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.

Keywords: ABCA1; ATP-binding cassette transporter A1; Atherosclerosis; Cholesterol; IL-6; Inflammation; NAFLD; Non-alcoholic fatty liver disease; TNF-α; miR-101; miRNA; microRNA.