title: Heme status affects human hepatic messenger RNA and microRNA expression.
abstract: OBJECTIVE
  To assess effects of heme on messenger RNA (mRNA) and microRNA (miRNA) profiles of liver cells derived from humans.
  
  
  METHODS
  We exposed human hepatoma cell line Huh-7 cells to excess iron protoporphyrin (heme) (10 μmol/L) or induced heme deficiency by addition of 4, 6-dioxoheptanoic acid (500 μmol/L), a potent inhibitor of aminolevulinic acid dehydratase, for 6 h or 24 h. We harvested total RNA from the cells and performed both mRNA and miRNA array analyses, with use of Affymetrix chips, reagents, and instruments (human genome U133 plus 2.0 and miRNA 2.0 arrays). We assessed changes and their significance and interrelationships with Target Scan, Pathway Studios, and Ingenuity software.
  
  
  RESULTS
  Changes in mRNA levels were most numerous and striking at 6 h after heme treatment but were similar and still numerous at 24 h. After 6 h of heme exposure, the increase in heme oxygenase 1 gene expression was 60-fold by mRNA and 88-fold by quantitative reverse transcription-polymerase chain reaction. We found striking changes, especially up-regulation by heme of nuclear erythroid-2 related factor-mediated oxidative stress responses, protein ubiquitination, glucocorticoid signaling, P53 signaling, and changes in RNAs that regulate intermediary metabolism. Fewer mRNAs were down-regulated by heme, and the fold decreases were less exuberant than were the increases. Notable decreases after 24 h of heme exposure were patatin-like phospholipase domain-containing protein 3 (-6.5-fold), neuronal PAS domain protein 2 (-1.93-fold), and protoporphyrinogen oxidase (-1.7-fold).
  
