Genetic Ablation of MiR-22 Fosters Diet-Induced Obesity and NAFLD Development

miR-22 is one of the most abundant miRNAs in the liver and alterations of its hepatic expression have been associated with the development of hepatic steatosis and insulin resistance, as well as cancer. However, the pathophysiological roles of miR-22-3p in the deregulated hepatic metabolism with obesity and cancer remains poorly characterized. Herein, we observed that alterations of hepatic miR-22-3p expression with non-alcoholic fatty liver disease (NAFLD) in the context of obesity are not consistent in various human cohorts and animal models in contrast to the well-characterized miR-22-3p downregulation observed in hepatic cancers. To unravel the role of miR-22 in obesity-associated NAFLD, we generated constitutive Mir22 knockout (miR-22KO) mice, which were subsequently rendered obese by feeding with fat-enriched diet. Functional NAFLD- and obesity-associated metabolic parameters were then analyzed. Insights about the role of miR-22 in NAFLD associated with obesity were further obtained through an unbiased proteomic analysis of miR-22KO livers from obese mice. Metabolic processes governed by miR-22 were finally investigated in hepatic transformed cancer cells. Deletion of Mir22 was asymptomatic when mice were bred under standard conditions, except for an onset of glucose intolerance. However, when challenged with a high fat-containing diet, Mir22 deficiency dramatically exacerbated fat mass gain, hepatomegaly, and liver steatosis in mice. Analyses of explanted white adipose tissue revealed increased lipid synthesis, whereas mass spectrometry analysis of the liver proteome indicated that Mir22 deletion promotes hepatic upregulation of key enzymes in glycolysis and lipid uptake. Surprisingly, expression of miR-22-3p in Huh7 hepatic cancer cells triggers, in contrast to our in vivo observations, a clear induction of a Warburg effect with an increased glycolysis and an inhibited mitochondrial respiration. Together, our study indicates that miR-22-3p is a master regulator of the lipid and glucose metabolism with differential effects in specific organs and in transformed hepatic cancer cells, as compared to non-tumoral tissue.


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Gene targeting in ES cells and blastocyst injection G4 embryonic stem cells (derived from mouse 129S6/C57BL/6Ncr) were cultured on neomycin-resistant primary embryonic fibroblast feeder layers, and 10 7 cells were electroporated with 30 µg of the linearized targeting construct. After electroporation, the cells were plated on 100-mm culture dishes and exposed to G418 (300 µg/ml; Sigma). Colonies were picked after 7-9 days selection and grown on 96-wells plate. DNA isolated from ES cell clones targeted with the construct was screened by long-range PCR, which produced a 2693 bp fragment with a primer pair corresponding to 5' end of the Mir22 gene (M22ges1: TCCCTATACCCCGAAGACCT) and 3'end of PGK-Neo cassette (Neoneoan: GGCGGATTTGTCCTACTCAGG). Up to 288 clones were picked and screened by PCR, and two clones containing a correct size fragment were then confirmed by sequencing. In order to delete the Neo-cassette included in the targeting vector, the once targeted ES cells were reelectroporated with the plasmid expressing the Frt-recombinase (pCAGGS-Flpo-IRES-puro).
After electroporation, the cells were plated on 100-mm culture dishes and colonies were picked up after 3-5 days growth and grown on 96-wells plate. Four correct clones were identified from the 96 clones analyzed, and the right clones were further confirmed by sequencing.
The targeted ES cells with the two loxP sites inserted were injected into C57BL/6N mouse blastocysts to generate chimeric mice. Germline transmission was achieved by crossbreeding male chimeras with C57BL/6N females. C57BL/6N mice used as blastocyst donors were obtained from Charles River Laboratories (Willmington, MA). The mice were maintained in a specific pathogen-free stage at Central Animal Laboratory at the University of Turku. All studies carried out with the mice were approved by The Finnish ethical committee for experimental animals, complying with international guidelines on the care and use of laboratory animals.

