Cells were collected 48?h post\infection for RNA and cell bioenergetics assays. 2.8. change 1 with a < 0.05. Abstract Cancer cell survival and metastasis are dependent on metabolic reprogramming that is capable of increasing resistance to oxidative and energetic stress. Targeting these two processes can be crucial for cancer progression. Herein, we describe the role of microRNA\661 (miR661) as epigenetic regulator of colon cancer (CC) cell metabolism. MicroR661 induces a global increase in reactive oxygen species, specifically in mitochondrial superoxide anions, which appears to be mediated by decreased carbohydrate metabolism and pentose phosphate pathway, and by a higher dependency on mitochondrial respiration. MicroR661 overexpression in non\metastatic human CC cells induces an epithelial\to\mesenchymal transition phenotype, and a reduced tolerance to metabolic stress. This seems to be a general effect of miR661 in CC, since metastatic CC cell metabolism is also compromised upon miR661 overexpression. We propose hexose\6\phosphate dehydrogenase and pyruvate kinase M2 as two key players related to the observed metabolic reprogramming. Finally, the clinical Ethoxzolamide relevance of miR661 expression levels in stage\II and III CC patients is discussed. In conclusion, we propose miR661 as a potential modulator of redox and metabolic homeostasis in CC. (for miR661) and expression. Relative expression was calculated by the 2 2???Ct method. 2.5. Invasion assays Invasion assays were performed with BD Biosciences Matrigel? (Madrid, Spain) invasion chambers following manufacture indications. Images were captured using an Olympus CKX41 microscope (Olympus, Tokyo, Japan), with a 20 objective and analysis getit software (Olympus). 2.6. Dual\luciferase assays (cloning and co\transfection) Short sequences from the 3UTR of and mutated (mut)\short 3 UTR and and mut\short 3 UTR 50?000) were transfected using Lipofectamine??2000 (Invitrogen, Madrid, Spain) accordingly to the manufacturer’s recommendations. For a directional cloning, or 3\UTR\short\mut\were co\transfected with 30?nM of LNA\miR661 in HEK293T cells. The translational repression of upon miR661 binding was determined after transfection using the Dual\Luciferase Reporter Assay System (Promega Biotech Ibrica S.L., Madrid, Spain). Relative luciferase activity (Renilla luminescence/Firefly luminescence) was represented. 2.7. Lentivirus\mediated transient overexpression of PKM2 and H6PD HEK293T cells were transfected using Lipofectamine? 2000 (Life Technologies) with lentiviral vectors expressing and/or (DNA 2.0) along with a set of packaging plasmids (Addgene). DLD1 cells were infected with supernatants produced upon 48\h post\transfection in HEK293T cells and 4?gL?1 polybrene (Merck Millipore, Madrid, Spain) as coadjutant. Cells were collected 48?h post\infection for RNA and cell bioenergetics assays. 2.8. List of antibodies for western blot Primary antibodies were N\cadherin (333900, Cell Signaling Technology Europe Invitrogen, Leiden, the Netherlands); AMPK\ (Cell Signaling #2532); P\AMPK\ (Thr172) (40H9, Cell Signaling #2535); GSK\3 (27C19, Cell Signaling #9315); P\GSK\3 /(Ser21/9) (Cell Signaling #9331); PKM2 (Cell Signaling #3198); H6PD (C\10: sc\377180). \actin or vinculin were used as a loading controls as indicated. 2.9. l\lactate quantification l\lactate quantification was done using Caymans Glycolysis cell\based assay (Cayman, Ethoxzolamide Ann Arbor, MI, USA, 600450) (were measured with H2DCFDA (2,7\dichlorodihydrofluorescein diacetate) and MitoSOx Red (Invitrogen Molecular Probes, Madrid, Spain; “type”:”entrez-nucleotide”,”attrs”:”text”:”M36008″,”term_id”:”214108″,”term_text”:”M36008″M36008), respectively. The membrane potential was assayed by TMRN staining. Briefly, 105 cells were seeded in a 12\well plate and treated with the probes for 30?min. The cells were then washed with PBS and collected as single cell suspensions. PI staining was done to discriminate dead cells. Fluorescence was detected by flow cytometry. 2.11. Global metabolomic profile DLD1\miR661 and DLD1\Control cells were prepared as indicated Rabbit Polyclonal to POLR1C by Metabolon Inc. for global metabolomic analysis (Reitman Vimentin, Snailand Slugand < 0.05, **< 0.01, ***< 0.001. We also checked cell sensibility to Ethoxzolamide glucose deprivation. Glucose starvation inhibits the pentose phosphate pathway (PPP) which is required for NADPH production to detoxify ROS (Jeon and (Hewitt and were not affected by miR661 overexpression in SW620 cells compared with control cells (expression was used as a validated described target for miR661) (Fig.?S3C). In addition, we monitored invasion through BD\coated Matrigel Chambers and found only a slight, not significant, decrease in invasiveness of SW620\miR661 compared with SW620\Control (Fig.?S3D). It seems that miR661 does not alter the expression of EMT markers or the invasiveness capability of SW620 metastatic cancer cell line. Quantitative analysis of metabolism of CC cell lines have revealed that their reliance on glycolysis and/or mitochondrial respiration is cell line\dependent, which might be a consequence of the mutational status of the cancer cells (Zaytseva bioinformatic prediction of miR661 targets related to cell metabolism, indicated that PKLR and H6PD were the two main candidates. (B) qRT\PCR analysis of and levels as predicted targets in DLD1 and SW620 overexpressing miR661 compared with the corresponding controls. (C).