MicroRNA-27a/b regulates cellular cholesterol efflux, influx and esterification/hydrolysis in THP-1 macrophages  Min Zhang, Jian-Feng Wu, Wu-Jun Chen, Shi-Lin Tang, Zhong-Cheng Mo, Yan-Yan Tang, Yuan Li, Jia-Lin Wang, Xiang-Yu Liu, Juan Peng, Kong Chen, Ping-Ping He, Yun-Cheng Lv, Xin-Ping Ouyang, Feng Yao, Deng-Pei Tang, Francisco S. Cayabyab, Da-Wei Zhang, Xi-Long Zheng, Guo-Ping Tian, Chao-Ke Tang  Atherosclerosis  Volume 234, Issue 1, Pages 54-64 (May 2014) DOI: 10.1016/j.atherosclerosis.2014.02.008 Copyright © 2014 Elsevier Ireland Ltd Terms and Conditions

Fig. 1 Predicted annealing of miR-27a and miR-27b to the ABCA1 3′ UTR. A, Schematic representation of miR-27a and miR-27b sequences including evolutionary conservation of the latter. B, The putative binding site of miR-27a/b to the ABCA1 3′ UTR in most animal species. C, Predicted annealing of human and murine miR-27a/b to the ABCA1 3′ UTR. D and E, Schematic alignment of the free energy scores (RNAhybrid and PicTar) for miR-27a/b–ABCA1 hybrids. Atherosclerosis 2014 234, 54-64DOI: (10.1016/j.atherosclerosis.2014.02.008) Copyright © 2014 Elsevier Ireland Ltd Terms and Conditions

Fig. 2 miR-27a/b directly targets the 3′ UTR of ABCA1. A, Luciferase activity assay of HEK 293 cells cotransfected with a luciferase reporter plasmid containing the ABCA1 3′ UTR (including WT, MUT1, MUT2 and MUT1+2) and miR-27a/b mimic for 24 h. B, C, D and E, THP-1 macrophage-derived foam cells were treated with miR-27a/b mimic in different concentrations (0, 10, 20, 40, 80, 100 nM) for 24 h or at different times (0, 6, 12, 24, 48 h) with 80 nM. B and C, Total RNA was extracted and qPCR was performed to determine the expression of ABCA1 mRNA. D and E, Western blot assays using antibodies against ABCA1 and β-actin were conducted. All results are expressed as mean ± S.D. from three independent experiments, each performed in triplicate. *P < 0.05 vs. 0, **P < 0.05 vs. control 0, ˆP < 0.05 vs. control. Atherosclerosis 2014 234, 54-64DOI: (10.1016/j.atherosclerosis.2014.02.008) Copyright © 2014 Elsevier Ireland Ltd Terms and Conditions

Fig. 3 MiR-27a/b regulate the endogenous levels of ABCA1 expression in different cells. THP-1 macrophage-derived foam cells (A and B) and HepG2 cells (C and D) were transfected with 80 nM miR-27a/b mimic or inhibitor for 24 h. The expression of ABCA1 mRNA (A and C) and protein (B and D) were determined using real-time quantitative PCR and western blotting assays, respectively. All the results are expressed as mean ± S.D. from three independent experiments, each performed in triplicate. *P < 0.05 and **P < 0.01 vs mimic-neg, #P < 0.05 relative to inhibitor-neg. Atherosclerosis 2014 234, 54-64DOI: (10.1016/j.atherosclerosis.2014.02.008) Copyright © 2014 Elsevier Ireland Ltd Terms and Conditions

