5p stood out clearly as potentially critical nodes, responsible for α cells resistance to cytokine-induced cell death.
in fact, besides being specifically expressed at steady state in α cells , but not in β cells , both mirnas were significantly co-expressed and downregulated in αtc1- <dig> during a time-course treatment with cytokines, compared to matched untreated controls .
our subsequent functional assays demonstrated that αtc1- <dig> transfected with mimics of mir-296-3p and mir-298-5p became susceptible to apoptosis when treated with the same cocktail of cytokines as compared to αtc1- <dig> transfected with scramble molecules.
although its biological effect was potentiated when also mir-296-3p was expressed, our results suggest that the role of mir-298-5p is more important in this process than that of mir-296-3p : this would highlight the role of one or a few specific mir-298-5p targets.
through western analysis, we confirmed our computational prediction that igf1rβ and tnfα are common targets to both mirnas and that mir-296-3p and mir-298-5p also control irs- <dig> and erk- <dig> within the igf1r signaling pathway.
igf1r is known to promote resistance to apoptosis by different mechanisms: it increases the levels of antiapoptotic proteins, as bcl <dig> and bcl-xl; it inactivates proapoptotic proteins, as bad and casp9; it stimulates mitogenic irs-1/mapks pathway  <cit> .
phospho-irs <dig>  and phospho-erk- <dig> , which we demonstrated to be controlled by mir-296-3p and mir-298-5p, are known to regulate the response to insulin and to be involved in survival and proliferation processes  <cit> .
decreased expression of mir-296-3p and mir-298-5p and the corresponding activation of survival and proliferation signals, mediated by igf1r and its downstream nodes , may thus explain why αtc1- <dig> cells are resistant to death induction by cytokines .
tnfα is known to be physiologically expressed by pancreatic endocrine cells and to contribute to maintain islet homeostasis  <cit> .
this protein is also known to decrease apoptosis levels of pancreatic acinar cells during acute pancreatitis by stimulating the synthesis of antiapoptotic proteins  <cit> .
CONCLUSIONS
ht mirna profiling data, functional analysis with synthetic mimics and molecular characterisation of modulated pathways strongly suggest that specific downregulation of mir-296-3p and mir-298-5p in pancreatic α cells, coupled to upregulation of their targets as igf1rβ and tnfα and activation of the corresponding signaling pathways, is a major determinant of their resistance to apoptosis induction by cytokines.
studies on pancreatic islet microorgan in toto will allow to experimentally confirm these results in a 3d natural system.
it also will permit to verify the hypothesis that increased tnfα synthesis by α cells during insulitis protects them by activating survival pathways, while priming a deadly regulatory loop and causing β cells apoptosis .

 insulitis is an inflammation of pancreatic langerhans islets, which is known to precede the onset of diabetes mellitus. post-insulitis decrease of pancreatic β cells is a major hallmark of both type and type diabetes mellitus. it is associated to α cells dysfunctions and high glucagon secretion, which contribute to chronic hyperglycemia and ensuing clinical outcomes in dm. accordingly, efforts to increase our knowledge on the biomolecular mechanisms regulating proliferation, physiopathological functions, and apoptosis of α cells will likely result in improved medical approaches to the disease. the pathways leading to mammalian β cell apoptosis, induced by proinflammatory cytokines, are complex but sufficiently known. on the contrary, the molecular bases of α cells higher resistance than β to the same cues are still largely uncharacterized. pancreatic α and β cells share common endocrine precursors, but upon differentiation they respond differently to external stimuli as proinflammatory cytokines, glucagon-like peptide, and inhibitors of dipeptidyl peptidase-. micrornas are small noncoding rnas, which control gene expression mainly at the post-transcriptional level: mirnas have been shown to be master regulators of cell networks. many mirnas are involved in dm physiopathology: for instance, mir- controls β cell mass and insulin secretion; mir- and mir-146a are involved in β-cell apoptosis; mir-30d promotes insulin synthesis and protects β cells from damage by proinflammatory cytokines; mir- is responsible for impaired angiogenetic signaling in dm patients. however, there are no high-throughput published data on α cells mirna transcriptome at steady state, its comparison with β cells steady state mirna transcriptome, and its alterations following treatment with cytokines. to discover new physiopathologic regulatory mechanisms, specific of cytokine-treated pancreatic cells, we profiled global mirnas expression in two mouse pancreatic cell lines at steady state and in αtc1- after treatment with proinflammatory cytokines ifn-γ, il-1β, tnf-α. this analysis brought to the identification of: differentially expressed mirnas between αtc1- and βtc at steady state; de mirnas between αtc1- treated with cytokines and matched untreated controls. to insert our data in the appropriate biological context, we reconstructed the molecular networks regulated at steady state by de mirnas in α and β cells. our results demonstrate that these networks are remarkably different: very likely, they increase α cells resistance to apoptosis and negatively interfere with pancreatic β cells viability after exposure to cytokines.  