title: MicroRNA-101 overexpression by IL-6 and TNF-α inhibits cholesterol efflux by suppressing ATP-binding cassette transporter A1 expression.
abstract: BACKGROUND
  MicroRNAs play key roles in regulating cholesterol homeostasis. Here, we investigated the role of microRNA-101 (miR-101) in regulating ATP-binding cassette transporter A1 (ABCA1) expression and cholesterol efflux under non-inflammatory and inflammatory conditions in human THP-1-derived macrophages and HepG2 hepatoblastoma cells.
  
  
  METHODS
  The cell lines were transfected with one of four lentiviral vectors: miR-101, miR-101 control, anti-miR-101, or anti-miR-101 control. A luciferase reporter assay was used to examine miR-101 binding to the 3' untranslated region (UTR) of ABCA1. Western blotting was conducted to assess ABCA1 protein expression. Cells were loaded with BODIPY-cholesterol and stained with oil red O to assess cholesterol efflux.
  
  
  RESULTS
  The luciferase activity assay revealed that wild-type miR-101 binding at site 2 significantly repressed ABCA1 3' UTR activity, suggesting that miR-101 directly targets the ABCA1 mRNA at site 2. In both cell lines, Western blotting revealed that miR-101 expression negatively regulates ABCA1 protein expression and significantly suppresses cholesterol efflux to ApoA1 under both low-density lipoprotein (LDL) and non-LDL conditions, which was confirmed by pronounced lipid inclusions visible by oil red O staining. In HepG2 cells, both IL-6 and TNF-α treatments produced significant miR-101 overexpression; however, in THP-1-derived macrophages, only IL-6 treatment produced significant miR-101 overexpression. Anti-mir-101 transfection under both IL-6 and TNF-α treatment conditions led to ABCA1 upregulation, indicating that miR-101 expression represses ABCA1 expression under inflammatory conditions.
  
  
  CONCLUSIONS
  miR-101 promotes intracellular cholesterol retention under inflammatory conditions through suppressing ABCA1 expression and suggests that the miR-101-ABCA1 axis may play an intermediary role in the development of NAFLD and vascular atherosclerosis.
authors.affiliation: Department of Cardiology, The First Affiliated Hospital of Chongqing Medical University, Chongqing, China.
authors.forename: Nan
authors.initials: N
authors.lastname: Zhang
authors.affiliation: Department of Cardiology, The First Affiliated Hospital of Chongqing Medical University, Chongqing, China.
authors.forename: JiaYan
authors.initials: J
authors.lastname: Lei
authors.affiliation: Department of Cardiology, The First Affiliated Hospital of Chongqing Medical University, Chongqing, China. Electronic address: zn127127@126.com.
authors.forename: Han
authors.initials: H
authors.lastname: Lei
authors.affiliation: Centre for Lipid Research, Key Laboratory of Metabolism on Lipid and Glucose, Chongqing Medical University, Chongqing, China; John Moorhead Research Laboratory, Centre for Nephrology, University College London Medical School, Royal Free Campus, London, United Kingdom.
authors.forename: Xiongzhong
authors.initials: X
authors.lastname: Ruan
authors.affiliation: Centre for Clinical Research, The First Affiliated Hospital, Chongqing Medical University, Chongqing, China.
authors.forename: Qing
authors.initials: Q
authors.lastname: Liu
authors.affiliation: Centre for Lipid Research, Key Laboratory of Metabolism on Lipid and Glucose, Chongqing Medical University, Chongqing, China.
authors.forename: Yaxi
authors.initials: Y
authors.lastname: Chen
authors.affiliation: Department of Cardiology, The First Affiliated Hospital of Chongqing Medical University, Chongqing, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Huang
mesh_terms: D020413:3' Untranslated Regions / Q000235:genetics; D064286:ATP Binding Cassette Transporter 1 / Q000235:genetics / Q000378:metabolism*; D015153:Blotting, Western; D006528:Carcinoma, Hepatocellular / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D002478:Cells, Cultured; D002784:Cholesterol / Q000378:metabolism*; D006801:Humans; D015850:Interleukin-6 / Q000494:pharmacology*; D008113:Liver Neoplasms / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D008156:Luciferases / Q000378:metabolism; D008264:Macrophages / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D035683:MicroRNAs / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
chemical_list: D020413:3' Untranslated Regions; C577836:ABCA1 protein, human; D064286:ATP Binding Cassette Transporter 1; D015850:Interleukin-6; C534072:MIRN101 microRNA, human; D035683:MicroRNAs; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D002784:Cholesterol; D008156:Luciferases
keywords: ABCA1; ATP-binding cassette transporter A1; Atherosclerosis; Cholesterol; IL-6; Inflammation; NAFLD; Non-alcoholic fatty liver disease; TNF-α; miR-101; miRNA; microRNA