A role for miR-296 in the regulation of lipoapoptosis by targeting PUMA

Abstract

Saturated free fatty acids (FFA) induce hepatocyte lipoapoptosis, a key mediator of liver injury in nonalcoholic fatty liver disease (NAFLD). Lipoapoptosis involves the upregulation of the BH3-only protein PUMA, a potent pro-apoptotic protein. Given that dysregulation of hepatic microRNA expression has been observed in NAFLD, we examined the role of miRNA in regulating PUMA expression during lipotoxicity. By in silico analysis, we identified two putative binding sites for miR-296-5p within the 3′ untranslated region (UTR) of PUMA mRNA. Enforced miR-296-5p levels efficiently reduced PUMA protein expression in Huh-7 cells, while antagonism of miR-296-5p function increased PUMA cellular levels. Reporter gene assays identified PUMA 3′UTR as a direct target of miR-296-5p. The saturated FFA, palmitate, repressed miR-296-5p expression; and Huh-7 cells were sensitized to palmitate-induced lipotoxicity by antagonism of miR-296-5p function using a targeted locked nucleic acid (LNA). Finally, miR-296-5p was reduced in liver samples from nonalcoholic steatohepatitis (NASH) patients compared with patients with simple steatosis (SS) or controls. Also miR-296-5p levels inversely varied with PUMA mRNA levels in human liver specimens. Our results implicate miR-296-5p in the regulation of PUMA expression during hepatic lipoapoptosis. We speculate that enhancement of miR-296-5p expression may represent a novel approach to minimize apoptotic damage in human fatty liver diseases. Saturated free fatty acids (FFA) induce hepatocyte lipoapoptosis, a key mediator of liver injury in nonalcoholic fatty liver disease (NAFLD). Lipoapoptosis involves the upregulation of the BH3-only protein PUMA, a potent pro-apoptotic protein. Given that dysregulation of hepatic microRNA expression has been observed in NAFLD, we examined the role of miRNA in regulating PUMA expression during lipotoxicity. By in silico analysis, we identified two putative binding sites for miR-296-5p within the 3′ untranslated region (UTR) of PUMA mRNA. Enforced miR-296-5p levels efficiently reduced PUMA protein expression in Huh-7 cells, while antagonism of miR-296-5p function increased PUMA cellular levels. Reporter gene assays identified PUMA 3′UTR as a direct target of miR-296-5p. The saturated FFA, palmitate, repressed miR-296-5p expression; and Huh-7 cells were sensitized to palmitate-induced lipotoxicity by antagonism of miR-296-5p function using a targeted locked nucleic acid (LNA). Finally, miR-296-5p was reduced in liver samples from nonalcoholic steatohepatitis (NASH) patients compared with patients with simple steatosis (SS) or controls. Also miR-296-5p levels inversely varied with PUMA mRNA levels in human liver specimens. Our results implicate miR-296-5p in the regulation of PUMA expression during hepatic lipoapoptosis. We speculate that enhancement of miR-296-5p expression may represent a novel approach to minimize apoptotic damage in human fatty liver diseases. Nonalcoholic fatty liver disease (NAFLD) is commonly associated with the metabolic syndrome and represents a growing epidemic afflicting up to 30% of the American population (1Browning J.D. Szczepaniak L.S. Dobbins R. Nuremberg P. Horton J.D. Cohen J.C. Grundy S.M. Hobbs H.H. Prevalence of hepatic steatosis in an urban population in the United States: impact of ethnicity.Hepatology. 