apoptosis induction by cytokines in αtc1- and βtc cells to verify the differential response of αtc1- and βtc cells to treatment with cytokines, we assayed their propensity to undergo apoptosis during a time course experiment. the number of apoptotic αtc1- cells did not significantly vary with respect to controls for the entire time course, demonstrating that in our system α cells appear to withstand apoptosis induction by cytokines; on the contrary, βtc cells are clearly more susceptible to treatment. steady state mirna transcriptome profiles of αtc1- and βtc cells to identify mirnas potentially responsible for αtc1- resistance to cytokines, we analyzed the steady state mirna transcriptome of αtc1- and βtc cells. at steady state, mirnas were exclusively expressed in αtc1-, while were expressed only in βtc; mirnas resulted significantly more expressed in αtc1- than in βtc whereas were significantly more expressed in βtc compared to αtc1-. their assignment to specific families is shown in additional files and treatment with cytokines alters αtc1- mirna transcriptome profiles after treatment with cytokines for h, mirnas were significantly differentially expressed in αtc1- as compared to matched untreated controls. their assignment to specific families is shown in additional file mirnas 296-3p and 298-5p are reliable candidates for involvement in αtc1- higher resistance than β cells to apoptosis induction by cytokines to identify mirnas whose functions could explain the differential response to cytokines of pancreatic α and β cells, we specifically focused our attention on mir-296-3p and mir-298-5p. both mirnas are expressed at steady state only in αtc1- but are not synthesized in βtc; both are part of the imprinted gnas/gnas clusters in mice and humans and share more targets than expected by chance, even though their seed regions are different: this suggests common regulatory functions. single taqman gene expression assays during a time course analysis showed a highly significant downregulation of mir-296-3p in αtc1- cells at and h pt, compared to matched untreated controls. mir-298-5p resulted significantly downregulated starting at h pt and reached a highly significant downregulation at h pt, compared to matched untreated controls: student’s t-test, bonferroni adjusted p-values, were < and <, respectively. both mirnas were co-expressed in αtc1- cells throughout the entire experimental time course. stas confirmed that they are not expressed either in βtc or ins- cells. genomics of genes encoding mir-296-3p, mir-298-5p, nespas and identification of upstream cpg islands genes encoding mirnas 296-3p and 298-5p are clustered in a genomic region, which also comprises the gene for the noncoding transcript nespas and is imprinted in mice and humans. sequences of mature mir-296-3p are 100% conserved between rodents and humans, whereas those of mir-298-5p are 74% identical. analysis of this region through ucsc browser revealed the presence of two clusters of cpg islands: one comprises two cpg islands, from to kb upstream the first nucleotide of pre-mir- and is located and kb downstream nespas transcription start site; the other is made of three cpg islands from to kb upstream the first nucleotide of pre-mir- and is located - kb upstream nespas tss. matinspector revealed a putative nespas promoter located bp upstream- bp downstream its tss. αtc1- transfection with mimics of mir-296-3p and mir-298-5p increases apoptosis levels induced by cytokines to precisely define mir-296-3p and mir-298-5p biological functions, we compared apoptosis levels of αtc1- cells, transfected with either one or both mirna mimics and treated with cytokines for h after transfection, with those of scramble-transfected αtc1- cells treated with cytokines by following a similar protocol. the percentage of apoptotic αtc1- cells after transfection with mimics of mir-296-3p was comparable to scramble-transfected controls during the entire time course treatment. on the other hand, transfection with mimics of mir-298-5p or of both mir-296-3p and mir-298-5p increased in a highly significant manner the number of αtc1- apoptotic cells, compared to matched scramble-transfected controls: and more than folds at h pt, respectively. identification of mir-296-3p and mir-298-5p targets to characterize the networks regulated by mirnas 296-3p and 298-5p, we computationally searched their validated and predicted targets. we identified validated target of mir-296-3p; validated targets of mir-298-5p; predicted targets of mir-296-3p; predicted