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. 2016 Jun;15(3):572-81.
doi: 10.1111/acel.12469. Epub 2016 Mar 17.

Metformin-mediated increase in DICER1 regulates microRNA expression and cellular senescence

Nicole Noren Hooten  1 , Alejandro Martin-Montalvo  2   3 , Douglas F Dluzen  1 , Yongqing Zhang  4 , Michel Bernier  2 , Alan B Zonderman  1 , Kevin G Becker  4 , Myriam Gorospe  4 , Rafael de Cabo  2 , Michele K Evans  1
Affiliations

Metformin-mediated increase in DICER1 regulates microRNA expression and cellular senescence

Nicole Noren Hooten et al. Aging Cell. 2016 Jun.

Abstract

Metformin, an oral hypoglycemic agent, has been used for decades to treat type 2 diabetes mellitus. Recent studies indicate that mice treated with metformin live longer and have fewer manifestations of age-related chronic disease. However, the molecular mechanisms underlying this phenotype are unknown. Here, we show that metformin treatment increases the levels of the microRNA-processing protein DICER1 in mice and in humans with diabetes mellitus. Our results indicate that metformin upregulates DICER1 through a post-transcriptional mechanism involving the RNA-binding protein AUF1. Treatment with metformin altered the subcellular localization of AUF1, disrupting its interaction with DICER1 mRNA and rendering DICER1 mRNA stable, allowing DICER1 to accumulate. Consistent with the role of DICER1 in the biogenesis of microRNAs, we found differential patterns of microRNA expression in mice treated with metformin or caloric restriction, two proven life-extending interventions. Interestingly, several microRNAs previously associated with senescence and aging, including miR-20a, miR-34a, miR-130a, miR-106b, miR-125, and let-7c, were found elevated. In agreement with these findings, treatment with metformin decreased cellular senescence in several senescence models in a DICER1-dependent manner. Metformin lowered p16 and p21 protein levels and the abundance of inflammatory cytokines and oncogenes that are hallmarks of the senescence-associated secretory phenotype (SASP). These data lead us to hypothesize that changes in DICER1 levels may be important for organismal aging and to propose that interventions that upregulate DICER1 expression (e.g., metformin) may offer new pharmacotherapeutic approaches for age-related disease.

Keywords: AUF1; RNA-binding proteins; aging; caloric restriction; diabetes mellitus; microRNA.

