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

Summary 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.

normalize mRNA levels from human cell lines. 18S was used for normalization in senescence experiments. Primer sequences are listed in Table S4.
For the analysis of mouse pri-miRNAs, RNA was reverse transcribed using the High-Capacity cDNA Reverse Transcription Kit (Life Technologies) according to the manufacturer's instructions.
Mouse primary miRNAs (pri-miRNAs) were quantified using Taqman Pri-miRNA primers and probes for mmu-miR-92a-1 (Assay ID Mm03306814_pri) and mmu-miR-130a (ID Mm03306263_pri). Taqman Gene Expression Master Mix (Life Technologies) was used for real-time RT-PCR on an Applied Biosystems 7500 Real-Time PCR machine. Primary miRNAs were normalized to Gapdh expression levels.

Cell fractionation and antibodies used for immunoblotting
To fractionate HeLa or WI-38 cell nuclear and cytoplasmic compartments, cells serum-starved for 18 hrs were pre-treated with 5 M Compound C or DMSO control for 1 hr and then treated for 1 hr with 500 M metformin or PBS and then fractionated into cellular and nuclear compartments using the NE-PER kit from Thermo Scientific according to manufacturer's instructions. An additional wash of the nuclear pellet with cold PBS was included to prevent cytoplasmic contamination into the nuclear fraction. Samples were analyzed by SDS-PAGE and immunoblotted as described above.

Immunoprecipitation of ribonucleoprotein (RNP) complexes
For ribonucleoprotein (RNP) immunoprecipitation (RIP) assays, cells were lysed for 10 min on ice in a buffer containing 20 mM Tris-HCl pH 7.5, 100 mM KCl, 5 mM MgCl 2 , 0.5% NP-40, RNaseOUT and protease inhibitors and then centrifuged at 10,000 g for 15 min at 4C. The supernatants were incubated with mouse IgG agarose beads (Sigma-Aldrich) that were precoated with anti-FLAG M2 antibodies or Gamma bind beads precoated with anti-AUF1 (Millipore; 07-260) or rabbit IgG (Santa Cruz Biotechnology) antibodies overnight at 4C. After extensive washing with ice-cold NT2 buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM MgCl 2 , 0.05% NP-40), the complexes were incubated with DNase I (RNase-free; Ambion) for 10 min at 30C and subsequently with 0.1% SDS, Proteinase K (0.5 mg/ml) for 15 min at 55°C to digest proteins present on the beads. RNA was extracted using acidic phenol, precipitated in the presence of glycoblue, and quantified by RT-qPCR analysis.

Immunofluorescence
HeLa cells plated on glass coverslips were serum-starved for 18 hrs and then treated for 1 hr with 500 M metformin or PBS. Cells were fixed in 3.7% formaldehyde in PBS for 15 min, permeabilized with 0.5% Triton X-100 in TBS for 3 min, washed in TBS, and then blocked for 1 hr in TBS containing 1% BSA and 10% goat serum. Cells were incubated with anti-AUF1 antibodies (Millipore) for 1 hr, washed, and incubated with secondary antibodies (Thermo Fisher Scientific) for 1 hr, washed and then stained with DAPI. Fluorescent images were taken on a Zeiss Observer D1 microscope with an AxioCam1Cc1 camera at a set exposure time.

microRNA microarray
We analyzed global microRNA expression from livers of the same cohort of mice (n=5 per group) whose global gene expression profile was previously reported (Accesssion Number: GSE40936)(Martin- Montalvo et al. 2013). Total RNA including miRNAs was isolated using the Absolutely RNA miRNA Kit (Agilent) and analyzed using the Agilent Mouse miRNA Microarray 15.0. Microarray was performed and analyzed as previously described (Noren Hooten et al. 2013). Individual miRNAs with pairwise z-test p value < 0.05, absolute value of Z ratio >1.5 , with fdr < 0.3 were considered significantly changed. The microRNA microarray data can be accessed at GEO (Accession Number: GSE73393). miRNA expression information from heat map are in Table S2.