260Identification of differential roles of microRNA-33a and -33b during atherosclerosis progression with genetically modified mice

Abstract

Abstract Background MicroRNAs (miRs) are small non-protein-coding RNAs that bind to specific mRNAs and inhibit translation or promote mRNA degradation. Recent reports, including ours, indicated that miR-33a located within the intron of sterol regulatory element-binding factor (SREBF) 2 targets cholesterol transporter ATP-binding cassette protein A1 (ABCA1) or other anti-atherogenic targets and contributes to atherogenesis. Its inhibition or deletion is known to result in the amelioration of atherosclerosis. However, mice lack the other member of miR-33 family, miR-33b, which exists in humans. Precise evaluation and comparison of the responsibilities of these two miRs during the progression of atherosclerosis are essential and need to be investigated. Methods and results The difference between miR-33a and miR-33b in vitro and in vivo were analyzed from multiple directions using genetically modified miR-33a knock-out (KO) and miR-33b knock-in (KI) humanized mice. At first, we performed transcriptomic analysis of primary cultured hepatocytes transfected with synthetic miR-33a, miR-33b, and a control miR and found similar potential target repression and targeting motif of miR-33a and miR-33b in vitro. However, we noticed distinct expression patterns of miR-33a and miR-33b in several organs. By crossing miR-33a KO and miR-33b KI mice, we established four strains with or without miR-33a and miR-33b. Comparison of these strains showed distinct distribution and regulation of miR-33 family. In particular, comparison between mice with only miR-33a (wild-type mice) and mice with only miR-33b (miR-33a−/− miR-33b+/+) revealed 4-fold predominant expression of miR-33b in the liver. Such differential expression resulted in a reduced expression of target genes such as ABCA1 and worsened serum cholesterol profile in mice with only miR-33b. On the contrary, in macrophages the expression levels of miR-33 family genes were similar and their effects on target genes and cholesterol efflux capacity to ApoA-I or HDL cholesterol (HDL-C) were almost comparable. To evaluate the whole body atherogenic potency, we developed ApoE−/− miR-33a+/+ miR-33b−/− mice and ApoE−/− miR-33a−/− miR-33b+/+ mice. ApoE−/− miR-33a−/− miR-33b+/+ mice developed increased atherosclerotic plaque compared with ApoE−/− miR-33a+/+ miR-33b−/− mice, in line with the predominant expression of miR-33b in the liver and decreased serum HDL-C levels whose lower cholesterol efflux capacity were confirmed in 3H-labeled macrophages. On the contrary, a bone marrow transplantation study showed no significant difference in atherosclerosis and serum cholesterol profile, and this was consistent with the relevant expression levels of miR-33a and miR-33b in bone marrow cells. Conclusions miR-33 family exhibited differences in distribution and regulation, and particularly in the progression of atherosclerosis, miR-33b would be more potent than miR-33a.