Generation of constitutive miR-22KO mice
To generate constitutive mutants for Mir22, we disrupted Mir22 in the male germline by combining a conditional Mir22 floxed allele with a germ-cell specific Ngn3 promoter-driven transgenic Cre line (1). The genotype of male gametes produced by this double transgenic line was miR-22 KO. Mice heterozygous for the Mir22 constitutive KO were viable and fertile and subsequent matings with these animals resulted in the expected ratio of 25% homozygous mutant mice, hereafter referred to as "miR-22KO".
Mice with constitutive Mir22 deletion and CTL mice were first generation from littermate couple in open cages in non-SPF facilities.

Quantitative PCR (RT-qPCR) analyses
Total RNA from the liver was extracted with Trizol reagent (Ambion, Thermo Scientific, USA) according to the manufacturer instructions. For microRNAs, reverse transcription was performed with 500ng of total RNA using Poly A polymerase and the Protoscript II Retrotranscriptase (New England Biolabs, USA), Sno-202, and miR-16 were used to normalize gene expression.

Biochemical analyses in plasma and tissue
Plasma levels of triglycerides (TG), cholesterol, and transaminases were determined by an automated Abott Architect analyzer (Abott Architect, Paris, France). Non-esterified fatty acids measurement was assessed using Cayman Free Fatty Acid Assay Kit. Insuline and C-peptide were determined using mouse insulin Elisa (Mercodia, Sweden) and mouse C-peptide Elisa (Merck-Millipore, USA) respectively. Triglycerides (TG) extraction from cryo-preserved hepatic samples or HepG2 cells was performed by hexane/isopropanol. TG content was then measured by colorimetric enzymatic analysis with commercial kits (TG Roche/Hitachi, Roche, Switzerland).

Metabolic phenotyping, EchoMRI analyses, and CT-scan imaging
An EchoMRI-700 quantitative nuclear magnetic resonance analyser (Echo Medical Systems, USA) was used to measure total fat and lean mass. Distribution, volume, and weight of fat depots were analysed by a multidetector CT-scan (Discovery 750 HD, GE Healthcare, Milwaukee, USA) and quantified with Osirix software (Switzerland). Metabolic phenotyping of mice was performed using a LabMaster system (GmbH Berlin, Germany) after 5 days of adaptation prior to recording.

Glucose tolerance tests
Glucose tolerance tests (GTT) were performed as previously described (2). Briefly, mice were starved overnight and a glucose load of 1.5g/kg was administered i.p. Blood samples were collected at indicated times, and the glucose level measured using Glucotrend Active (Roche, Switzerland).

Histological Analyses
Liver samples were fixed in 4% paraformaldehyde and after dehydration and embedment in paraffin, 5-µm-thick sections were stained with hematoxylin/eosin (H&E) for morphological investigations.

Sample preparation for LC-MS/MS
Liver tissues were resuspended in four volumes of PBS, homogenized with a dounce homogenizer and centrifuged at 1'400 x g for five min. at 4°C. The supernatant was centrifuged at 100'000 x g for one hour at 4°C yielding a soluble and membrane proteome. Proteins were precipitated by the addition of four volumes of methanol, one volume of chloroform, and three volumes of water. Samples were centrifuged at 20'000 x g for five min. at 4°C and the methanol/water and chloroform layers were removed. Proteins were washed with four volumes of methanol and centrifuged once more. Proteins were dried and resuspended in PBS.
Proteomes (15 µg) were denatured with 6 M urea in 50 mM NH4HCO3, reduced with 10 mM TCEP for 30 min. and alkylated with 25 mM iodoacetamide for 30 min. in the dark. Samples were diluted to 2 M Urea with 50 mM NH4HCO3, and digested with trypsin (Promega, 1 µL of 0.5 µg/µL) in the presence of 1 mM CaCl2 for 12 hours at 37 °C. Samples were acidified to a final concentration of 5% acetic acid, desalted over a self-packed C18 spin column, and dried.
Samples were analyzed by LC-MS/MS (see below) and the MS data was processed with MaxQuant (see below).