Fig. 4 Effects of miR-27a/b on cholesterol efflux and the expression of apoA1. A, ApoA1- or HDL-mediated cholesterol efflux of THP-1 macrophage-derived foam cells were analyzed by liquid scintillation counting assays. B, THP-1 macrophage-derived foam cells were transfected with control or ABCA1 siRNA, and then incubated with miR-27a/b (80 nM) for 24 h ApoA1 mediated cholesterol efflux was analyzed. C and D, HepG2 cells were transfected with 80 nM miR-27a/b mimic or inhibitor for 24 h. Total RNA was extracted and qPCR was performed to determine the expression of apoA1 mRNA (C), and then Western blot assays using antibody against human apoA1 and β-actin were conducted (D). All results are expressed as mean ± S.D. from three independent experiments, each performed in triplicate. *P < 0.05 and **P < 0.01 vs. mimic-neg, #P < 0.05 and ##P < 0.01 vs. inhibitor-neg, ˆP < 0.05 and ˆˆP < 0.01 vs. control, @P < 0.05 and @@P < 0.01 vs. ABCA1 siRNA, &P < 0.05 vs. miR-27a mimic, $P < 0.05 vs. miR-27b mimic. Atherosclerosis 2014 234, 54-64DOI: (10.1016/j.atherosclerosis.2014.02.008) Copyright © 2014 Elsevier Ireland Ltd Terms and Conditions

Fig. 5 miR-27a/b regulates ox-LDL uptake by effecting LPL and CD36 expression in THP-1 macrophages. A, THP-1 macrophages were treated with 80 nM miR-27a/b mimic/inhibitor for 24 h and then incubated with Dil-oxLDL (10 μg/ml) for an additional 4 h at 4 °C. After PBS washes, cell lysates were collected for measurement of fluorescence. B, Sequence alignment of the miR-27a/b mature sequence with the binding sites of the LPL 3′ UTR in most animal species. C, Schematic alignment between the mature miR-27a/b seed sequence and the mRNA target region sequence in the 3′ UTR of human and murine LPL. D, E, G and H, THP-1 macrophage-derived foam cells were transfected with 80 nM miR-27a/b mimic or inhibitor for 24 h. The expression of LPL mRNA (D) and protein (E) or CD36 mRNA (G) and protein (H) were determined using qPCR and Western blot assays. F, HEK 293 cells were transfected with miR-27a/b mimic and WT, MUT1, MUT2 and MUT1+2 LPL and Luc activity was measured at 24 h posttransfection. WT, Luc reporter vector with wild-type LPL 3′ UTR; MUT1, Luc reporter vector with site 1 mutation; MUT2, Luc reporter vector with site 2 mutation; MUT1+2, Luc reporter vector with sites 1 and 2 mutations. All results are expressed as mean ± S.D. from three independent experiments, each performed in triplicate. *P < 0.05 and **P < 0.01 vs. mimic-neg, #P < 0.05 and ##P < 0.01 vs. inhibitor-neg, ˆP < 0.05 and ˆˆP < 0.01 vs. control. Atherosclerosis 2014 234, 54-64DOI: (10.1016/j.atherosclerosis.2014.02.008) Copyright © 2014 Elsevier Ireland Ltd Terms and Conditions

Fig. 6 miR-27a/b regulates the CE:FC ratio by targeting ACAT1. A and B, THP-1 macrophage-derived foam cells were treated with respective controls or miR-27a/b mimic or inhibitor. A, Total RNA was extracted and qPCR was performed to determine the expression of ACAT1 mRNA. B, Western blot assays using antibody against ACAT1 and β-actin were conducted. C, Sequence alignment of the ABCA1 3′ UTR and the mature miR-27a/b sequence (area inside red-dotted line is the seed sequence). D, Luciferase activity assay of HEK 293 cells cotransfected with a luciferase reporter plasmid containing the ACAT1 3′ UTR (including WT and MUT) and miR-27a/b mimic for 24 h. E, THP-1 macrophage-derived foam cells were treated with miR-27a/b mimic or inhibitor and CE formation was measured by incubation with [14C]-oleic acid. Lipids were then extracted and separated by thin-layer chromatography (TLC). Spots corresponding to cholesteryl oleate and oleic acid were scraped and radioactivity was measured. CE formation was calculated as a percentage of [14C]-oleate incorporated into cholesterol. All results are expressed as mean ± S.D. from three independent experiments, each performed in triplicate. *P < 0.05 and **P < 0.01 vs. mimic-neg, #P < 0.05 vs. inhibitor-neg, ˆP < 0.05 and ˆˆP < 0.01 vs. control. Atherosclerosis 2014 234, 54-64DOI: (10.1016/j.atherosclerosis.2014.02.008) Copyright © 2014 Elsevier Ireland Ltd Terms and Conditions