miR-33a and -a* and miR-33b and -b* are differentially expressed in human tissues. (A) Schematic representation of hsa-miR-33a and hsa-miR-33b stem-loop structures. Mature sequences for the 5′ (5p) and 3′ (3p) strands are highlighted in blue and orange, respectively. (B) Assessment of miR-33a and miR-33a* primer efficiencies. Cycle threshold (C_t) values were plotted against the log of six sequential 10-fold dilutions of human miR-33a or -33a* mimics. Efficiency (E) = 10(−1/slope). (C) Assessment of miR-33b and miR-33b* primer efficiencies. Cycle threshold values were plotted against the log of six sequential 10-fold dilutions of human miR-33b or -33b* mimics. E = 10(−1/slope). (D) Expression profile of miR-33a and miR-33a* in selected human tissues (Life Technologies). (E) Expression profile of miR-33b and miR-33b* in selected human tissues (Life Technologies). (F) qRT-PCR analysis of miR-33a, miR-33a*, miR-33b, miR-33b*, miR-148a, and miR-451 in Huh7 cells transfected with nonsilencing control siRNA (NS) or Dicer siRNA (siDicer). Western blot analysis of Dicer expression is shown in the right panel. (G) Assessment of miR-33a and miR-33a* relative expression levels in Huh7 cells transfected with control mimic (CM), miR-33a, or miR-33a* after Ago2 immunoprecipitation (Ago2-IP). The relative expression levels of miR-33a and miR-33a* were normalized to cells transfected with CM. In panels D, E, and G, data are means ± SEM and representative of 2 experiments in duplicate. In panel F, data are means ± SEM from 3 experiments in triplicate. *, P ≤ 0.05.

miR-33a and -a* and miR-33b and -b* are coexpressed with their host genes, Srebp-2 and Srebp-1. (A) Quantitative real-time fluorescence PCR (qRT-PCR) analysis of miR-33a, miR-33a*, and SREBP-2 in human hepatic (Huh7) cells loaded with cholesterol by nLDL (120 μg/ml) and depleted of cholesterol by statin treatment (5 μM). (B) qRT-PCR analysis of miR-33b, miR-33b*, and SREBP-1c in Huh7 cells treated with the LXR agonist T0901317 (T090) for 12 h. (C) qRT-PCR analysis of SREBP-2 and miR-33a and miR-33a* in the livers of C57BL6 mice (n = 5 per group) fed a chow diet, high-fat diet (HFD), or rosuvastatin-supplemented diet (statin). (D) qRT-PCR analysis of SREBP-1c, miR-33b, and miR-33b* in the livers of rhesus monkeys (n = 6 per treatment) after 1 h of glucose stimulation. In panels A to D, the data are means ± SEM and representative of ≥2 experiments in triplicate. *, P ≤ 0.05.

miR-33a* specifically targets the 3′ UTR of human genes Npc1, Crot, Irs2, Src1, Src3, and Nyfc, while miR-33b* specifically targets the 3′ UTR of human Nfyc. (A through F) Luciferase reporter activity in COS7 cells transfected with control mimic (CM) or miR-33a* mimic and the human or mouse (A) Npc1, (B) Crot, (C) Irs2, (D) Src1, (E) Src3, or (F) Nfyc 3′ UTRs containing the indicated point mutations (PM) in the miR-33a* target sites. (G) Luciferase reporter activity in COS7 cells transfected with control mimic (CM) or miR-33b* mimic and the human Nfyc 3′ UTR containing the indicated point mutations (PM) in the miR-33b* target site. (H) qRT-PCR analysis of CROT and NPC1 in Huh7 cells transfected with the control mimic (CM), miR-33a, or miR-33a* after Ago2 immunoprecipitation (Ago2-IP). In panels A to G, the data are expressed as mean percentages of 3′-UTR activity of CM ± SEM and are representative of ≥3 experiments in triplicate. *, P ≤ 0.05. n.s., not significant. In panel H, the data are means ± SEM and representative of 2 experiments in duplicate.