LC-MS/MS analysis
Peptides were resuspended in water with 0.1% formic acid (FA) and analyzed using Proxeon Dynamic exclusion was set to 10 s, peptide match to prefer and isotope exclusion was enabled.

MaxQuant analysis
The MS data was analysed with MaxQuant(3) (V1.5.2.8) and searched against the mouse proteome (Uniprot) and a common list of contaminants (included in MaxQuant). The first peptide search tolerance was set to 20 ppm, 7 ppm was used for the main peptide search and fragment mass tolerance was set to 0.02 Da. The false discovery rate for peptides, proteins, and sites identification was set to 1%. The minimum peptide length was set to 6 amino acids and peptide re-quantification, label-free quantification (MaxLFQ), and "match between runs" were enabled. The minimal number of peptides per protein was set to two. Oxidized methionines and N-terminal acetylation were searched as variable modifications and carbamidomethylation of cysteines was searched as a fixed modification.

Identification of lipid/glucose metabolism-associated proteins differentially expressed in the proteomic analysis and potentially regulated by miR-22-3p
The list of upregulated proteins in miR22KO mice identified in our proteomic analysis was crossreferenced with a list of potential mouse miR-22-3p targets retrieved from the miRWalk 2.0 database. The 54 candidates identified were then segregated by biological processes based on literature. Finally, protein candidates involved in lipid or glucose metabolism were represented in a heatmap (Log2 fold Change) with a hierarchical clustering for the samples based on the Euclydian distance, using Multiple Viewer experiment (MeV) software.

GEO Datasets Analyses
The Gene Expression Omnibus database (http://www.ncbi.nlm.nih.gov/gds/?term=hCC) was used to retrieve transcriptomic datasets of human or mouse models of NAFLD. Fold Change differences and statistical differences between experimental groups were calculated with the GEO2R tool. The results were annotated significant if the p-value was lower to 0.05.

Gene Ontology enrichment analysis
Overrepresented GO terms (biological processes or tissue expression) were identified using DAVID 6.7 database (https://david.ncifcrf.gov/). Functional enrichment analyses were performed on potential miR-22-3p targets or differentially expressed proteins between control and miR22KO mice fed HFD. Enrichments are represented with an EASE score (Modified Fisher's exact test). GO terms having a p-value<0.05 were considered significant.

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Expression of miRNA in liver tissues and hepatic cell lines.
The miRmine database (http://guanlab.ccmb.med.umich.edu/mirmine/) was used to retrieve the expression levels of miRNAs in human liver tissues and HepG2 cells. Relative miRNA expressions are expressed as Reads Per Million Reads (RPM).

List of predicted and validated miR-22-3p targets
The miRWalk 2.0 database (http://zmf.umm.uniheidelberg.de/apps/zmf/mirwalk2/custom.html) was used to retrieve the list of predicted and validated miR-22-3p targets in human and mouse. Only targets recognized by at least 3 different databases were considered for further analyses.

Interactome analysis.
The interactions between proteins deregulated between control and miR-22KO mice fed HFD (proteomic analysis) were determined using the STRING (http://string-db.org) database.

Western Blot Analyses
Tissues were homogenized with tissue lyser II (Qiagen®, Germany) and cells were lysed in ice-cold RIPA buffer. Equal amounts of proteins were resolved by 5-20% gradient SDS-PAGE and blotted to nitrocellulose membranes. Proteins were detected with specific primary antibodies and HRP-conjugated secondary antibodies using enhanced chemoluminescence (ECL select, Amersham, GE healthcare, UK) (List of antibodies in Supplementary Table).
Chemoluminescence signal was detected using PXi/PXi Touch from Syngene (Synoptics group, Cambridge, UK). Quantifications were performed using the Syngene Software.