2004; 40: 1387-1395Crossref PubMed Scopus (2955) Google Scholar). NAFLD covers a wide spectrum of chronic liver diseases, ranging from simple steatosis (SS), to nonalcoholic steatohepatitis (NASH), to fibrosis, and ultimately, to cirrhosis (2Adams L.A. Lymp J.F. St. Sauver J. Sanderson S.O. Lindor K.D. Feldstein A. Angulo P. The natural history of nonalcoholic fatty liver disease: a population-based cohort study.Gastroenterology. 2005; 129: 113-121Abstract Full Text Full Text PDF PubMed Scopus (2326) Google Scholar). This syndrome is characterized by elevated levels of circulating free fatty acids (FFA) and hepatocyte apoptosis, which increase in relationship to NAFLD severity (3Nehra V. Angulo P. Buchman A.L. Lindor K.D. Nutritional and metabolic considerations in the etiology of nonalcoholic steatohepatitis.Dig. Dis. Sci. 2001; 46: 2347-2352Crossref PubMed Scopus (140) Google Scholar–5Kahraman A. Schlattjan M. Kocabayoglu P. Yildiz-Meziletoglu S. Schlensak M. Fingas C.D. Wedemeyer I. Marquitan G. Gieseler R.K. Baba H.A. et al.Major histocompatibility complex class I-related chains A and B (MIC A/B): a novel role in nonalcoholic steatohepatitis.Hepatology. 2010; 51: 92-102Crossref PubMed Scopus (87) Google Scholar). This relationship has generated the concept that the pathogenesis of NAFLD is related to the lipotoxic effect of FFA on liver cells. Saturated FFAs are directly hepatotoxic and induce lipoapoptosis (6Barreyro F.J. Kobayashi S. Bronk S.F. Werneburg N.W. Malhi H. Gores G.J. Transcriptional regulation of Bim by FoxO3A mediates hepatocyte lipoapoptosis.J. Biol. Chem. 2007; 282: 27141-27154Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar–8Cazanave S.C. Elmi N.A. Akazawa Y. Bronk S.F. Mott J.L. Gores G.J. CHOP and AP-1 cooperatively mediate PUMA expression during lipoapoptosis.Am. J. Physiol. Gastrointest. Liver Physiol. 2010; 299: G236-G243Crossref PubMed Scopus (157) Google Scholar). Lipoapoptosis in vitro requires upregulation of the pro-apoptotic BH3-only protein PUMA. PUMA knockdown in Huh-7 cells or genetic deficiency of Puma in mouse primary hepatocytes partially protects liver cells from lipotoxic insults (7Cazanave S.C. Mott J.L. Elmi N.A. Bronk S.F. Werneburg N.W. Akazawa Y. Kahraman A. Garrison S.P. Zambetti G.P. Charlton M.R. et al.JNK1-dependent PUMA expression contributes to hepatocyte lipoapoptosis.J. Biol. Chem. 2009; 284: 26591-26602Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). Hepatic PUMA mRNA and protein levels are increased in NAFLD patients (7Cazanave S.C. Mott J.L. Elmi N.A. Bronk S.F. Werneburg N.W. Akazawa Y. Kahraman A. Garrison S.P. Zambetti G.P. Charlton M.R. et al.JNK1-dependent PUMA expression contributes to hepatocyte lipoapoptosis.J. Biol. Chem. 2009; 284: 26591-26602Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar), and overexpression of cellular PUMA promotes cell death (9Jabbour A.M. Heraud J.E. Daunt C.P. Kaufmann T. Sandow J. O'Reilly L.A. Callus B.A. Lopez A. Strasser A. Vaux D.L. et al.Puma indirectly activates Bax to cause apoptosis in the absence of Bid or Bim.Cell Death Differ. 2009; 16: 555-563Crossref PubMed Scopus (63) Google Scholar). The increased cellular levels of PUMA protein directly promote mitochondrial dysfunction, resulting in caspase 3/7 activation and culminating in cellular demise (10Kim H. Tu H.C. Ren D. Takeuchi O. Jeffers J.R. Zambetti G.P. Hsieh J.J. Cheng E.H. Stepwise activation of BAX and BAK by tBID, BIM, and PUMA initiates mitochondrial apoptosis.Mol. Cell. 2009; 36: 487-499Abstract Full Text Full Text PDF PubMed Scopus (474) Google Scholar). During the lipotoxic insult, the mechanisms resulting in upregulation of PUMA mRNA are complex and mediated by multiple p53-independent