Effects of overexpression of miR-33a-5P and Anti-miR-33a-5P on intracellular lipid accumulation in THP-1 macrophages in the absence or presence of LDL.

THP-1 macrophages were infected using Con-miR, miR-33a-5P, Con-Anti-miR, and Anti-miR-33a-5P, respectively, after 24 h PMA stimulation. After 48 h infection, THP-1 macrophages we incubated in serum-free medium at 37°C for 24 h. Then, the medium was respectively replaced by fresh serum-free medium (0.2% BSA) containing blank control (A, I, Con-miR), blank control (A, II, miR-33a-5P), 40 ng/ml IL-6 (A, III, Con- Anti-miR plus IL-6), 40 ng/ml IL-6 (A, IV, anti-miR-33a-5P plus IL-6), 25 µg/ml LDL (B, I, Con-miR plus LDL), 25 µg/ml LDL (B, II, miR-33a-5P plus LDL), 25 µg/ml LDL plus 40 ng/ml IL-6 (B, III, Con-Anti-miR plus IL-6 plus LDL), or 25 µg/ml LDL plus 40 ng/ml IL-6 (B, IV, Anti-miR-33a-5P plus IL-6 plus LDL), followed by incubation at 37°C for 24 h. (A and B) The cells were examined for lipid inclusions by oil red O staining. The results are representative of those observed in six separate experiments (×400). (C and D) Semi-quantitative analysis of oil red O positive staining. Data are means ± SD from 6 separate fields. (E and F) Quantification of levels of intracellular cholesterol contents. Data are means ± SD of duplicate wells from 6 experiments. *, P<0.05 compared with Con-miR or Con-miR plus LDL; **, P<0.01 compared with Con-miR plus LDL; #, P<0.05 compared with Con-Anti-miR plus IL-6 or Con-Anti-miR plus IL-6 plus LDL; ##, P<0.01 compared with Con- Anti-miR plus IL-6 or Con-Anti-miR plus IL-6 plus LDL.