  
  CONCLUSIONS
  Heme excess exhibits several toxic effects on liver and kidney, which deserve study in humans and in animal models of the human porphyrias or other disorders.
authors.affiliation: Liver, Digestive and Metabolic Disorders Laboratory, Cannon Research Center, Carolinas Medical Center, 1542 Garden Terrace, Charlotte, NC 28203, USA. herbert.bonkovsky@carolinas.org
authors.forename: Herbert L
authors.initials: HL
authors.lastname: Bonkovsky
authors.forename: Weihong
authors.initials: W
authors.lastname: Hou
authors.forename: Nury
authors.initials: N
authors.lastname: Steuerwald
authors.forename: Qing
authors.initials: Q
authors.lastname: Tian
authors.forename: Ting
authors.initials: T
authors.lastname: Li
authors.forename: Judy
authors.initials: J
authors.lastname: Parsons
authors.forename: Alicia
authors.initials: A
authors.lastname: Hamilton
authors.forename: Sunil
authors.initials: S
authors.lastname: Hwang
authors.forename: Laura
authors.initials: L
authors.lastname: Schrum
mesh_terms: D045744:Cell Line, Tumor; D020869:Gene Expression Profiling / Q000379:methods; D005786:Gene Expression Regulation; D006418:Heme / Q000172:deficiency / Q000378:metabolism*; D006537:Heptanoates / Q000494:pharmacology; D006801:Humans; D008099:Liver / Q000187:drug effects / Q000378:metabolism*; D035683:MicroRNAs / Q000378:metabolism*; D020411:Oligonucleotide Array Sequence Analysis; D011524:Protoporphyrins / Q000378:metabolism / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism*; D013997:Time Factors
chemical_list: D006537:Heptanoates; D035683:MicroRNAs; D011524:Protoporphyrins; D012333:RNA, Messenger; D006418:Heme; C020804:succinylacetone
keywords: Delta-aminolevulinic acid synthase; Heat shock proteins; Heme; Hepatotoxicity; Messenger RNA; MicroRNA
references.citation: Br J Haematol. 1990 Aug;75(4):617-8
references.pmid: 2207013
references.citation: Proc Natl Acad Sci U S A. 1997 Sep 30;94(20):10925-30
references.pmid: 9380736
references.citation: J Clin Invest. 1994 Aug;94(2):649-54
references.pmid: 8040318
references.citation: Methods Enzymol. 1990;186:301-9
references.pmid: 2233302
references.citation: Hepatology. 2010 May;51(5):1494-504
references.pmid: 20127796
references.citation: Transplantation. 2002 Sep 15;74(5):591-8
references.pmid: 12352873
references.citation: Ann Intern Med. 2005 Mar 15;142(6):439-50
references.pmid: 15767622
references.citation: Transplantation. 1999 Mar 27;67(6):922-8
references.pmid: 10199745
references.citation: J Biol Chem. 2011 Jul 29;286(30):26424-30
references.pmid: 21659532
references.citation: Compr Physiol. 2013 Jan;3(1):365-401
references.pmid: 23720291
references.citation: Trends Immunol. 2003 Aug;24(8):449-55
references.pmid: 12909459
references.citation: Curr Med Chem. 2012;19(1):70-6
references.pmid: 22300078
references.citation: J Lab Clin Med. 1970 Mar;75(3):410-21
references.pmid: 4392030
references.citation: Gastroenterology. 2007 Oct;133(4):1166-74
references.pmid: 17919492
references.citation: Gastroenterology. 2010 May;138(5):1909-19
references.pmid: 19909748
references.citation: Eur J Biochem. 1996 Aug 15;240(1):112-7
references.pmid: 8797843
references.citation: J Clin Invest. 2005 Nov;115(11):3007-14
references.pmid: 16239966
references.citation: J Clin Invest. 1999 Jan;103(1):129-35
references.pmid: 9884342
references.citation: Antioxid Redox Signal. 2005 Nov-Dec;7(11-12):1761-6
references.pmid: 16356137
references.citation: Transplantation. 2002 Mar 27;73(6):911-5
references.pmid: 11923691
references.citation: Int J Obes (Lond). 2012 Feb;36(2):244-53
references.pmid: 21467998
references.citation: Cell. 1993 Jan 15;72(1):19-28
references.pmid: 8380757
references.citation: FASEB J. 2006 Dec;20(14):2651-3
references.pmid: 17065227
references.citation: Pharmacol Rev. 2008 Mar;60(1):79-127
references.pmid: 18323402
references.citation: Proc Natl Acad Sci U S A. 1974 Feb;71(2):278-82
references.pmid: 4521800
references.citation: Proc Natl Acad Sci U S A. 1997 Sep 30;94(20):10919-24
references.pmid: 9380735
references.citation: Proc Natl Acad Sci U S A. 1997 Dec 23;94(26):14848-53
references.pmid: 9405702
references.citation: EMBO J. 2002 Oct 1;21(19):5216-24
references.pmid: 12356737
references.citation: N Engl J Med. 1971 Jan 7;284(1):17-20
references.pmid: 5538584
references.citation: Gut. 2011 May;60(5):671-9
references.pmid: 21159893
references.citation: Semin Liver Dis. 1982 May;2(2):108-24
references.pmid: 6753161
references.citation: Prog Liver Dis. 1994;12:47-62
references.pmid: 7746877
references.citation: Transplantation. 2002 Feb 15;73(3):469-72
references.pmid: 11884947
references.citation: Clin Liver Dis. 2004 Nov;8(4):807-38, viii
references.pmid: 15464657
references.citation: Cell. 2005 Jan 14;120(1):15-20
references.pmid: 15652477
references.citation: Proc Natl Acad Sci U S A. 1971 Nov;68(11):2725-9
references.pmid: 5288250
references.citation: Biochim Biophys Acta. 2008 Mar;1779(3):195-203
references.pmid: 18325350
references.citation: Biochem J. 2005 Nov 15;392(Pt 1):173-80
references.pmid: 16033334
references.citation: FASEB J. 2007 Sep;21(11):2639; author reply 2640
references.pmid: 17766326
references.citation: Methods. 2001 Dec;25(4):402-8
references.pmid: 11846609
references.citation: Clin Exp Rheumatol. 2012 Jan-Feb;30(1):73-8
references.pmid: 22325451
references.citation: Mol Cell Biol. 1996 Nov;16(11):6083-95
references.pmid: 8887638
references.citation: Bioinformatics. 2003 Jan 22;19(2):185-93
references.pmid: 12538238
references.citation: Int J Biochem Cell Biol. 2002 Mar;34(3):216-20
references.pmid: 11849989