transcriptional processes, including those regulated by activator protein (AP)-1 and C/EBP homologous protein (CHOP) (7Cazanave S.C. Mott J.L. Elmi N.A. Bronk S.F. Werneburg N.W. Akazawa Y. Kahraman A. Garrison S.P. Zambetti G.P. Charlton M.R. et al.JNK1-dependent PUMA expression contributes to hepatocyte lipoapoptosis.J. Biol. Chem. 2009; 284: 26591-26602Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, 8Cazanave S.C. Elmi N.A. Akazawa Y. Bronk S.F. Mott J.L. Gores G.J. CHOP and AP-1 cooperatively mediate PUMA expression during lipoapoptosis.Am. J. Physiol. Gastrointest. Liver Physiol. 2010; 299: G236-G243Crossref PubMed Scopus (157) Google Scholar). The cellular regulation of PUMA is, however, multifaceted and also includes posttranscriptional processes (11Yu J. Zhang L. PUMA, a potent killer with or without p53.Oncogene. 2008; 27: S71-S83Crossref PubMed Scopus (449) Google Scholar). For example, only approximately 50% of PUMA induction by FFA can be accounted for by transcriptional regulation (8Cazanave S.C. Elmi N.A. Akazawa Y. Bronk S.F. Mott J.L. Gores G.J. CHOP and AP-1 cooperatively mediate PUMA expression during lipoapoptosis.Am. J. Physiol. Gastrointest. Liver Physiol. 2010; 299: G236-G243Crossref PubMed Scopus (157) Google Scholar). Given its cytotoxic potential, additional insights into the different levels of PUMA regulation during lipoapoptosis may help develop novel approaches to treating human fatty liver diseases. A recent study has reported altered hepatic expression of several microRNAs in human NASH (12Cheung O. Puri P. Eicken C. Contos M.J. Mirshahi F. Maher J.W. Kellum J.M. Min H. Luketic V.A. Sanyal A.J. Nonalcoholic steatohepatitis is associated with altered hepatic microRNA expression.Hepatology. 2008; 48: 1810-1820Crossref PubMed Scopus (545) Google Scholar). MicroRNAs are short noncoding RNAs of approximately 22 nucleotides long. MicroRNAs negatively regulate protein expression via binding to complementary sequences within the 3′untranslated region (UTR) of target mRNA. The bound mRNA remains untranslated, leading to reduced levels of the corresponding protein, or it can be resulting in levels of the corresponding mRNA and 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, H. microRNAs to target mRNA 2010; PubMed Scopus Google Scholar). of in the of human fatty liver disease are and a role for microRNAs in regulating PUMA expression during lipoapoptosis has been the we identified microRNA miR-296-5p as a direct of PUMA expression during hepatocyte lipoapoptosis, to of PUMA expression in liver NASH hepatic miR-296-5p levels were and its was associated with increased PUMA for the of targeted to minimize apoptotic damage in patients with fatty liver diseases. Huh-7 and cells, a human cell and a human cell were in and cells were only as cellular to study the of PUMA 3′UTR by in a gene acid and acid St. were as (7Cazanave S.C. Mott J.L. Elmi N.A. Bronk S.F. Werneburg N.W. Akazawa Y. Kahraman A. Garrison S.P. Zambetti G.P. Charlton M.R. et al.JNK1-dependent PUMA expression contributes to hepatocyte lipoapoptosis.J. Biol. Chem. 2009; 284: 26591-26602Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). The of FFA in the were to the FFA observed in human nonalcoholic steatohepatitis R. C. J. J. A. F. J. et of in with nonalcoholic J. PubMed Scopus Google Scholar, A.J. C. Mirshahi F. Contos M.J. R.K. Luketic V.A. Nonalcoholic of and mitochondrial 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). The of the was in we two and that miR-296-5p is a putative the 3′UTR of PUMA. The of was