Posttranscriptional regulation of ABCA1, NPC1, CROT, CPT1a, IRS2, SRC1, SRC3, NFYC, and RIP140 by miR-33a and miR-33a* in Huh7 cells. (A) qRT-PCR analysis of SREBP-2, SREBP-1c, ABCA1, NPC1, CROT, CPT1a, IRS2, SRC1, SRC3, NYFC, and RIP140 in Huh7 cells transfected with a control mimic (CM), miR-33a mimic, or miR-33a* mimic. (B) Western blot analysis of ABCA1, NPC1, CROT, CPT1a, IRS2, SRC1, SRC3, NFYC, and RIP140 in Huh7 cells transfected with CM, miR-33a, or miR-33a*. Quantification of protein relative to the loading control (HSP90) is shown in the right panel. a.u., arbitrary units. (C) qRT-PCR analysis of SREBP-2, SREBP-1c, ABCA1, NPC1, CROT, CPT1a, IRS2, SRC1, SRC3, NYFC, and RIP140 in Huh7 cells transfected with a control inhibitor (CI), inhibitor of miR-33a (Inh-33a), or inhibitor of miR-33a* (Inh-33a*). (D) Western blot analysis of ABCA1, NPC1, CROT, CPT1a, IRS2, SRC1, SRC3, NFYC, and RIP140 in Huh7 cells transfected with CI, Inh-33a, or Inh-33a*. Quantification of protein relative to the loading control (HSP90) is shown in the right panel. In panels A to D, the data are means ± SEM and representative of ≥3 experiments in triplicate. *, P ≤ 0.05.

Posttranscriptional regulation of ABCA1, NPC1, SRC1, SRC3, NFYC, and RIP140 by miR-33a and miR-33a* in THP1 cells. (A) qRT-PCR analysis of ABCA1, NPC1, SRC1, SRC3, NYFC, and RIP140 in THP1 cells transfected with a control mimic (CM), miR-33a mimic, or miR-33a* mimic. (B) Western blot analysis of ABCA1, NPC1, SRC1, SRC3, and NFYC in THP1 cells transfected with CM, miR-33a, or miR-33a*. Quantification of protein relative to the loading control (HSP90) is shown in the right panel. (C) qRT-PCR analysis of ABCA1, NPC1, SRC1, SRC3, NYFC, and RIP140 in THP1 cells transfected with a control inhibitor (CI), inhibitor of miR-33a (Inh-33a), or inhibitor of miR-33a* (Inh-33a*). (D) Western blot analysis of ABCA1, NPC1, SRC1, SRC3, and NFYC in THP1 cells transfected with CI, Inh-33a, or Inh-33a*. Quantification of protein relative to the loading control (HSP90) is shown in the right panel. In panels A to D, the data are means ± SEM and representative of ≥3 experiments in triplicate. *, P ≤ 0.05.

Posttranscriptional regulation of ABCA1, NFYC, and RIP140 by miR-33b and miR-33b* in Huh7 cells. (A) qRT-PCR analysis of SREBP-2, SREBP-1c, ABCA1, RIP140, and NFYC in Huh7 cells transfected with a control mimic (CM), miR-33b mimic, or miR-33b* mimic. (B) Western blot analysis of ABCA1, RIP140, and NFYC in Huh7 cells transfected with CM, miR-33b, or miR-33b*. Quantification of protein relative to the loading control (HSP90) is shown in the right panel. (C) qRT-PCR analysis of SREBP-2, SREBP-1c, ABCA1, RIP140, and NFYC in Huh7 cells transfected with a control inhibitor (CI), inhibitor of miR-33b (Inh-33b), or inhibitor of miR-33b* (Inh-33b*). (D) Western blot analysis of ABCA1, RIP140, and NFYC in Huh7 cells transfected with CI, Inh-33b, or Inh-33b*. Quantification of protein relative to the loading control (HSP90) is shown in the right panel. In panels A to D, the data are means ± SEM and representative of ≥3 experiments in triplicate. *, P ≤ 0.05.