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Figures

Figure 1
Figure 1
DICER1 levels are altered by human age and by metformin treatment in mice and humans. (A) DICER1 and DROSHA mRNA levels were quantified from PBMCs from young (~30 years) and old (~64 years) individuals (n = 14/group) from the HANDLS study using RT–qPCR. (B) DICER1 mRNA and DICER protein (C) levels were quantified by RT–qPCR and immunoblotting, respectively, from livers of mice on standard diet (SD), metformin‐treated (Met), or on calorie restriction (CR). For protein levels in C, a representative immunoblot is shown and Dicer levels from 8 mice per group were quantified from immunoblots and normalized to actin. (D) DICER1 mRNA levels were quantified from PBMCs of nondiabetics, diabetics taking sulfonylureas, and diabetics taking metformin (n = 20/group). Demographic information for cohorts in (A) and (D) is in Table 1. The histograms represent the mean + SEM. *P < 0.05, **P ≤ 0.01.
Figure 2
Figure 2
Metformin stabilizes DICER1 expression through modulating the binding of the RNA‐binding protein AUF1. (A) Serum‐starved HepG2 cells were treated with 500 μm metformin or PBS for 1 h and ribonucleoprotein (RNP) immunoprecipitation (RIP) assays followed by RT–qPCR analysis was used to measure the enrichment of DICER1 mRNA in AUF1 immunoprecipitates. Each IP was compared to control IgG IP and normalized to GAPDH mRNA levels. (B) HeLa cells were transfected with either AUF1 or Control (Con) siRNA and 24 h later, cells were treated with metformin for 24 h. The levels of HNRNPD mRNA (which encodes AUF1) and DICER1 mRNA were measured by RT–qPCR analysis and normalized to GAPDH mRNA levels. (C–E) Cells were transfected with either AUF1 siRNA‐1 (Qiagen), AUF1 siRNA‐2 (Santa Cruz) or the indicated FLAG‐tagged plasmids were treated as in (B), and lysates were analyzed by immunoblotting to assess protein levels of DICER1, AUF1 and actin using specific antibodies. Histograms represent the mean + SEM from three independent experiments. ***P < 0.001 or *P < 0.05 by Student's t‐test.
Figure 3
Figure 3
Metformin induces AUF1 nuclear retention. (A) Serum‐starved HeLa cells were treated with 500 μm metformin for 1 h. Cells were fixed and stained with an AUF1 antibody and DAPI. (B) AUF1 staining in the nucleus and cytoplasm was quantified and normalized to DAPI‐stained nuclei. (C) HeLa cells were pretreated with Compound C for 1 h or vehicle and were then subsequently treated with 500 μm metformin for 1 h. Cells were fractionated into cytoplasmic and nuclear fractions and analyzed by immunoblotting with anti‐AUF1, anti‐HSP70, anti‐Lamin B1 (nuclear marker), and anti‐GAPDH antibodies (cytoplasmic marker). (D) HeLa cells were transfected with AMPK siRNA or control and treated with metformin as described above. (E) HeLa cells transfected with individual isoforms of AUF1 were treated with or without metformin, and RIP assays were performed to measure the enrichment of DICER1 mRNA in FLAG immunoprecipitates. Each IP was compared to control FLAG IP, normalized to GAPDH mRNA levels, and then normalized to mock‐treated cells. (F) AUF1 immunoprecipitates from HeLa cells treated with 500 μm metformin for the indicated time points were probed with anti‐phospho‐AMPK substrate, anti‐phospho‐serine/threonine, anti‐HSP70, and anti‐AUF1 antibodies. Black arrows indicate AUF1 phosphorylation, and red arrow indicates HSP70 phosphorylation. Histograms in B and E represent the mean + SEM from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 by Student's t‐test.
Figure 4
Figure 4
miRNA expression changes in mice treated with metformin or caloric restriction. (A) miRNAs were isolated from livers of mice on standard diet (SD), metformin (Met; 0.1%), or on caloric restriction (CR). Using total liver RNA, miRNA microarray analysis was performed. Heat map indicating miRNAs significantly changed in any of the indicated comparisons (z ratio). (B) RNA was isolated from livers from mice on standard diet (SD), treated with metformin (Met), or on caloric restriction (CR). Mature miRNA (B) or pri‐miRNA levels (C) were quantified by RT–qPCR analysis. The histograms represent the mean + SEM for SD (n = 5), Met (n = 5), and CR (n = 6). *P < 0.05 and **P < 0.01 compared with SD by Student's t‐test.
Figure 5
Figure 5
Inhibition of cellular senescence by metformin requires DICER1. Presenescent WI‐38 or IMR‐90 cells were transfected with either the indicated siRNAs or transfected with pDEST‐DICER1 and then either treated with PBS or with 500 μm metformin (Met) for 48 h and stained for SA‐β‐gal activity (A,B,D). (C) IDH4 cells cultured without dex were incubated with metformin. (D) WI‐38 and IMR‐90 cells exposed to ionizing radiation (IR) were treated with metformin. SA‐β‐gal‐positive cells were counted and normalized to the total number of cells. Representative phase‐contrast images from WI‐38 cells from (B) are shown, and the histograms represent the mean + SEM from 3 independent experiments. *P < 0.05, **P < 0.01 and **P < 0.01 by Student's t‐test. (F) Lysates from the indicated cell lines and treatments were analyzed for protein levels of senescence markers and actin for a protein loading control.
Figure 6
Figure 6
SASP mRNA levels are decreased by metformin. RNA was isolated from the indicated cell lines and treatments as detailed in Experimental procedures. mRNA levels of the indicated genes were quantified by RT–qPCR. IR, ionizing radiation; (−), mock‐treated (+), metformin‐treated.
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