by from human using the The was into the to of the and into the sites of the expression expression of Huh-7 cells miR-296-5p were generated by cells with of the expression using were in and were by for miR-296-5p. The human PUMA 3′UTR to two miR-296-5p target sequences binding was from using the A and were the of the and human PUMA 3′UTR was in of the gene and as the and of the of the miR-296-5p target was generated by using the nucleotides are and to the miR-296-5p target by were we a different to on miR-296-5p sequences in the PUMA 3′UTR a for the of which was in the generated on a of the miR-296-5p target was generated by of the with by with to and of the using This in of a of The was as The expression and were using and assays or were of levels was for with microRNA for miR-296-5p was as a was using for human PUMA was as in (7Cazanave S.C. Mott J.L. Elmi N.A. Bronk S.F. Werneburg N.W. Akazawa Y. Kahraman A. Garrison S.P. Zambetti G.P. Charlton M.R. et al.JNK1-dependent PUMA expression contributes to hepatocyte lipoapoptosis.J. Biol. Chem. 2009; 284: 26591-26602Abstract Full Text Full Text PDF PubMed Scopus (161) Google using For microRNA of was into using for or the and the was using for the different microRNAs or expression levels were to the to the was on a was examined in cells or cells in the of the a was on the cell were as (7Cazanave S.C. Mott J.L. Elmi N.A. Bronk S.F. Werneburg N.W. Akazawa Y. Kahraman A. Garrison S.P. Zambetti G.P. Charlton M.R. et al.JNK1-dependent PUMA expression contributes to hepatocyte lipoapoptosis.J. Biol. Chem. 2009; 284: 26591-26602Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). of protein was by on a to and with primary were from the and were with was using and was to and protein in were using the Reporter and was in a (7Cazanave S.C. Mott J.L. Elmi N.A. Bronk S.F. Werneburg N.W. Akazawa Y. Kahraman A. Garrison S.P. Zambetti G.P. Charlton M.R. et al.JNK1-dependent PUMA expression contributes to hepatocyte lipoapoptosis.J. Biol. Chem. 2009; 284: 26591-26602Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). The results are reported as protein. were with for and cells were by cells with the of and were apoptotic (7Cazanave S.C. Mott J.L. Elmi N.A. Bronk S.F. Werneburg N.W. Akazawa Y. Kahraman A. Garrison S.P. Zambetti G.P. Charlton M.R. et al.JNK1-dependent PUMA expression contributes to hepatocyte lipoapoptosis.J. Biol. Chem. 2009; 284: 26591-26602Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). was as a of cells For caspase 3/7 cells were in The was using the as (7Cazanave S.C. Mott J.L. Elmi N.A. Bronk S.F. Werneburg N.W. Akazawa Y. Kahraman A. Garrison S.P. Zambetti G.P. Charlton M.R. et al.JNK1-dependent PUMA expression contributes to hepatocyte lipoapoptosis.J. Biol. Chem. 2009; 284: 26591-26602Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). The study was by the and patients for in Our cohort of patients liver the of for The of NAFLD was on liver patients a were into liver simple steatosis and NASH and with NAFLD of steatohepatitis or for and patients with of chronic liver disease liver liver or were from was from using from represent and are as of the were compared using or with The miR-296-5p levels and PUMA mRNA levels was by a was the different we identified miR-296-5p as a putative to regulate PUMA expression on the of its multiple with the 3′UTR of PUMA. two miR-296-5p binding sites were identified and of PUMA 3′UTR binding sites and using the of et A. P. in Cell. 2007; 27: Full Text Full Text PDF PubMed Scopus Google Scholar). miR-296-5p PUMA