﻿OCR and ECAR data were then analyzed and plotted based upon the averages and standard deviations of all measurements

﻿OCR and ECAR data were then analyzed and plotted based upon the averages and standard deviations of all measurements. here support the hypothesis that miR-211 loss in melanoma cells causes abnormal regulation of energy metabolism, which in turn allows cancer cells to survive under low oxygen concentrationsa condition that generally kills normal cells. These findings highlight a novel mechanism of melanoma formation: miR-211 is a molecular switch that is turned off in melanoma cells, raising the hope that in the future we might be able to turn the switch back on, thus providing a Fluocinonide(Vanos) better treatment option for melanoma. INTRODUCTION Melanoma is the leading cause of skin cancer deaths in the United States. Invasive melanoma is recalcitrant to most existing chemotherapies, CD246 and there is an urgent need to understand the molecular regulatory pathways that contribute to melanoma formation and progression. A hallmark of most cancer cells, including melanoma cells, is their ability to reroute energy provision and consumption to support the demands associated with pathological growth and survival (1,C8). For instance, Scott and colleagues (9) subjected normal melanocytes and melanoma cell lines to a partial systems level metabolic analysis and confirmed that melanoma cell lines exhibit the Warburg effect, that is, increased glycolysis and lactic acid fermentation in favor of aerobic glycolysis. Here we present evidence for a novel molecular switch driven by a microRNA (miRNA), which controls the Warburg effect in melanocytes and melanoma cells. We and others have identified several miRNAs responsible for the development and progression of melanomas, with miRNA 211 (miR-211) as an important tumor suppressor candidate (10,C16): miR-211 expression is significantly lower in nonpigmented melanoma cells and clinical melanoma samples than in normal melanocytes, and ectopic expression of miR-211 in melanoma cells reverses the high growth rate and invasiveness of melanoma cells (10, 13, 14). miR-211 has several putative target genes, including the calcium-activated potassium channel subunit -1 gene ((10, 13, 17, 18). We hypothesized that miR-211 might exert some of its effects by altering melanoma cell metabolism, such that when this miRNA is expressed the melanoma cells might lose some aspects of their cancer-specific metabolic state. To explore this, we characterized melanoma cells that ectopically expressed miR-211 using deep sequencing and mass spectrometry (MS). We report that miR-211-expressing melanoma cells exhibit increased oxygen consumption and contain elevated numbers of mitochondria compared Fluocinonide(Vanos) to melanoma cells that do not express miR-211. The metabolic alterations are causally related to downregulation of a previously unidentified miR-211 target gene, that for pyruvate dehydrogenase (PDH) kinase 4 (expression. Thus, miR-211 is likely to be an important regulator of melanocyte metabolism, and its loss of expression appears to be an epochal event during melanomagenesis and melanoma progression. MATERIALS AND METHODS Cell lines and tissue culture conditions. Cell lines examined in this study included the melanoma cell lines A375 (melanoma stage 4; Fluocinonide(Vanos) American Type Culture Collection) and WM1552C (melanoma stage 3; ATCC CRL-2808), as well as the human epidermal melanocyte cell line HEM-l (catalog no. 2200; ScienCell). All cell lines were maintained and selected as previously described by Mazar et al. (14). Western blot analysis. Western blot assays were performed using cell lysates under the same conditions as those described by Mazar et al. (14). Blots were probed with the following primary antibodies and dilutions: anti-HIF-1 (catalog no. NB100-105; Novus Biologicals) at 1/500, anti-PDK4 (catalog no. AP7041b; Abgent) at 1/250, anti-ERR (catalog no. D-1:sc-393969; Santa Cruz), anti-RUNX2 (catalog.

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