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Genome-wide identification of microRNAs regulating cholesterol and triglyceride homeostasis

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Abstract

Genome-wide association studies (GWASs) have linked genes to various pathological traits. However, the potential contribution of regulatory noncoding RNAs, such as microRNAs (miRNAs), to a genetic predisposition to pathological conditions has remained unclear. We leveraged GWAS meta-analysis data from >188,000 individuals to identify 69 miRNAs in physical proximity to single-nucleotide polymorphisms (SNPs) associated with abnormal levels of circulating lipids. Several of these miRNAs (miR-128-1, miR-148a, miR-130b, and miR-301b) control the expression of key proteins involved in cholesterol-lipoprotein trafficking, such as the low-density lipoprotein (LDL) receptor (LDLR) and the ATP-binding cassette A1 (ABCA1) cholesterol transporter. Consistent with human liver expression data and genetic links to abnormal blood lipid levels, overexpression and antisense targeting of miR-128-1 or miR-148a in high-fat diet–fed C57BL/6J and Apoe-null mice resulted in altered hepatic expression of proteins involved in lipid trafficking and metabolism, and in modulated levels of circulating lipoprotein-cholesterol and triglycerides. Taken together, these findings support the notion that altered expression of miRNAs may contribute to abnormal blood lipid levels, predisposing individuals to human cardiometabolic disorders.

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Figure 1: Identification of miRNAs located in genomic loci that are enriched for SNPs associated with abnormal circulating total cholesterol, triglyceride, LDL-C and HDL-C levels.
Figure 2: Expression of LDLR and ABCA1 is regulated by miRNAs located in genomic loci associated with abnormal circulating blood lipid levels.
Figure 3: miR-128-1, miR-148a, miR-130b and miR-301b regulate ABCA1 expression and cholesterol efflux in mouse macrophages.
Figure 4: Overexpression of miR-128-1 and miR-148a in C57BL/6J mice decreases circulating levels of HDL-C, whereas antisense antagonism of these miRNAs increases LDL-C clearance.
Figure 5: Antisense inhibition of miR-148a in Apoe−/− mice fed a Western-type diet results in altered circulating lipoprotein/lipid levels.
Figure 6: Antisense inhibition of miR-128-1 in Apoe−/− mice fed a Western-type diet results in altered circulating lipoprotein/lipid levels, improved glucose homeostasis/insulin signaling and decreased hepatic steatosis.

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Acknowledgements

This work was supported by the following grants: US National Institutes of Health (NIH) grants R21DK084459 and R01DK094184 (A.M.N); R37DK048873 and R01DK056626 to D.E.C.; K24DK078772 to R.T.C.; and R01HL107953 and R01HL106063 to C.F.-H. A.M.N. was supported by a scholar award from the Massachusetts General Hospital (MGH). R.E.G. was supported by an Established Investigator Award from the American Heart Association. C.F.-H. was supported by a Fondation Leducq Transatlantic Network of Excellence in Cardiovascular Research Award. T.H. acknowledges the support of NIH grant R01HL49094 and a Fondation Leducq Transatlantic Network of Excellence in Cardiovascular Research Award. N.S. and J.S.T. were supported by the Intramural Research Program of the US National Institute of Allergy and Infectious Diseases. C.M.R. was supported by the American Heart Association SDG Grant 15SDG23000025. A.W. was supported by a fellowship from the MGH Executive Committee on Research. We thank C. Molony of Merck Research Laboratories for help with genotyping data quality assessment. We thank the Harvard Medical School Neurobiology Department and the Neurobiology Imaging Facility for consultation and instrument availability that supported this work. This facility is supported in part by the Neural Imaging Center as part of a National Institutes on Neurological Disorders and Stroke P30 Core Center grant no. NS072030, and by the Harvard Digestive Diseases Center (P30 DK034854).

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Contributions

S.H.N.-S. and A.M.N. conceived and carried out the initial miR-128-1 studies that provided support for the expanded project. A.W. and A.M.N. conceived and designed the expanded, published studies, interpreted the data, and wrote the manuscript, which was commented on by all authors. A.W., S.H.N.-S., L.W., S.S., Y. Li, F.K., N.P., D.E.C. and R.E.G. performed experiments and analyzed data in Apoe deficient mice and in C57BL/6 mice. L.G., C.M.R. and C.F.-H. quantified miRNAs in Rhesus monkeys and mice fed with different diets and performed efflux experiments in mouse peritoneal macrophages. N.S., Y. Lu, J.S.T., E.S., R.T., I.H., P.C.S. and L.M.K. contributed to the human liver miR-QTL analysis. Y.-C.L. and T.H. performed the Ago2 PAR-CLIP analysis in BMDMs. A.S.d. and R.T.C. performed the liver histology analysis. V.V. injected the lentivirus in C57BL/6 mice. J.C.B. and J.W. performed DNA microarray analysis from mouse liver samples prepared by A.W. S.K. and A.B. analyzed the GWAS data. R.d.C. carried out the non-human primate studies.

Corresponding author

Correspondence to Anders M Näär.

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Competing interests

A.M.N., A.W. and S.H.N.-S. have issued and pending patents (US Patent nos. 9,045,749 and US 61/865,327) on microRNA-targeting therapeutics for the treatment of cardiometabolic diseases.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–8 (PDF 3650 kb)

Supplementary Table 1

69 miRNAs are associated with abnormal levels of total cholesterol (TC), LDL-C, HDL-C, and TAGs. (XLSX 65 kb)

Supplementary Table 2

Gene Ontology analysis of miRNA target genes. (XLSX 37 kb)

Supplementary Table 3

List of SNPs associated with total cholesterol, LDL-C, HDL- C and triglycerides in the miR-128-1, miR-148a, miR-130b and miR-301b loci. (XLSX 121 kb)

Supplementary Table 4

Selected metabolism genes predicted to be targeted by miR- 128-1, miR-148a, miR-130b and miR-301b. (XLSX 46 kb)

Supplementary Table 5

Liver cis miR-QTL data. (XLSX 1002 kb)

Supplementary Table 6

Expression changes of predicted miR-128-1 targets in the livers of mice treated with antimiR-128-1 versus control scrambled LNA antimiR over 5 days. (XLSX 91 kb)

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Wagschal, A., Najafi-Shoushtari, S., Wang, L. et al. Genome-wide identification of microRNAs regulating cholesterol and triglyceride homeostasis. Nat Med 21, 1290–1297 (2015). https://doi.org/10.1038/nm.3980

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