Huh-7 cells were with a the of by the of miR-296-5p in cells with was efficiently increased compared with Huh-7 cells miR-296-5p expression in a of PUMA cellular mRNA and protein levels This effect was as the protein levels of a BH3-only protein that miR-296-5p binding in the Huh-7 cells miR-296-5p This overexpression of miR-296-5p cellular levels identified that miR-296-5p PUMA in an in a miR-296-5p function was by Huh-7 cells with a locked nucleic acid that to its of miR-296-5p was associated with a increase in PUMA mRNA levels and a increase in protein levels compared with cells with a an effect of miR-296-5p on PUMA and protein results that miR-296-5p negatively PUMA cellular that PUMA mRNA is a direct target for the PUMA 3′UTR was of the of the of Huh-7 cells with the the expression antagonism of miR-296-5p by increased a of levels using the expression or the for miR-296-5p Huh-7 cells were with of was observed of or miR-296-5p binding sites within PUMA 3′UTR and were results that miR-296-5p to PUMA 3′UTR binding were in a different cellular using the cell cells cells of compared with Huh-7 cells was by expression of and increased antagonism of miR-296-5p function The in by miR-296-5p levels were the in PUMA mRNA and a effect of miR-296-5p on PUMA protein in to an effect on PUMA mRNA miR-296-5p directly PUMA cellular expression by binding to target sequences within the PUMA PUMA expression contributes to saturated apoptosis (7Cazanave S.C. Mott J.L. Elmi N.A. Bronk S.F. Werneburg N.W. Akazawa Y. Kahraman A. Garrison S.P. Zambetti G.P. Charlton M.R. et al.JNK1-dependent PUMA expression contributes to hepatocyte lipoapoptosis.J. Biol. Chem. 2009; 284: 26591-26602Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, 8Cazanave S.C. Elmi N.A. Akazawa Y. Bronk S.F. Mott J.L. Gores G.J. CHOP and AP-1 cooperatively mediate PUMA expression during lipoapoptosis.Am. J. Physiol. Gastrointest. Liver Physiol. 2010; 299: G236-G243Crossref PubMed Scopus (157) Google Scholar). PUMA mRNA levels with By of with palmitate, a increase in PUMA mRNA expression was by an increase in PUMA cellular protein levels This increase in PUMA mRNA and protein levels with a 50% in miR-296-5p levels with the saturated FFA a of Huh-7 cells with the FFA which PUMA mRNA expression (7Cazanave S.C. Mott J.L. Elmi N.A. Bronk S.F. Werneburg N.W. Akazawa Y. Kahraman A. Garrison S.P. Zambetti G.P. Charlton M.R. et al.JNK1-dependent PUMA expression contributes to hepatocyte lipoapoptosis.J. Biol. Chem. 2009; 284: 26591-26602Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar), miR-296-5p levels into the processes by which we examined was in cells, and the of the saturated increase in PUMA mRNA and protein levels with a of miR-296-5p cellular levels to the of cell of miR-296-5p to be to microRNA microRNA Given the we to in miR-296-5p levels saturated with (6Barreyro F.J. Kobayashi S. Bronk S.F. Werneburg N.W. Malhi H. Gores G.J. Transcriptional regulation of Bim by FoxO3A mediates hepatocyte lipoapoptosis.J. Biol. Chem. 2007; 282: 27141-27154Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar–8Cazanave S.C. Elmi N.A. Akazawa Y. Bronk S.F. Mott J.L. Gores G.J. CHOP and AP-1 cooperatively mediate PUMA expression during lipoapoptosis.Am. J. Physiol. Gastrointest. Liver Physiol. 