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. 2010 Jul;2(7):415-31.
doi: 10.18632/aging.100176.

miRNAs Regulate SIRT1 Expression During Mouse Embryonic Stem Cell Differentiation and in Adult Mouse Tissues

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Free PMC article

miRNAs Regulate SIRT1 Expression During Mouse Embryonic Stem Cell Differentiation and in Adult Mouse Tissues

Laura R Saunders et al. Aging (Albany NY). .
Free PMC article

Abstract

SIRT1 is increasingly recognized as a critical regulator of stress responses, replicative senescence, inflammation, metabolism, and aging. SIRT1 expression is regulated transcriptionally and post-transcriptionally, and its enzymatic activity is controlled by NAD+ levels and interacting proteins. We found that SIRT1 protein levels were much higher in mouse embryonic stem cells (mESCs) than in differentiated tissues. miRNAs post-transcriptionally downregulated SIRT1 during mESC differentiation and maintained low levels of SIRT1 expression in differentiated tissues. Specifically, miR-181a and b, miR-9, miR-204, miR-199b, and miR-135a suppressed SIRT1 protein expression. Inhibition of mir-9, the SIRT1-targeting miRNA induced earliest during mESC differentiation, prevented SIRT1 downregulation. Conversely, SIRT1 protein levels were upregulated post-transcriptionally during the reprogramming of mouse embryonic fibroblasts (MEFs) into induced pluripotent stem (iPS) cells. The regulation of SIRT1 protein levels by miRNAs might provide new opportunities for therapeutic tissue-specific modulation of SIRT1 expression and for reprogramming of somatic cells into iPS cells.

Conflict of interest statement

The authors of this manuscript have no conflict of interests to declare.

