Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2015 Aug 6:5:12911.
doi: 10.1038/srep12911.

Inhibition of miR-29 has a significant lipid-lowering benefit through suppression of lipogenic programs in liver

C Lisa Kurtz  1 , Emily E Fannin  1 , Cynthia L Toth  2 , Daniel S Pearson  3 , Kasey C Vickers  2 , Praveen Sethupathy  4
Affiliations

Inhibition of miR-29 has a significant lipid-lowering benefit through suppression of lipogenic programs in liver

C Lisa Kurtz et al. Sci Rep. .

Abstract

MicroRNAs (miRNAs) are important regulators and potential therapeutic targets of metabolic disease. In this study we show by in vivo administration of locked nucleic acid (LNA) inhibitors that suppression of endogenous miR-29 lowers plasma cholesterol levels by ~40%, commensurate with the effect of statins, and reduces fatty acid content in the liver by ~20%. Whole transcriptome sequencing of the liver reveals 883 genes dysregulated (612 down, 271 up) by inhibition of miR-29. The set of 612 down-regulated genes are most significantly over-represented in lipid synthesis pathways. Among the up-regulated genes are the anti-lipogenic deacetylase sirtuin 1 (Sirt1) and the anti-lipogenic transcription factor aryl hydrocarbon receptor (Ahr), the latter of which we demonstrate is a direct target of miR-29. In vitro radiolabeled acetate incorporation assays confirm that pharmacologic inhibition of miR-29 significantly reduces de novo cholesterol and fatty acid synthesis. Our findings indicate that miR-29 controls hepatic lipogenic programs, likely in part through regulation of Ahr and Sirt1, and therefore may represent a candidate therapeutic target for metabolic disorders such as dyslipidemia.