targets of mir-298-5p. we focused our attention on targets of mir-296-3p, of mir-298-5p, common to both mirnas: they were chosen according to their involvement in apoptosis, cell cycle progression, cell differentiation and hormone secretion. among them, bcl ccna irs nr4a are transcriptionally regulated by creb which is a validated target of mir-296-3p; tnf and vdr are validated targets of mir-298-5p. all other targets were computationally predicted, including igf1rβ that we validated through western analysis. modulation of mir-296-3p and mir-298-5p also alters expression of their targets to verify whether in vitro modulation of mir-296-3p and mir-298-5p affected the expression of their targets, we performed transient transfection experiments of αtc1- cells with their mimics. transfection efficiency at h at was in all cases higher than 90%. in αtc transfected with mimics of mir-296-3p or mir-298-5p, real-time pcr showed altered expression of different genes with respect to scramble-transfected cells, including igf1r, tnf, vdr. expression of igf1rβ and tnfα is controlled by mir-296-3p and mir-298-5p in αtc1- cells in αtc1- treated with cytokines for h, levels of proteins igf1rβ and tnfα increased and folds with respect to untreated controls, respectively, while mirnas 296-3p and 298-5p decreased of and folds with respect to untreated controls, respectively. we further demonstrated through western analysis that protein igf1rβ decreased about folds in steady-state αtc1- after transfection with mimics of mir-296-3p or mir-298-5p, as compared to scramble-transfected controls; this decrease was higher than folds when cells were transfected with both mimics. the decrease of igf1rβ was not detectable in αtc1- transfected with either mimic of mir-296-3p or mir-298-5p and then treated with cytokines for h. in αtc1- treated with cytokines for h, after transfection with mimics of both mirnas 296-3p and 298-5p, it was instead lower than at steady state. this could also be due to the decrease of mirnas 296-3p and 298-5p following treatment with cytokines. following transfection of αtc1- with either mimics of mir-296-3p or mir-298-5p, tnfα protein was about folds less expressed with respect to scramble-transfected controls; this decrease was more pronounced when cells were transfected with both mimics. each of the two mirna mimics decreased the expression of tnfα more than folds after h of treatment with cytokines, but the silencing effect was not potentiated when the two mimics were transfected together. activation of irs- and erk- is also under control of mir-296-3p and mir-298-5p to verify if the igf1r signaling pathway was controlled by mir-296-3p and mir-298-5p through igf1rβ, we assayed the phosphorylation levels of irs- and erk in αtc1- transfected with mimics of each one or both mirnas; this analysis was performed both after treatment with cytokines for h or on untreated cells, exploiting as controls matched scramble-transfected αtc1- cells. the expression of phospho-irs- didn’t change in untreated αtc1- transfected with mimics of each mirna alone, while it decreased about folds in αtc1- transfected with mimics of both mir-296-3p and mir-298-5p. by using the same controls, we detected a slight decrease of phospho-irs- in αtc1- transfected with mimics of both mir-296-3p and mir-298-5p and treated with cytokines. interestingly, also the activation of erk- appears to be regulated by mir-296-3p and mir-298-5p: in the absence of treatment with cytokines, αtc1- cells transfected with mimics of mir-296-3p showed levels of phospho-erk- similar to those found in scramble-transfected αtc1- cells; the transfection with mimics of mir-298-5p or of both mirnas led instead to a decrease of the protein. under cytokine treatment, the amount of phospho-erk- did not change in αtc1- transfected with either mimics of mir-296-3p or of mir-298-5p alone, compared to scramble-transfected cells; in αtc1- simultaneously transfected with mimics of both mirnas, we detected a decrease of folds of phospho-erk- levels, compared to scramble-transfected cells. identification of tfs regulating de mirnas in the genomic region comprising the genes encoding mir-296-3p, mir-298-5p and nespas, matinspector identified transcription factor binding sites for sixty seven transcription factors. four of them are known to be expressed in the islets of langerhans, and mafb is known to be expressed only in rodent islet α cells within adult pancreas; the presence of binding sites for this tf within the promoter of the genes encoding mir-296-3p and mir-298-5p suggests that it may regulate the expression of both mirnas. multi experiment matrix showed a statistically highly significant negative correlation between mafb and igf1r. interestingly, microrna.org predicts two binding sites for mmu-mir-296-3p and mmu-mir-298-5p on mafb mrna 3’ utr. differences between αtc1- and βtc steady state regulatory networks of mirnas, more abundantly or exclusively expressed at steady state in αtc1- with respect to βtc have validated targets known to be expressed