Posttranscriptional regulation of ABCA1, NFYC, and RIP140 by miR-33b and miR-33b* in THP1 cells. (A) qRT-PCR analysis of ABCA1, NYFC, and RIP140 in THP1 cells transfected with a control mimic (CM), miR-33b mimic, or miR-33b* mimic. (B) Western blot analysis of ABCA1 and NFYC in THP1 cells transfected with CM, miR-33b, or miR-33b*. Quantification of protein relative to the loading control (HSP90) is shown in the right panel. (C) qRT-PCR analysis of ABCA1, NYFC, and RIP140 in THP1 cells transfected with a control inhibitor (CI), inhibitor of miR-33b (Inh-33b), or inhibitor of miR-33b* (Inh-33b*). (D) Western blot analysis of ABCA1 and NFYC in THP1 cells transfected with CI, Inh-33b, or Inh-33b*. Quantification of protein relative to the loading control (HSP90) is shown in the right panel. In panels A to D, the data are means ± SEM and representative of ≥3 experiments in triplicate. *P ≤ 0.05.

Both arms of the miR-33 duplex contribute to the posttranscriptional regulation of target gene expression. (A to B) Huh7 cells were transduced with a lentivirus encoding the miR-33a precursor (pre-miR-33) or empty vector control. Transduction efficiency was confirmed 48 h after culturing by measuring GFP expression using fluorescence microscopy (A) and flow cytometry (B). (C) qRT-PCR analysis of miR-33a (top panel) and miR-33a* (bottom panel) in empty vector-transduced or pre-miR-33-transduced Huh7 cells. (D) qRT-PCR analysis of SREBP-2, SREBP-1c, ABCA1, NPC1, CROT, CPT1a, IRS2, SRC1, SRC3, RIP140, and NFYC in Huh7 cells transduced with pre-miR-33. Values were compared to those from empty vector-transduced cells. (E) qRT-PCR analysis of ABCA1, NPC1, CROT, CPT1a, IRS2, SRC1, SRC3, RIP140, and NFYC in pre-miR-33-transduced Huh7 cells transfected with a control inhibitor (CI), inhibitor of miR-33a (Inh-miR-33a), or inhibitor of miR-33a* (Inh-miR-33a*). (F) Western blot analysis of ABCA1, NPC1, CROT, CPT1a, IRS2, SRC1, SRC3, NFYC, and RIP140 in pre-miR-33-transduced Huh7 cells transfected with CI, Inh-miR-33a, or Inh-miR-33a*. Quantification of protein relative to the loading control (HSP90) is shown in the right panel. FS, forward scatter. In panels A to F, the data are means ± SEM and representative of ≥2 experiments in triplicate. *, P ≤ 0.05.

miR-33* cooperates with miR-33 to regulate fatty acid oxidation in human hepatic cells. (A) Neutral lipid accumulation in Huh7 cells loaded with 0.25% BSA or 1 mM oleic acid and stained with Bodipy (green) and DAPI (blue). Scale bar, 200 μm. (B) Neutral lipid accumulation in Huh7 cells transfected with a control mimic (CM), miR-33a mimic, or miR-33a* mimic and loaded with 1 mM oleic acid. Following 0 h and 24 h of starvation, cells were washed, fixed, and stained with Bodipy (green) and DAPI (blue). Scale bar, 200 μm. (C) Analysis of β-hydroxybutyrate in Huh7 cells transfected with CM, miR-33a, miR-33a*, or miR-33 and miR-33a* at 24 h of starvation. (D) Analysis of triglyceride (TG) content in Huh7 cells transfected with CM, miR-33a, or miR-33a* at 0 h and 24 h of starvation. (E) Analysis of free fatty acid (FFA) content in Huh7 cells transfected with CM, miR-33a or miR-33a* at 0 h and 24 h of starvation. In panels A to E, the data are means ± SEM and representative of ≥2 experiments in triplicate. *, P ≤ 0.05 compared to CM (C) and compared to CM, miR-33, and miR-33* at time (t) 0 h (D and E). #, P ≤ 0.05 compared to CM at 24 h (D and E).

Proposed model of miR-33 arm-specific processing and target gene regulation. miR-33a* and -b* work collectively with their sister strands, miR-33a and -b, to boost intracellular cholesterol and fatty acid levels by balancing the posttranscriptional repression of genes involved in cellular cholesterol efflux (ABCA1 and NPC1), fatty acid oxidation (CROT, CPT1a, HADHB, and AMPK), glucose metabolism (SIRT6 and IRS2), and the transcriptional regulation of lipid metabolism (SRC1, SRC3, NFYC, and RIP140). Pol II, polymerase II.