MicroRNA-146b promotes adipogenesis by suppressing the SIRT1-FOXO1 cascade
Version of Record online: 6 SEP 2013
Copyright © 2013 The Authors. Published by John Wiley and Sons, Ltd on behalf of EMBO
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
EMBO Molecular Medicine
Volume 5, Issue 10, pages 1602–1612, October 2013
How to Cite
Ahn, J., Lee, H., Jung, C. H., Jeon, T. I. and Ha, T. Y. (2013), MicroRNA-146b promotes adipogenesis by suppressing the SIRT1-FOXO1 cascade. EMBO Mol Med, 5: 1602–1612. doi: 10.1002/emmm.201302647
- Issue online: 2 OCT 2013
- Version of Record online: 6 SEP 2013
- Manuscript Accepted: 2 AUG 2013
- Manuscript Revised: 31 JUL 2013
- Manuscript Received: 18 FEB 2013
- Korea Food Research Institute
As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.
|emmm201302647.reviewer_comments.pdf||PDF document||306K||Review Process File|
Figure S1. Target analysis predicts that SIRT1 is a potential target of miR-146b.
MicroRNA target prediction databases, such as microRNA.org, showed alignment between miR-146b and SIRT1.
Figure S2. Knockdown of SIRT1 increased lipid accumulation in differentiated 3T3-L1 cells.
A. SIRT1 was knocked down by lentiviral shRNA-SIRT1 (Lenti-sh SIRT1) and confirmed by Western blot analysis. Lenti-sh NC, lentiviral shRNA negative control.
B. Preadipocytes transduced with Lenti-sh NC- or Lenti-sh were stimulated to differentiation. At day 8, differentiated cells were stained with Oil red O.
C. Intracellular lipid accumulation was quantified by measuring optical absorbance at 500 nm (n = 3). **p < 0.01. Values are means ± SD.
D. Preadipocytes that were transduced with Lenti-sh NC or Lenti-sh SIRT1 were transfected with miR-146b In or miR In CTL and stimulated to differentiate. Expression levels of miR-146b and SIRT1 were quantified by qRT-PCR on day 8 of differentiation (n = 3). Values are means ± SD. *p < 0.05 versus miR In CTL.
Figure S3. SIRT1 mediates the adipogenic effect of miR-146b in 3T3-L1 cells.
A. The effect of EX-527 on the anti-adipogenic activity of miR-146b In. Cells were pretreated with 10 μM EX-527 for 6 h. Media were then replaced with differentiation media containing 10 μM EX-527. Cells were stained with Oil red O on day 8 of differentiation, and lipid droplets were quantified (n = 3). Values are means ± SD. *p < 0.05 versus miR In CTL.
B. The effect of EX-527 on miR-146b expression in the experiment described in (A). Expression of miR-146b was measured by qRT-PCR (n = 3). Values are means ± SD. *p < 0.05 versus miR In CTL.
C. The effect of SA-3 on miR-146b expression. 3T3-L1 cells were transfected with miR-146b Ac or miR-Ac CTL. After 2 days, cells were maintained in growth media containing 5 μM SA-3 for 8 days. Expression levels of miR-146b were measured by qRT-PCR (n = 3). Values are means ± SD. *p < 0.05 versus miR Ac CTL.
D. The effect of SA-3 on SIRT1 mRNA expression in the experiment described in (C). Expression levels of SIRT1 mRNA were measured by qRT-PCR (n = 3). Values are means ± SD. *p < 0.05 versus miR Ac CTL.
Figure S4. Expression of miR-146b and its direct target, SIRT1 in WAT from various obese mice.
A–C. The increase in miR-146b and the resulting decrease in SIRT1 are correlated with hypertrophy of adipose tissue from obese mice, such as ob/ob (A), db/db (B) and diet-induced obese mice (C). qRT-PCR analysis was performed to measure miR-146b and SIRT1 mRNA expression levels in epididymal adipose tissue (n = 5). Values are means ± SEM. *p < 0.05 versus corresponding control mice.
Figure S5. Efficacy and toxicity of LNA-miR-146b antagomir.
A. Expression levels of miR-146b were measured by qRT-PCR in various tissues to test the efficacy of LNAmiR-146b antagomir in mice. Expression of miR-146b was compared between mice treated with LNA-miR-146b and those treated with LNA-scrambled negative control (n = 5). Values are means ± SEM. EF, epididymal fat pad; PF, perirenal fat; BF, brown fat; SF, subcutaneous fat; SI, small intestine; LI, large intestine.
B, C. Effect of LNA-miR146b injection on AST and ALT, which are biomarkers of liver injury (n = 5). Values are means ± SEM.
Figure S6. Knockdown of miR-146b reduced adiposity and whole body mass in high fat-fed obese mice.
A. Knockdown of miR-146b by LNA-miR-146b significantly decreased the acetylated FOXO1(Ac-FOXO1)/total FOXO1 ratio of white adipose tissue. Ac-FOXO1/FOXO1 ratio was based on densitometric quantifications of Ac-FOXO1 and total FOXO1 levels on Western blots (n = 5). Values are means ± SEM. *p < 0.05 versus LNA-scramble.
B. Knockdown of miR-146b by LNA-miR-146b reduced white adipose fat mass. The weight of perirenal white adipose tissue was measured 72 h after the last injection (n = 5). Values are means ± SEM. *p < 0.05 versus LNA-scramble.
C. Administration of LNA-miR-146b inhibitor (LNA-miR-146b) significantly decreased whole body mass (n = 5).
Values are means ± SEM. ns, not significant; *p < 0.05.
D. LNA-miR-146b significantly reduced total body fat mass compared to LNA-scramble (n = 5). Values are means ±SEM. ns, not significant; *p < 0.05.
Figure S7. Knockdown of miR-146b improved hepatic steatosis via upregulation of SIRT1 in high fat-fed obese mice.
A. Administration of LNA-miR-146b reduced hepatic hypertrophy (n = 5). Values are means ± SEM. *p < 0.05 versus LNA-scramble.
B. Injection of LNA-miR-146b decreased intracellular lipid accumulation in hepatocytes. Hepatic tissues were stained with H&E for histological examination. Scale bar = 100 μm.
C. Immunoblot showing SIRT1 and β-actin expressions.
D. Densitometric analyses for (C). Values are means ± SD. **p < 0.01.
Figure S8. Knockdown of miR-146b ameliorated insulin resistance in high fat-fed obese mice.
A. Glucose tolerance test in LNA-scramble and LNA-miR146b injected mice (n = 6). Values are means ± SEM. *p < 0.05 versus LNA-scramble.
B. Insulin tolerance test in LNA-scramble and LNA-miR146b injected mice (n = 6). Values are means ± SEM. *p < 0.05 versus LNA-scramble.
Table S1. Changes in the expression levels of various miRNAs after adipogenesis in 3T3-L1.
Table S2. Measurement of body weight and white adipose tissue (WAT) weight.
Table S3. Serum lipids profile in LNA-injected mice.
Table S4. Blood glucose and hormones levels in LNA-injected mice.
Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.