in mammalian pancreatic α cells. by using these data, we generated a network comprised of nodes and edges: from this network, we inferred a subnetwork made of α-mirnas for which validated targets have been identified, consisting of nodes and edges. in the case of βtc cells, we found that of mirnas more abundantly or exclusively expressed with respect to αtc1- have validated targets known to be expressed in pancreatic β cells: a network of nodes and edges was generated from them. the subnetwork made of β-mirnas consisted of nodes and edges. the enrichment in specific biological processes was calculated in pancreatic αtc1- versus βtc cells subnetworks. interestingly, in the network of genes regulated by α-mirnas, pancreatic α cell fate commitment is a biological process overrepresented with respect to the network regulated by β-mirnas. within the network of genes regulated by α-mirnas, insulin-like growth factor receptor signaling pathway is a biological process significantly enriched among the genes interacting with targets of mir-296-3p and mir-298-5p, with respect to the genes linked to the targets of the other mirnas. biological processes are ranked based on increasing adjusted p-values. discussion the data reported in this paper and those from the literature confirm that mammalian pancreatic α cells are more resistant than β cells to apoptosis induced by cytokines. our results suggest that mir-296-3p and mir-298-5p play a pivotal role in determining this trait. due to dm epidemic spreading, its molecular characterization has become an important biological and translational problem in biomedicine. our ht and network analyses of mirna transcriptome in α and β cells highlighted very different profiles at steady state. computational analysis of these data allowed us to identify a set of mirnas, specifically synthesized in pancreatic α cells, which appear to also regulate pancreatic α cell commitment. specific enrichment of pathways, regulated by α-mirnas within this biological process, allowed the identification of mirnas that could be involved in the acquisition of the trait resistance to cytokines-induced cell death upon pancreatic α cells differentiation. among them, mir-296-3p and mir-298-5p stood out clearly as potentially critical nodes, responsible for α cells resistance to cytokine-induced cell death. in fact, besides being specifically expressed at steady state in α cells, but not in β cells, both mirnas were significantly co-expressed and downregulated in αtc1- during a time-course treatment with cytokines, compared to matched untreated controls. our subsequent functional assays demonstrated that αtc1- transfected with mimics of mir-296-3p and mir-298-5p became susceptible to apoptosis when treated with the same cocktail of cytokines as compared to αtc1- transfected with scramble molecules. although its biological effect was potentiated when also mir-296-3p was expressed, our results suggest that the role of mir-298-5p is more important in this process than that of mir-296-3p: this would highlight the role of one or a few specific mir-298-5p targets. through western analysis, we confirmed our computational prediction that igf1rβ and tnfα are common targets to both mirnas and that mir-296-3p and mir-298-5p also control irs- and erk- within the igf1r signaling pathway. igf1r is known to promote resistance to apoptosis by different mechanisms: it increases the levels of antiapoptotic proteins, as bcl and bcl-xl; it inactivates proapoptotic proteins, as bad and casp9; it stimulates mitogenic irs-1/mapks pathway. phospho-irs and phospho-erk-, which we demonstrated to be controlled by mir-296-3p and mir-298-5p, are known to regulate the response to insulin and to be involved in survival and proliferation processes. decreased expression of mir-296-3p and mir-298-5p and the corresponding activation of survival and proliferation signals, mediated by igf1r and its downstream nodes, may thus explain why αtc1- cells are resistant to death induction by cytokines. tnfα is known to be physiologically expressed by pancreatic endocrine cells and to contribute to maintain islet homeostasis. this protein is also known to decrease apoptosis levels of pancreatic acinar cells during acute pancreatitis by stimulating the synthesis of antiapoptotic proteins.  ht mirna profiling data, functional analysis with synthetic mimics and molecular characterisation of modulated pathways strongly suggest that specific downregulation of mir-296-3p and mir-298-5p in pancreatic α cells, coupled to upregulation of their targets as igf1rβ and tnfα and activation of the corresponding signaling pathways, is a major determinant of their resistance to apoptosis induction by cytokines. studies on pancreatic islet microorgan in toto will allow to experimentally confirm these results in a 3d natural system. it also will permit to verify the hypothesis that increased tnfα synthesis by α cells during insulitis protects them by activating survival pathways, while priming a deadly regulatory loop and causing β cells apoptosis. 
