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Robert M. Lavker, Robert Hamanaka, Navdeep S. Chandel, Han Peng; microRNA-31 Targets Factor-inhibiting Hypoxia-inducible Factor 1 (FIH-1) to Positively Regulate Glycogen in the Corneal Epithelium. Invest. Ophthalmol. Vis. Sci. 2011;52(14):4389.
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Corneal epithelial cells have large stores of glycogen, which serve as their primary energy source. Our knowledge of corneal epithelial energy metabolism comes primarily from gene regulation and protein analyses. MicroRNAs (miRNAs), which function at the interface of these two systems, have only recently begun to receive attention. Herein, we report that miRNA-31 (miR-31), which is routinely expressed in unperturbed corneal epithelium, functions to regulate glycogen metabolism.
Potential targets of miR-31 identified by bioinformatics were validated by luciferase reporter assays in HeLa cells. To confirm these findings, we conducted gain- and loss-of- function experiments with miR-31 in human corneal epithelial keratinocytes (HCEKs), in conjunction with Northern, Western, immunostaining, and real time PCR analyses. These investigations were complemented by metabolic studies, which focused on cellular respiration.
Luciferase assays confirmed that FIH-1 is a direct target of miR-31. Suppression of miR-31 in HCEKs by treatment with an antagomir (Antago-31) increased FIH-1 protein, whereas ectopic expression of miR-31 reduced FIH-1 protein. One of the functions of FIH-1 is to regulate the stability of HIF-1α so that in normoxia, HIF-1α function is repressed. Consistent with such repression, transcriptional activity of HIF-1α and its gene target, Glut1, also decreased. When levels of FIH-1 were increased in HCEKs by Antago-31 treatment, glycogen stores were reduced by ~50%. Retroviral transduction of HECKs with a miR-31-resistant FIH-1 similarly reduced glycogen stores, confirming a miR-31/FIH-1/glycogen relationship. To determine the effects of miR-31 on metabolic activity, we measured extracellular acidification rates (ECAR) resulting from glycolysis and glucose utilization. Accordingly, ECARs of Antago-31 treated HCEKs were significantly lower than irrelevant Antago-treated HCEKs, indicative of lower glycolytic activity.
By defining miR-31 as a positive regulator of glycogen stores via a FIH-1 interaction, our results provide new information on how the corneal epithelium regulates its energy supply.
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