type 2 diabetes ( t2 dm ) is a global public health problem and the number of affected individuals is increasing at an alarming rate [ 1 , 2 ] . the pathogenesis of t2 dm is characterized by a progressive insulin resistance with eventual insulin deficiency . both environmental and genetic factors are known to contribute to the development of the disease . thus , insulin secretion increases to maintain normal glucose concentration in the face of insulin resistance and/or decreasing -cell mass [ 4 , 5 ] . although exact prodiabetic mechanisms are still largely unknown , it has been implicated that increased serum level of free fatty acids ( ffas ) , alone or combined with hyperglycemia , may contribute to the pathophysiology of the disease . accumulating evidences have demonstrated that chronic high levels of circulating ffas play a pivotal role in -cell failure characterized with impaired glucose - stimulated insulin secretion ( gsis ) and enhanced apoptotic level . many signaling factors , such as sterol regulatory element - binding protein ( srebp)-1c , farnesoid x receptor ( fxr ) , and forkhead box - containing protein o ( foxo ) 1 , have been identified to be associated with ffas - induced  cell dysfunction [ 9 , 10 ] . thus , delineating the molecular mechanisms underlying ffa - triggered pancreatic -cell apoptosis would put new insights into our understanding of t2 dm and open new avenues for therapy . ( death receptor - induced ) and  intrinsic  ( bcl-2-regulated or mitochondrial ) pathways , have been demonstrated to be contributing significantly to apoptosis . the b - cell lymphoma-2 ( bcl-2 ) family is closely related to the intrinsic pathway of apoptosis . bcl-2 family proteins are a large family , which may either facilitate cell survival ( bcl-2 , bcl - xl , bcl - w , and others ) or promote cell death ( bax , bak , bad , and others ) . bcl-2 , one of antiapoptosis genes , was reduced significantly when treated with fatty acid [ 13 , 14 ] . accumulated evidence showed that ffa - induced pancreatic -cell apoptosis was accompanied by downregulating the apoptosis inhibitor bcl-2 expression [ 15 , 16 ] . moreover , bcl-2 overexpression does prevent  cell death induced by staurosporine , fasl , and some intrinsic signals . many endeavors have been made to elucidate the mechanisms that participate in dysregulation of genes in response to palmitate in -cells . besides transcription factors , such as the forkhead box o ( foxo ) transcription factor foxo1 , micrornas ( mirnas ) have been shown to represent another layer of gene regulation in palmitate - induced alteration of the gene expression [ 4 , 17 ] . mirnas , endogenous noncoding small rnas , bind to specific sites at the 3-untranslated region ( 3-utr ) of target genes , thereby suppressing translation and/or inducing degradation of target mrnas [ 18 , 19 ] . deregulated expression of mirnas is correlated with various pathological processes including cancer and diabetes [ 18 , 20 ] . recent studies by lovis et al . have showed that mir-34a is one of the significantly upregulated mirnas in the -cell treated with palmitate . moreover , elevated expression of mir-34a enhanced the -cell apoptosis [ 4 , 21 , 22 ] . however , the possible role of mir-34a in palmitate - induced -cell apoptosis is largely unknown . here , we found that downregulation of bcl-2 expression by mir-34a accounts for palmitate - induced increased apoptosis of pancreatic -cell min6 pancreatic -cells were maintained in dmem medium ( gibco , carlsbad , ca ) containing 25  mm glucose supplemented with 10% fetal bovine serum ( fbs ) , 10  nm 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid ( hepes ) , 2  mm l - glutamine , 100 unit / ml penicillin , 100  g / ml streptomycin , and 50 m -mercaptoethanol at 37c in 5% co2 . a stock solution ( 100  nm ) of palmitate ( sigma , st . louis , mo ) was prepared by dissolving the fatty acid in 50% ethanol and then diluted in culture medium with 0.5% fatty acid - free bsa ( sigma , st . cloning of the mouse bcl-2 3-utr containing mir-34a binding site was performed by pcr using the following primers : forward : 5-gcgccgcggtcgacaaacctgccccaaac-3 ; reverse : 5-gcgctgcagacgtttcttggcaattcctg-3. the pcr product was then cloned into a modified pgl3 control vector where sacii and psti sites were introduced into the original xba i site downstream of the luciferase gene . to mutate the binding sites within the 3-utr , quikchange site - directed mutagenesis kit from stratagene ( la jolla , ca ) cloning of the mouse bcl-2 orf was performed by pcr using following primers : forward : 5-ataggtaccatggcgcaagccgggagaac-3 ; reverse : 5-cggtctagatcacttgtggcccaggtatg-3. the pcr product and the expression vector pcdna3 ( invitrogen , carlsbad , ca ) were cut with kpni and xbai and ligated to generate the construct pcdna3-m - bcl-2 . real - time pcr was performed using sybr premix ex taq ( takara , dalian , china ) in an abi7500 system ( applied biosystems , carlsbad , ca , usa ) . bulge - loop mirna qpcr primers for mir-34a were synthesized by ribobio ( guangzhou , china ) . the following primary antibodies were used : anti - bcl-2 and anti - cleaved - caspase-3 ( cell signaling , beverly , ma ) , and anti--tubulin ( sigma - aldrich , st louis , mo ) . mirna mimic , mirna inhibitor , and their corresponding control oligonucleotides ( purchased from ribobio , guangzhou , china ) were transfected using lipofectamine 2000 following the manufacturer 's instruction ( invitrogen , carlsbad , ca ) . sense - strand sequences of bcl-2 sirnas are 5-aaguacauacauuauaagcug-3 and 5-guacauccauuauaagcug-3. the number of apoptotic cells was evaluated by scoring cells displaying pycnotic nucleus and/or fragmented nucleus visualized with hoechst 33342 as previously described . a commercial caspase 3 activity assay kit ( roche , mannheim , germany ) was used to measure the intracellular caspase 3 activity according to manufacturer 's instruction . for the reporter assay , cells were seeded in triplicate in 24-well plates and allowed to settle for 24  h. indicated plasmids and oligonucleotides were transfected into the cells using the lipofectamine 2000 reagent ( invitrogen , carlsbad , ca ) . forty - eight hours after transfection , dual - luciferase reporter assays were performed according to the manufacturer 's instructions ( promega , madison , wi ) . results are presented as means  standard deviation ( sd ) of three independent experiments performed in triplicate . all values represent at least three independent experiments and are expressed as the means  sd . the differences between two experimental conditions were compared on a one - to - one basis using student 's t - tests . statistical significance within groups as shown in figure 1(a ) , the expression level of mir-34a was significantly elevated by palmitate treatment in min6 cells . next , we investigated whether change in the level of mir-34a induced by palmitate was able to influence apoptotic rate of the cells . as shown in figure 1(b ) , ectopic expression of mir-34a increased the number of dying cells . as the caspase-3 activity is a key marker of apoptosis , the expressions of cleaved caspase-3 and caspase-3 activity were investigated to confirm the proapoptotic role of mir-34a . as shown in figures 1(c ) and 1(d ) , together , these data suggested that upregulated mir-34a contributes to proapoptotic effects of palmitate on pancreatic  cell . bcl-2 , an antiapoptotic protein involved in  cell apoptosis , is of specific interest as the computational tool ( targetscan   http://www.targetscan.org/ ) predicted a mir-34a - binding site within the bcl-2 3-utr ( figure 2(a ) ) . to verify the potential targeting of bcl-2 by mir-34a , we measured the protein level of bcl-2 in mir-34a transfected min6 cells . as shown in figure 2(b ) , the expression of bcl-2 was significantly suppressed by mir-34a transfection . to further confirm that the suppression of bcl-2 expression is directly mir-34a - driven , we generated a luciferase construct containing the bcl-2 3-utr . meanwhile , a construction with the mutation at the putative binding site ( figure 2(a ) ) was also generated as control . cotransfection of the wild - type bcl-2 3-utr and mir-34a mimic led to a significant inhibition of luciferase activity ( figure 2(c ) ) . in contrast , no significant alteration of luciferase activity was detected in cells transfected with mutated construct . consistently , palmitate treatment also suppressed the luciferase activity of the wild - type bcl-2 3-utr and had no effect on mutated construct ( figure 2(d ) ) . taken together , these data suggest that mir-34a suppresses bcl-2 expression via directly binding to its 3-utr . considering the apparent increase in the expression of mir-34a in response to palmitate , it is expected that pretreatment of mir-34a inhibitor may prevent the palmitate imposed effects . indeed , we showed that the increased apoptotic rate and decreased bcl-2 expression induced by palmitate in pancreatic  