altogether, high-throughput microrna profiling, functional analysis with synthetic mimics and molecular characterization of modulated pathways strongly suggest that specific downregulation of mir-296-3p and mir-298-5p, coupled to upregulation of their targets as igf1rβ and tnfα, is a major determinant of mammalian pancreatic α cells resistance to apoptosis induction by cytokines. the molecular bases of mammalian pancreatic α cells higher resistance than β to proinflammatory cytokines are very poorly defined. we also characterized the alterations of αtc1- cells microrna transcriptome after treatment with proinflammatory cytokines. mammalian pancreatic α and β cellsmicrorna transcriptomeproinflammatory cytokinesapoptosiscellular networksdiabetes mellitus through high-throughput real-time pcr, we analyzed the steady state microrna transcriptome of murine pancreatic α and β cells: their comparison demonstrated significant differences. we focused our study on two micrornas, mir-296-3p and mir-298-5p, which were: specifically expressed at steady state in αtc1- but not in βtc or ins- cells; significantly downregulated in αtc1- cells after treatment with cytokines in comparison to untreated controls. micrornas are master regulators of cell networks, but only scanty data are available on their transcriptome in these cells and its alterations in diabetes mellitus.
 the molecular bases of mammalian pancreatic α cells higher resistance than β to proinflammatory cytokines are very poorly defined. micrornas are master regulators of cell networks, but only scanty data are available on their transcriptome in these cells and its alterations in diabetes mellitus.  through high-throughput real-time pcr, we analyzed the steady state microrna transcriptome of murine pancreatic α and β cells: their comparison demonstrated significant differences. we also characterized the alterations of αtc1- cells microrna transcriptome after treatment with proinflammatory cytokines. we focused our study on two micrornas, mir-296-3p and mir-298-5p, which were: specifically expressed at steady state in αtc1- but not in βtc or ins- cells; significantly downregulated in αtc1- cells after treatment with cytokines in comparison to untreated controls. these micrornas share more targets than expected by chance and were co-expressed in αtc1- during a 6– h time course treatment with cytokines. the genes encoding them are physically clustered in the murine and human genome. by exploiting specific microrna mimics, we demonstrated that experimental upregulation of mir-296-3p and mir-298-5p raised the propensity to apoptosis of transfected and cytokine-treated αtc1- cells with respect to αtc1- cells, treated with cytokines after transfection with scramble molecules. both micrornas control the expression of igf1rβ, its downstream targets phospho-irs- and phospho-erk, and tnfα. our computational analysis suggests that mafb controls the expression of mir-296-3p and mir-298-5p.  altogether, high-throughput microrna profiling, functional analysis with synthetic mimics and molecular characterization of modulated pathways strongly suggest that specific downregulation of mir-296-3p and mir-298-5p, coupled to upregulation of their targets as igf1rβ and tnfα, is a major determinant of mammalian pancreatic α cells resistance to apoptosis induction by cytokines. mammalian pancreatic α and β cellsmicrorna transcriptomeproinflammatory cytokinesapoptosiscellular networksdiabetes mellitus 