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MicroRNA-148a regulates LDL receptor and ABCA1 expression to control circulating lipoprotein levels

Abstract

The hepatic low-density lipoprotein receptor (LDLR) pathway is essential for clearing circulating LDL cholesterol (LDL-C). Whereas the transcriptional regulation of LDLR is well characterized, the post-transcriptional mechanisms that govern LDLR expression are just beginning to emerge. Here we develop a high-throughput genome-wide screening assay to systematically identify microRNAs (miRNAs) that regulate LDLR activity in human hepatic cells. From this screen we identified and characterized miR-148a as a negative regulator of LDLR expression and activity and defined a sterol regulatory element–binding protein 1 (SREBP1)-mediated pathway through which miR-148a regulates LDL-C uptake. In mice, inhibition of miR-148a increased hepatic LDLR expression and decreased plasma LDL-C. Moreover, we found that miR-148a regulates hepatic expression of ATP-binding cassette, subfamily A, member 1 (ABCA1) and circulating high-density lipoprotein cholesterol (HDL-C) levels in vivo. These studies uncover a role for miR-148a as a key regulator of hepatic LDL-C clearance through direct modulation of LDLR expression and demonstrate the therapeutic potential of inhibiting miR-148a to ameliorate an elevated LDL-C/HDL-C ratio, a prominent risk factor for cardiovascular disease.

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Figure 1: Genome-wide miRNA screen identifies novel regulators of LDLR activity.
Figure 2: Post-transcriptional regulation of LDLR expression and activity by miR-148a in human hepatic cells.
Figure 3: LDLR-GFP overexpression rescues LDLR activity in miR-148a–transfected cells.
Figure 4: SREBP1c modulates miR-148a expression in vitro and in vivo.
Figure 5: Inhibition of miR-148a alters plasma cholesterol levels in vivo.
Figure 6: miR-148a post-transcriptionally regulates ABCA1 expression.

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Acknowledgements

We thank C. Yun, J. Recio, S. Katz and R. DasGupta at the New York University (NYU) RNAi Core for their advice and assistance with the miRNA screen, K. Harry and members of the Yale University Liver Center for primary mouse hepatocyte isolation, members of the Iwakiri laboratory for reagents and advice on primary hepatocyte culture, the Yale University School of Medicine Mouse Metabolic Phenotyping Center (MMPC) for liver toxicity measurements, P. Tontonoz (UCLA) for generously providing the LDLR-GFP plasmid, the Nonhuman Primate Core of the National Institute on Aging for providing liver samples and E. Fisher (NYU School of Medicine) for kindly providing the human hepatocellular carcinoma cell line (Huh7) and mouse hepatic cell line (Hepa1–6). This work was supported through grants by the US National Institutes of Health (NIH; grants R01HL107953, R01HL107953-04S1 and R01HL106063 (C.F.-H.); grant R01HL105945 (Y.S.); grant 1F31AG043318 (L.G.); grant P30KD034989 (Yale University Liver Center), the American Heart Association (grant 15SDG23000025 (C.M.R.)), the Howard Hughes Medical Institute International Student Research Fellowship (E.A.), the Foundation Leducq Transatlantic Network of Excellence in Cardiovascular Research (C.F.-H.) and the Ministerio de Industria y Comercio, Spain (grant SAF2011-29951 (M.A.L.)). Centro de Investigación Biomédica en Red Fisiopatología de Obesidad y Nutrición (CIBERobn) is an initiative of Instituto de Salud Carlos III (ISCIII), Spain. R.d.C. is supported by the Intramural Research Program of the NIH, National Institute of Aging. A.M.N. and A.W. are supported by NIH grant R01DK 094184. The NYU RNAi core is supported by the Laura and Isaac Perlmutter Cancer Center (NIH, National Cancer Institute grant P30CA16087) and the New York State Stem Cell Science (NYSTEM) contract C026719. The Yale University School of Medicine MMPC is supported by NIH grant U24 DK059635.

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Contributions

L.G. and C.F.-H. conceived and designed the study. L.G. optimized and performed the miRNA screen. J.F.A. and A.C.-D. performed confocal experiments, assisted with cloning and analyzed data. L.G. and A.C.-D. performed in vitro experiments and analyzed data. L.G., N.R., C.M.R. and A.C.-D. performed mouse experiments and analyzed data. N.R. performed western blotting for plasma lipoproteins and assessed lipoprotein distribution by FPLC. E.A. cloned the MIR148A promoter. C.-S.L. assisted with western blotting. N.N.A. analyzed gene expression in the livers of fed and fasted wild-type mice and ob/ob mice. R.d.C. designed the nonhuman primate experiments and J.D.H. provided the mouse samples. M.A.L. provided DiI-LDL and native LDL. A.W. and A.M.N. assisted with mouse experiments. Y.S. and C.F.-H. assisted with experimental design and data interpretation. L.G. and C.F.-H. wrote the manuscript, which was commented on by all authors.

Corresponding author

Correspondence to Carlos Fernández-Hernando.

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Competing interests

C.F.-H. and L.G. have filed a patent (United States PCT/US2014/042196) for the therapeutic use of miR-148 inhibitors in treating cardiometabolic diseases.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–10 and Supplementary Table 2 (PDF 18771 kb)

Supplementary Table 1

Raw data from primary miRNA screen. (XLS 347 kb)

Supplementary Table 3

Functional annotation clusters of human miR-148a targets identified by DAVID. (XLS 768 kb)

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Goedeke, L., Rotllan, N., Canfrán-Duque, A. et al. MicroRNA-148a regulates LDL receptor and ABCA1 expression to control circulating lipoprotein levels. Nat Med 21, 1280–1289 (2015). https://doi.org/10.1038/nm.3949

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