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Introduction
Atherosclerosis is a chronic lipid-associated inflammatory disease (1). Hyperlipidemia triggers multiple metabolic disturbances and has been considered as an important pathogenic factor. Oxidized low-density lipoprotein (oxLDL) engulfed by macrophages activates a variety of genes and the expression of microRNAs (miRNAs/miRs). miRNAs are a class of single-stranded nucleotides of ~22 bases in length, which induce the formation of silencing complexes in a sequence-specific manner, leading to translational repression of target genes (2). miRNAs are involved in numerous cellular biological activities associated with pathological processes, including cardiogenesis, oncogenesis, immunological diseases and hematopoietic differentiation (3). miR-146a modulates innate immunity (4), neoplasia (5) and infection (6) by targeting Toll-like receptor (TLR) signaling. Previous studies have indicated that miR-146a significantly controls diverse aspects of autoimmune diseases, including systemic lupus erythematosus (7), rheumatoid arthritis (8–10) and psoriasis (11). In spite of several studies that established the involvement of miR-146a in immune-inflammatory diseases, the underlying mechanism of its regulatory function has largely remained elusive. A previous study by our group showed that oxLDL activates miR-146a expression, which in turn stimulates various inflammatory factors (12). The present study investigated the regulation of miR-146a by oxLDL in cells of the immune system with the aim of identifying novel therapeutic targets for the treatment of atherosclerosis. Due to its known association with atherosclerosis, the THP-1 human macrophage cell line was used as a model (13).
Materials and methods
Cell culture
The THP-1 human monocytic cell line was a gift from the State Key Laboratory for Diagnosis and Treatment of Infectious Diseases (Zhejing University, Hangzhou, China). The cells were cultured in RPMI 1640 medium (Gibco; Thermo Fisher Scientific, Waltham, MA, USA) containing 10% fetal calf serum (Gibco). Prior to each experiment, the cells were treated for 24 h with 100 nmol phorbol myristate acetate (Sigma-Aldrich, St. Louis, MO, USA) to acquire a macrophage-like adherent phenotype. Pre-treatment with chemical inhibitors, including c-jun N-terminal kinase (JNK) inhibitor SP600125, p38/mitogen-activated protein kinase (MAPK) inhibitor SB203580, MAPK kinase inhibitor UO126 or nuclear factor (NF)-κB inhibitor pyrrolidine dithiocarbamate (PDTC) (all from Beyotime Institute of Biotechnology, Inc., Haimen, China) was performed for 30 min prior to addition of 40 µg/ml oxLDL (Guangzhou Yiyuan Biological Technology Co., Ltd., Guangzhou, China). The cells were harvested after 24 h of oxLDL treatment.
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