2010; 299: G236-G243Crossref PubMed Scopus (157) Google Scholar), the of apoptosis in Huh-7 cells. as by and caspase 3/7 expression of miR-296-5p in Huh-7 cells the increase of PUMA mRNA levels with lipoapoptosis the of with the in miR-296-5p levels and increase in PUMA mRNA a with the of the FFA in Y. D. M.J. saturated fatty and cell death in liver Cell. 2009; PubMed Scopus Google Scholar). antagonism of miR-296-5p function by increased Huh-7 cell apoptosis compared with cells with a a with a miR-296-5p as an microRNA A.M. J. of human and for an of miRNA in cell and 2005; PubMed Scopus Google Scholar). of miR-296-5p function the of miR-296-5p levels by and cell death with a of and also been reported to negatively regulate PUMA expression Zhang Zhang A.L. L. T. et and target PUMA to induce cell in 2010; PubMed Scopus Google A. L. J. R. M. F. H. G. L. et role of the 2010; PubMed Scopus Google Scholar). we the levels of and during lipotoxicity. to the effect on miR-296-5p of Huh-7 cells with the or with in NASH patients (12Cheung O. Puri P. Eicken C. Contos M.J. Mirshahi F. Maher J.W. Kellum J.M. Min H. Luketic V.A. Sanyal A.J. Nonalcoholic steatohepatitis is associated with altered hepatic microRNA expression.Hepatology. 2008; 48: 1810-1820Crossref PubMed Scopus (545) Google Scholar), and levels were increased or by palmitate, microRNAs to be related to increased PUMA expression in of lipotoxicity. are with saturated dysregulation of which cell to apoptosis by PUMA FFAs and hepatic PUMA expression are increased in NAFLD patients (3Nehra V. Angulo P. Buchman A.L. Lindor K.D. Nutritional and metabolic considerations in the etiology of nonalcoholic steatohepatitis.Dig. Dis. Sci. 2001; 46: 2347-2352Crossref PubMed Scopus (140) Google Scholar, S.C. Mott J.L. Elmi N.A. Bronk S.F. Werneburg N.W. Akazawa Y. Kahraman A. Garrison S.P. Zambetti G.P. Charlton M.R. et al.JNK1-dependent PUMA expression contributes to hepatocyte lipoapoptosis.J. Biol. Chem. 2009; 284: 26591-26602Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar), we to the levels of miR-296-5p in human liver liver to those with simple steatosis and of The of miR-296-5p was in liver from patients with NASH compared with liver from patients with simple steatosis and This in miR-296-5p levels was associated with an increase in PUMA mRNA and levels as reported (7Cazanave S.C. Mott J.L. Elmi N.A. Bronk S.F. Werneburg N.W. Akazawa Y. Kahraman A. Garrison S.P. Zambetti G.P. Charlton M.R. et al.JNK1-dependent PUMA expression contributes to hepatocyte lipoapoptosis.J. Biol. Chem. 2009; 284: 26591-26602Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). levels of PUMA mRNA and corresponding to the of the and in the were to results into the of of of simple steatosis and of NASH patients levels of PUMA mRNA and levels of miR-296-5p an PUMA mRNA expression and miR-296-5p in liver from patients with or without NAFLD was observed that of miR-296-5p expression in NAFLD may to the pathogenesis of fatty liver The of the study insights the regulation of PUMA expression during hepatocyte Our results the a microRNA to target PUMA PUMA expression by PUMA and PUMA protein of PUMA expression by miR-296-5p is direct and and palmitate, the saturated FFA, a in miR-296-5p which contributes to its associated in the upregulation of PUMA with in vitro also that miR-296-5p levels are in the liver of patients with NASH and that an hepatic miR-296-5p and PUMA levels in with or without are The study that the saturated FFA levels of miR-296-5p in liver cells. microRNA levels are regulated by and miR-296-5p transcriptional by a effect of on or of miR-296-5p. During apoptotic processes, the that the microRNA into a can be by with of function A. Y. S. D. of into a 2010; PubMed Scopus Google Scholar, F. H.H. of during apoptosis in cells to and of Death Differ. 