Figure 3.

miR-33 regulates lipidation and level of ApoE in the brain. A, miR-33 impaired cellular cholesterol efflux in N2a and astrocytes. N2a and astrocytes were transfected with miR-33 or scrambled negative control for 24 h and then incubated the cells with radioactively labeled ³H-cholesterol. After 24 h, cells were incubated with human ApoE3 as a cholesterol acceptor. Data are shown as a percentage of total cellular ³H-cholesterol content (total effluxed ³H-cholesterol + cell-associated ³H-cholesterol). Values are mean ± SEM. **p < 0.01 (t test). ***p < 0.001 (t test). n = 3. B, mir-33 deletion increased highly lipidated ApoE-containing particles. PBS-soluble fractions were analyzed by native PAGE and ApoE-specific Western blot. Total ApoE levels applied for each lane were shown below. C, D, PBS-soluble apoE levels were decreased in mir-33-deficient mice, compared with WT. Extracellular protein-enriched PBS-soluble fraction from cortex of mir-33-deficient (n = 6) and WT mice (n = 8) were analyzed by Western blots. Total protein levels (Ponceau staining) were used for normalization. Data are shown as a percentage of control, and values are mean ± SEM. ***p < 0.001 (t test).