Fig 4.

miR-33b binds to the 3′UTRs of PCK1, G6PC, SRC1, RORα, and CREB1. (A) PCK1, G6PC, SRC1, RORα, and CREB1 3′UTR sequences. Underlined sequences indicate the miR-33-binding sites. Nucleotides highlighted in red indicate the point mutations in the miR-33-binding sites. (B) Luciferase reporter activity in COS-7 cells transfected with control miRNA (CM) or miR-33 mimic (miR-33b) and the PCK1, G6PC, SRC1, RORα, or CREB1 3′UTR (wild type [WT]) or the constructs containing the indicated point mutations (MUT). DM indicates that the two miR-33-binding sites are mutated in the same construct. Data are expressed as relative luciferase activity compared to the activity in control samples cotransfected with an equal concentration of CM and correspond to the means ± SEM of 3 experiments performed in duplicate. ∗, significantly different from cells cotransfected with CM; #, significantly different from cells transfected with CM or miR-33b and the wild-type 3′UTR (P ≤ 0.05). (C) G6PC, PCK1, SRC1, CREB1, and ABCA1 mRNA levels determined by Ago IP relative to IgG IP were quantified by ribonucleoprotein immunoprecipitation followed by RT-qPCR analysis in Huh-7 cells transfected for 24 h with miR-33b. Results are the means ± SEM of 2 experiments performed in duplicate.