Figure 2.

miR-33 suppresses ABCA1 expression in the brain. A, Schematic diagram of the conserved target sites of miR-33 in the 3′UTR of ABCA1 mRNA. The mir-33 gene is located within the intron of Sterol regulatory element binding transcription factor 2 (SREBF2) gene. The genomic sequences around mir-33 are highly conserved in mammals. The mature sequence of miR-33 is shown in red within pre-mir-33. miR-33 has three potential targeting sites in the ABCA1. Blue underlines indicate 3′UTR. *Conserved nucleotides. B, miR-33 directly targets the 3′UTR of ABCA1 mRNA. (n = 6 per group). Luciferase reporter assays were performed with the reporter construct containing the full-length 3′UTR of hABCA1 mRNA downstream of Renilla luciferase. Renilla luciferase activity was normalized with the corresponding firefly luciferase activity. C, D, miR-33 decreased ABCA1 levels in N2a cells (n = 6) and mouse primary astrocytes (n = 5). Cells were transfected with miR-33 or scrambled negative control (Ctl). At 48 h after transfection, cells were harvested for Western blot analyses. ABCA1 protein levels were normalized by actin levels and quantified as a percentage of control. E, miR-33 is selectively deleted in cortex of miR-33 (−/−) mice without altering the expression of its host gene, SREBF2, in cortex. GAPDH levels were used for normalization. Data are shown as a percentage of WT. F, G, ABCA1 levels were increased in cortex of mir-33 knock-out mice (miR-33 (−/−), n = 5), compared with WT mice (miR-33 (+/+), n = 5). ABCA1 levels were analyzed by Western blot at 4 months of age. Actin levels were used for normalization. Data are shown as a percentage of control, and values are mean ± SEM. **p < 0.01 (t test). ***p < 0.001 (t test).