MicroRNA-101 overexpression by IL-6 and TNF-α inhibits cholesterol efflux by suppressing ATP-binding cassette transporter A1 expression

• Published in: Experimental cell research Vol. 336; no. 1; pp. 33 - 42
• Main Authors: Zhang, Nan, Lei, JiaYan, Lei, Han, Ruan, Xiongzhong, Liu, Qing, Chen, Yaxi, Huang, Wei
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.08.2015
• Subjects: 3' Untranslated Regions - genetics; ABCA1; Atherosclerosis; ATP Binding Cassette Transporter 1 - genetics; ATP Binding Cassette Transporter 1 - metabolism; ATP-binding cassette transporter A1; Blotting, Western; Carcinoma, Hepatocellular - drug therapy; Carcinoma, Hepatocellular - genetics; Carcinoma, Hepatocellular - metabolism; Carcinoma, Hepatocellular - pathology; Cells, Cultured; Cholesterol; Cholesterol - metabolism; Humans; IL-6; Inflammation; Interleukin-6 - pharmacology; Liver Neoplasms - drug therapy; Liver Neoplasms - genetics; Liver Neoplasms - metabolism; Liver Neoplasms - pathology; Luciferases - metabolism; Macrophages - cytology; Macrophages - drug effects; Macrophages - metabolism; microRNA; MicroRNAs - genetics; miR-101; miRNA; NAFLD; Non-alcoholic fatty liver disease; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger - genetics; TNF-α; Tumor Necrosis Factor-alpha - pharmacology; ABCA1; NAFLD; TNF-α; miR-101; Atherosclerosis; microRNA; miRNA; Inflammation; ATP-binding cassette transporter A1; Non-alcoholic fatty liver disease; Cholesterol; IL-6
• ISSN: 0014-4827; 1090-2422
• DOI: 10.1016/j.yexcr.2015.05.023

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. 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. 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. 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. •Here, we investigated miR-101's role in regulating ABCA1 and cholesterol efflux.•Luciferase assays suggest that miR-101 directly targets the ABCA1 mRNA at site 2.•miR-101 promotes cholesterol retention under inflammatory conditions.•This cholesterol retention is achieved through miR-101's suppression of ABCA1.