PubMed Disclaimer

Figures

Figure 1
Figure 1. In vivo LNA29 administration effectively inhibits the miR-29 family in the liver.
(a,b) RT-qPCR analysis of LNA29-treated (20 mg/kg) C57BL/6 J female mice (n = 14) with saline-treated age-, gender-, and strain-matched controls (n = 12) shows that endogenous expression of hepatic miR-29 is dramatically reduced (a); expression of Col1a1, a validated target of miR-29, is significantly elevated in several tissues including liver (b); U6 and Rps9 were used as expression normalizers for miRNA and gene analysis, respectively. (c) Plasma alanine transaminase (ALT) measurements (IU/L) are shown for LNA29-treated animals (n = 6) and saline treated controls (n = 5). *p < 0.05; **p < 0.01; ***p < 0.005; p-values were calculated by two-tailed unpaired Student’s t-test. Error bars represent standard error of the mean.
Figure 2
Figure 2. Inhibition of miR-29 leads to a significant reduction of plasma cholesterol and triglycerides.
(a,c–e) Plasma was isolated from whole blood collected via submandibular bleed one week post-treatment with either LNA29 (n = 14) or saline (n = 12) and was analyzed for levels of total cholesterol (a), triglycerides (c), glucose (d), and β-hydroxybutyrate (e). (b) Changes in total plasma cholesterol (∆mg/dL) between pre- and post-dosing are shown for each mouse treated with either LNA29 (n = 14) or saline (n = 12). *p < 0.05; ***p < 0.005; p-values were calculated by two-tailed unpaired Student’s t-test. Error bars represent standard error of the mean.
Figure 3
Figure 3. RNA-seq identifies lipid synthesis as the most enriched pathway among genes down-regulated in mouse liver upon inhibition of miR-29.
(a) Deep sequencing of total RNA revealed 612 down-regulated genes (blue) and 271 up-regulated genes (purple) in the livers of LNA29-treated mice (n = 6) compared to saline-treated (n = 6) controls. (b,c) Results of miRNA target site enrichment analysis shown for down-regulated (b) and up-regulated genes (c). (d,e) Results of gene ontology (GO) term enrichment analysis using NIH David shown for down-regulated (d) and up-regulated genes (e). Vertical dashed lines represent corrected p < 0.05. RNA-seq p-values were calculated by one-tailed unpaired Student’s t-test. Percentages represent the number of genes from the gene set involved in the pathway divided by the total number of genes in the gene set.
Figure 4
Figure 4. Inhibition of miR-29 represses the fatty acid synthesis pathway and reduces fatty acid content in the liver.
(a) Expression fold-change (FC) by RNA-seq in LNA29-treated mice (n = 6) relative to saline-treated controls (n = 6) is shown for key genes in the hepatic fatty acid synthesis pathway. p-values were calculated by one-tailed unpaired Student’s t-test. (b) RT-qPCR validation of RNA-seq results shown for Srebf1, Mlxipl/ChREBP, Acly, Acaca, Fasn and Scd1. Relative abundance (2^-dCt) was compared between LNA29-treated (n = 14) and saline-treated (n = 12) mice. Rsp9 was used as the normalizer. (c) Densitometry analysis shown for Scd1 protein in livers from LNA29-treated mice (n = 10) compared to saline-treated controls (n = 8). β-actin was used as the loading control. Immunoblot results also shown for two representative mice from each treatment group. (d) Quantification of radiolabeled acetate incorporation into fatty acids in vitro (Huh7 cells) after transfection with LNA29a (10 nM, n = 6) or transfection reagent alone (Mock, n = 6). (e) Measurement of total fatty acid levels in the livers of LNA29-treated animals (n = 14) compared to saline-treated controls (n = 12). (f) Levels of individual long-chain fatty acids in LNA29-treated relative to saline-treated mice. C14:0 (Myristic acid), C16:0 (Palmitic acid), C18:0 (Stearic acid), C18:1 (Oleic acid), C18:2 (Linoleic acid), C18:3 (Linolenic acid), C20:1 (Gondoic acid), C20:2 (Eicosadienoic acid), C20:3 (Homogamalinolenic acid), C20:4 (Arachidonic acid), C20:5 (Eicosapentaenoic acid), C24:0 (Lignoceric acid), C22:5 (Docosapentaenoic acid), C22:6 (Docosahexaenoic acid). *p < 0.05; **p < 0.01; ***p < 0.005; p-values were calculated by two-tailed unpaired Student’s t-test. Error bars represent standard error of the mean.
Figure 5
Figure 5. Inhibition of miR-29 represses the cholesterol synthesis pathway but slightly increases total hepatic cholesterol levels.
(a) Expression fold-change (FC) by RNA-seq in LNA29-treated mice (n = 6) relative to saline-treated controls (n = 6) is shown for key genes in the hepatic cholesterol synthesis pathway. p-values were calculated by one-tailed unpaired Student’s t-test. (b,c) RT-qPCR results shown for Srebf2, Mvk, Cyp51, Sqle and Idi1 (b) as well as for miR-33a (c), which is co-transcribed with Srebf2. (d) Quantification of radiolabeled acetate incorporation into cholesterol in vitro (Huh7 cells) after transfection with LNA29a (10 nM, n = 6) or transfection reagent alone (Mock, n = 6). (e) Measurement of total cholesterol levels in the livers of LNA29-treated mice (n = 14) compared to saline-treated controls (n = 12). *p < 0.05; **p < 0.01; ***p < 0.005; p-values were calculated by two-tailed unpaired Student’s t-test. Error bars represent standard error of the mean.
Figure 6
Figure 6. Inhibition of miR-29 alleviates miR-29-mediated repression of the anti-lipogenic transcription factor Ahr.
(a) Densitometry analysis for hepatic Ahr protein from LNA29-treated mice (n = 5) compared to saline-treated controls (n = 3). β-actin was used as the loading control. Immunoblot results also shown for two representative mice from each treatment group. (b) Expression fold-change (FC) by RNA-seq in LNA29-treated mice (n = 6) relative to saline-treated controls (n = 6) is shown for genes known to be transcriptionally activated by Ahr. p-values were calculated by one-tailed unpaired Student’s t-test. (c) A diagram of the predicted base pairing between miR-29 and the Ahr 3′ UTR sequence of both mouse and human is shown. (d) Effects of miR-29a mimic (10 nM) in HEK293T cells on the activity of Firefly (FL) luciferase with or without the Ahr 3′ UTR normalized to Renilla luciferase (RL) are shown. ***p < 0.005; *p < 0.05; p-values were calculated by two-tailed unpaired Student’s t-test. Error bars represent standard error of the mean.
Figure 7
Figure 7. Schematic of the putative molecular mechanism underlying the effects of LNA29 treatment.
In this model, under normal conditions, miR-29 may promote lipogenesis through suppression of Ahr, Sirt1 and Foxo3. Upon LNA29 treatment, miR-29 activity is suppressed, and Ahr, Sirt1, and Foxo3 levels are elevated, leading to reduced cholesterol and fatty acid synthesis.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Ling J., Lewis J., Douglas D., Kneteman N. M. & Vance D. E. Characterization of lipid and lipoprotein metabolism in primary human hepatocytes. Biochim. Biophys. Acta. 1831, 387–397 (2013). - PubMed
    1. Donnelly K. L. et al. Sources of fatty acids stored in liver and secreted via lipoproteins in patients with nonalcoholic fatty liver disease. J. Clin. Invest. 115, 1343–1351 (2005). - PMC - PubMed
    1. Fernandez-Hernando C., Suarez Y., Rayner K. J. & Moore K. J. MicroRNAs in lipid metabolism. Curr. Opin. Lipidol. 22, 86–92 (2011). - PMC - PubMed
    1. Rottiers V. & Naar A. M. MicroRNAs in metabolism and metabolic disorders. Nat. Rev. Mol. Cell Biol. 13, 239–250 (2012). - PMC - PubMed
    1. Sacco J. & Adeli K. MicroRNAs: emerging roles in lipid and lipoprotein metabolism. Curr. Opin. Lipidol. 23, 220–225 (2012). - PubMed

Publication types