cell could be counteracted by pretreatment with mir-34a inhibitor ( figures 3(a ) and 3(b ) ) . therefore , upregulated mir-34a expression participates in the palmitate - induced effects on pancreatic  cell . to confirm the functional role of bcl-2 in pancreatic  cell , inhibition of bcl-2 expression by sirnas wb assays were performed and confirmed that bcl-2 expression was significantly silenced ( figure 4(a ) ) . as expected , data presented in figure 4(b ) showed that inhibition of bcl-2 caused increased min6 cell apoptosis . moreover , reexpression of bcl-2 in mir-34a - expressing min6 cells ( figure 4(c ) ) partially antagonized the mir-34a - induced apoptosis . taken as a whole , these data indicate that bcl-2 is a functionally relevant target of mir-34a 's effect in response to palmitate treatment . it has been previously demonstrated that bcl-2 expression is suppressed during palmitate - induced apoptosis   and mir-34a expression was upregulated in response to palmitate treatment . this raised the possibility that suppression of bcl-2 in apoptotic cells may be regulated by mir-34a . we evaluated this hypothesis in min6 pancreatic  cell , where we showed that bcl-2 is directly targeted by mir-34a . mir-34a - induced suppression of bcl-2 is associated with palmitate - associated pancreatic  cell apoptosis . t2 dm is a metabolic disorder that is driven by the interaction between genetic and acquired factors . the impact of ectopic fat and lipotoxicity on pancreatic -cell dysfunction and insulin resistance in humans has been paid great attention . accumulated evidence shows that lipotoxic stress - induced -cell death ( lipotoxicity ) is a key contributor to the development of t2 dm by inducing insulin resistance . lipotoxicity is caused by circulating free fatty acids increased , changes in lipoprotein profiles and body fat distribution . it is widely accepted that a sustained exposure to fatty acids impairs glucose - stimulated insulin secretion ( gsis ) , promotes apoptosis , and alters insulin gene expression in vitro and in vivo . bcl-2 , an antiapoptotic bcl-2 family member , is a crucial controller of the mitochondrial pathway of pancreatic -cell apoptosis induced by lipotoxicity . although previous studies suggest that the suppression of bcl-2 is involved in lipotoxicity induced pancreatic  cell apoptosis , the mechanisms involved in the decreased expression of bcl-2 in response to palmitate remains to be clarified . in researching the mechanism of palmitate - induced downregulated expression of bcl-2 , we noted that mir-34a , as palmitate , can dramatically induce its expression . it has been demonstrated that mir-34a was involved in various cell apoptosis conditions , including pancreatic beta cells . roggli et al . have demonstrated that anti - mir-34a can protect pancreatic  cell from proinflammatory cytokines - induced cell death and mir-34a - triggered bcl-2 downregulation might be associated with cytokine - mediated  cell apoptosis . if bcl-2 was inhibited by mir-34a overexpression , it is likely that the effect of palmitate on  cell apoptosis is mediated through suppression of bcl-2 by mir-34a . here an in silico approach identified a potential mir-34a binding site within the 3-utr of bcl-2 gene . furthermore , the protein level of bcl-2 is significantly suppressed by ectopic expression of mir-34a . intriguingly , anti - mir-34a pretreatment was able to attenuate palmitate - induced bcl-2 downregulation , as well as  cell apoptosis , strongly indicating the functional role of mir-34a in the  cell in response to lipotoxicity . we also investigated the importance of bcl-2 for palmitate - induced cell apoptosis in min6 pancreatic  cell lines by loss- and gain - of - function experiments . given that one specific mirna can regulate a group of target genes , together with the fact that repressing bcl-2 could not fully recapitulate mir-34a phenotype in pancreatic  cell , it is expected that other target genes regulated by mir-34a are involved in palmitate - induced  cell dysfunction . future work is still needed in the area of defining possible target genes of mir-34a that contribute to apoptosis of  cell exposed to palmitate . in conclusion , suppression of bcl-2 by mir-34a accounts for palmitate - induced increased apoptotic rate in pancreatic  cell . our findings reveal a new mechanistic aspect of the proapoptotic effect of palmitate on pancreatic  cell and raise the possibility of influencing mir-34a as a therapeutic strategy for diabetes .