2009; 16: PubMed Scopus Google Scholar). activation may resulting in of the and miR-296-5p. Our (8Cazanave S.C. Elmi N.A. Akazawa Y. Bronk S.F. Mott J.L. Gores G.J. CHOP and AP-1 cooperatively mediate PUMA expression during lipoapoptosis.Am. J. Physiol. Gastrointest. Liver Physiol. 2010; 299: G236-G243Crossref PubMed Scopus (157) Google the AP-1 and CHOP in the transcriptional regulation of PUMA expression during lipoapoptosis. we observed that of AP-1 and CHOP PUMA induction by by 50% (8Cazanave S.C. Elmi N.A. Akazawa Y. Bronk S.F. Mott J.L. Gores G.J. CHOP and AP-1 cooperatively mediate PUMA expression during lipoapoptosis.Am. J. Physiol. Gastrointest. Liver Physiol. 2010; 299: G236-G243Crossref PubMed Scopus (157) Google Scholar), mechanisms that regulate PUMA miR-296-5p which results in increase in PUMA expression with associated and overexpression of miR-296-5p palmitate-induced PUMA expression also by approximately that miR-296-5p represents an additional in the regulation of PUMA expression during lipotoxicity. in miR-296-5p levels in to compared with the activation of CHOP and AP-1 (7Cazanave S.C. Mott J.L. Elmi N.A. Bronk S.F. Werneburg N.W. Akazawa Y. Kahraman A. Garrison S.P. Zambetti G.P. Charlton M.R. et al.JNK1-dependent PUMA expression contributes to hepatocyte lipoapoptosis.J. Biol. Chem. 2009; 284: 26591-26602Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar), it with a increase in PUMA mRNA and protein are with an effect of miR-296-5p on PUMA and protein it is that and transcriptional regulation of PUMA may for of PUMA mRNA by palmitate, dysregulation of miR-296-5p during lipoapoptosis for a increase in PUMA mRNA and protein levels. Our that PUMA expression is in by AP-1 and CHOP the transcriptional and by the microRNA miR-296-5p the posttranscriptional and of processes may regulate PUMA during lipoapoptosis. elevated FFA, increased PUMA and hepatocyte apoptosis are of human NAFLD (1Browning J.D. Szczepaniak L.S. Dobbins R. Nuremberg P. Horton J.D. Cohen J.C. Grundy S.M. Hobbs H.H. Prevalence of hepatic steatosis in an urban population in the United States: impact of ethnicity.Hepatology. 2004; 40: 1387-1395Crossref PubMed Scopus (2955) Google Scholar, V. Angulo P. Buchman A.L. Lindor K.D. Nutritional and metabolic considerations in the etiology of nonalcoholic steatohepatitis.Dig. Dis. Sci. 2001; 46: 2347-2352Crossref PubMed Scopus (140) Google Scholar, A. Angulo P. M. Lindor K.D. Gores G.J. apoptosis and expression are of human nonalcoholic Full Text Full Text PDF PubMed Scopus Google Scholar, S.C. Mott J.L. Elmi N.A. Bronk S.F. Werneburg N.W. Akazawa Y. Kahraman A. Garrison S.P. Zambetti G.P. Charlton M.R. et al.JNK1-dependent PUMA expression contributes to hepatocyte lipoapoptosis.J. Biol. Chem. 2009; 284: 26591-26602Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). NAFLD only with an increase in hepatocyte apoptosis A. Angulo P. M. Lindor K.D. Gores G.J. apoptosis and expression are of human nonalcoholic Full Text Full Text PDF PubMed Scopus Google also with a dysregulation of the hepatic expression of several microRNAs (12Cheung O. Puri P. Eicken C. Contos M.J. Mirshahi F. Maher J.W. Kellum J.M. Min H. Luketic V.A. Sanyal A.J. Nonalcoholic steatohepatitis is associated with altered hepatic microRNA expression.Hepatology. 2008; 48: 1810-1820Crossref PubMed Scopus (545) Google Scholar), a role for microRNAs in regulating cell death in the of human fatty liver of in disease are For example, which is the of NAFLD (12Cheung O. Puri P. Eicken C. Contos M.J. Mirshahi F. Maher J.W. Kellum J.M. Min H. Luketic V.A. Sanyal A.J. Nonalcoholic steatohepatitis is associated with altered hepatic microRNA expression.Hepatology. 