Figures

Figure 1.
Figure 1.. SIRT1 expression is regulated post-transcriptionally in adult mouse tissues and during mESC differentiation.
(A-B) Protein and RNA were extracted from mESC and tissues from ~6-week-old mice. (A) Western blot analysis with antibodies against SIRT1 (Frye antiserum top blot; Upstate antiserum lower blot), HDAC1, HDAC2, and tubulin. (B) qRT-PCR analysis of SIRT1, HDAC1, and HDAC2 normalized to GAPDH levels. Data are mean ± s.d. for four samples. (C-D) Protein and RNA were isolated from mESCs differentiated in vitro for up to 20 days (EBs d2-20). (C) Western blots analysis of expression of SIRT1, various HDACs, markers of pluripotent embryonic stem cells, and markers of differentiation. Data are representative of four experiments. (D) qRT-PCR analysis of SIRT1, HDAC2, markers of pluripotent embryonic stem cells, and markers of differentiation. Data were normalized to GAPDH and plotted as expression relative to the mean of four mESC samples. Data are mean ± s.d. for four samples.
Figure 2.
Figure 2.. miRNAs post-transcriptionally regulate SIRT1.
(A) mESCs were differentiated and treated on d8 with the proteasome inhibitor MG-132 (10 μM, 3-7 h), and protein lysates were analyzed on western blots. Data are representative of four experiments. (B) Protein levels of SIRT1 and REST relative to tubulin levels were quantified by densitometry with NIH Image. (C-E) The consequences of Dicer inactivation and loss of small RNAs were assessed in protein lysates and RNA from livers of control and Dicerflox/flox mice injected with the AAV8 vector expressing cre at the indicated times. (C) Western blotting was used to analyze 70 μg of liver lysate and 10 μg of mESC lysate. (D) SIRT1 protein levels relative to tubulin or GAPDH were quantified by densitometry. (E) SIRT1 and Dicer mRNA levels were measured by qRT-PCR. Data are mean ± s.d. for four samples. (F-H) Lung fibroblasts were cultured from DicerFlox/Flox mice and infected with adenoviral Cre or GFP. (F) SIRT1 protein levels were measured by western blotting 72 h after Cre inactivation of Dicer. (G) SIRT1 protein levels relative to tubulin were quantified by densitometry. (H) mRNA levels of SIRT1 and Dicer were measured by qRT-PCR. Data are mean ± s.d. for three samples. (I-K) siRNAs were transfected into NIH3T3 cells to knockdown DGCR8, Dicer, or GL3 luciferase as a control. (I) DGCR8 knockdown and increased SIRT1 protein levels were analyzed by western blotting 72 h after siRNA transfection. Data are representative of three experiments. (G) qRT-PCR analysis confirmed Dicer knockdown and no significant change in SIRT1 mRNA levels. Data are mean ± s.d. for three samples.
Figure 3.
Figure 3.. Expression profiling of miRNAs that potentially target the SIRT1 3'-UTR during mESC differentiation.
(A) 18 miRNAs from nine miRNA families that potentially target the 3'-UTR of SIRT1 were induced during mESC differentiation at the time SIRT1 protein was downregulated. Their fold induction in d20 embryoid bodies above their expression in undifferentiated mESCs was plotted on the y-axis, and the location of their seed binding site in the 3'-UTR of mSIRT1 was plotted on the x-axis. (B-C), qRT-PCR of miRNA expression relative to miR-16 from undifferentiated mESCs and differentiating embryoid bodies of specific miRNAs that potentially target SIRT1. Data are mean ± s.d. for four samples.
Figure 4.
Figure 4.. miRNAs post-transcriptionally regulate the 3'-UTR of SIRT1 mRNA.
(A) Luciferase assays were performed 24 h after transfection of the full-length 1.6 kb SIRT1 3'-UTR downstream of luciferase (SIRT1 3'-UTR) or constructs with 4 bp in the seed-binding regions mutated (SIRT1 3'-UTR 181mt, left panel; SIRT1 3'-UTR 9mt, right panel) and control, miR-181a, b, and c miRNA mimics (left panel) or pSuper and pSuper miR-9 expression constructs (right panel). Data are mean ± s.d. for eight experiments. (B-C) mESCs were transfected with individual miRNA expression constructs; protein and RNA were isolated 48 h later. (B) Repression of SIRT1 protein was analyzed by western blotting. Data are representative of six experiments. (C) qRT-PCR analysis of SIRT1 mRNA levels and mature miRNA levels. Data are mean ± s.d. for four samples.
Figure 5.
Figure 5.. Inhibition of miR-9 prevents downregulation of SIRT1 during mESC differentiation.
(A-C) mESCs were differentiated and transfected at d4 and d7 with LNA probes. Protein and RNA were isolated on indicated days. (A) qRT-PCR of miR-9 shows the expected upregulation during differentiation and 35% inhibition when embryoid bodies were transfected with LNA-miR-9 but not with LNA-SCR. (B) qRT-PCR show no significant change in SIRT1 mRNA levels. Data are mean ± s.d. for four samples and representative of three experiments. (C) Western blot analysis shows that the downregulation of SIRT1 protein during mESC differentiation was specifically inhibited in cells transfected with LNA-miR-9 but not by transfection of LNA-SCR or untransfected controls. Data are representative of four experiments. (D-F) EBs were dissociated and transfected at d6 with LNA probes. Protein and RNA were isolated on d11. (D) Western blot analysis shows upregulation of SIRT1 protein in EBs transfected with LNA-miR-9 but not LNA-SCR. (E) qRT-PCR analysis shows inhibition of miR-9 in EBs transfected with LNA-miR-9, but not with LNA-SCR, and no significant change in SIRT1 mRNA levels (F). Data are mean ± s.d. for four samples.
Figure 6.
Figure 6.. SIRT1 protein levels are upregulated during reprogramming.
(A) mESCs, MEFs, and iPS cells were subject to western blot analysis with antibodies to the indicated proteins. (B) SIRT1, HDAC1, and HDAC2 protein levels relative to tubulin were quantified by densitometry. (C) qRT-PCR analysis of SIRT1 mRNA levels in mESCs, MEFs, and iPS cells were measured relative to GAPDH. (D) qRT-PCR analysis of miRNA expression relative to miR-16 in mESCs, MEFs, and iPS. Data are mean ± s.d. for three samples.

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