2008; 48: 1810-1820Crossref PubMed Scopus (545) Google Scholar), the expression of key hepatic C. S. S.F. M. L. M. R. et regulation of by in Full Text Full Text PDF PubMed Scopus Google Scholar). which is increased in NAFLD, was to in disease by the in the of and J. expression by microRNAs in and metabolic 2010; PubMed Scopus Google and the regulation of cellular apoptosis M. M. of Sci. 2008; PubMed Scopus Google Scholar). We that PUMA mRNA is in human NASH we that miR-296-5p is in liver from patients with NASH and associated with the increase in PUMA mRNA. miR-296-5p levels in patients with or without NAFLD with increased expression of PUMA, PUMA expression with increased hepatocyte apoptosis in human with miR-296-5p levels are also in the of patients with NAFLD M. D. H. A. V. expression of in the of patients with fatty liver 2010; PubMed Scopus Google and may to the apoptosis associated with human A. S. S. Feldstein apoptosis, a and hepatic Biol. Chem. 2010; Full Text Full Text PDF PubMed Scopus Google Scholar). Finally, the of miR-296-5p in fatty liver disease is also by the that a region on human including the for miR-296-5p is to human L. J. A. A.J. et of an on mouse and of to and on the human homologous region PubMed Scopus Google Scholar). The study by et a in miR-296-5p expression in NASH while study identified microRNA as repressed by saturated FFA and in NASH liver This is to as in the study involves a of in of The for an are to and and the for the as a are a The miR-296-5p of and the and for microRNA are to from the for the The of targeted by as in the the in and is and for target of NASH patients levels of PUMA approximately of the patients with simple steatosis also increased levels of PUMA that PUMA may be the of in the of BH3-only as also to lipoapoptosis (6Barreyro F.J. Kobayashi S. Bronk S.F. Werneburg N.W. Malhi H. Gores G.J. Transcriptional regulation of Bim by FoxO3A mediates hepatocyte lipoapoptosis.J. Biol. Chem. 2007; 282: 27141-27154Abstract Full Text Full Text PDF PubMed Scopus (166) Google and with PUMA in cell death processes M. V. C. G. A. G. Strasser A. A. Puma with the BH3-only protein in cell death during in apoptosis PubMed Scopus Google Scholar). NASH patients increased expression of death as and death which commonly mediate hepatocyte apoptosis A. Angulo P. M. Lindor K.D. Gores G.J. apoptosis and expression are of human nonalcoholic Full Text Full Text PDF PubMed Scopus Google Scholar, A. Schlattjan M. Kocabayoglu P. Yildiz-Meziletoglu S. Schlensak M. Fingas C.D. Wedemeyer I. Marquitan G. Gieseler R.K. Baba H.A. et al.Major histocompatibility complex class I-related chains A and B (MIC A/B): a novel role in nonalcoholic steatohepatitis.Hepatology. 2010; 51: 92-102Crossref PubMed Scopus (87) Google Scholar, H. F.J. H. Bronk S.F. Gores G.J. fatty acids hepatocytes to mediated 2007; PubMed Scopus Google Scholar). NAFLD is the of a and which of is in the pathogenesis of the disease remains to be of miR-296-5p levels represents an additional resulting in PUMA upregulation in the of human fatty liver disease to miR-296-5p expression to minimize liver damage in human Given the of the in microRNA L. and microRNA regulation for and 2009; PubMed Scopus Google Scholar), the concept of liver study in NAFLD The for with activator binding C/EBP homologous locked nucleic acid nonalcoholic fatty liver disease nonalcoholic steatohepatitis mediator of apoptosis simple steatosis untranslated region