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MicroRNA-31 targets FIH-1 to positively regulate corneal epithelial glycogen metabolism.

ABSTRACT: Corneal epithelium relies on abundant glycogen stores as its primary energy source. MicroRNA-31 (miR-31), a corneal epithelial-preferred miRNA, negatively regulates factor inhibiting hypoxia-inducible factor-1 (FIH-1). Since HIF-1? is involved in anaerobic energy production, we investigated the role that miR-31 and FIH-1 play in regulating corneal epithelial glycogen. We used antagomirs (antago) to reduce the level of miR-31 in primary human corneal epithelial keratinocytes (HCEKs), and a miR-31-resistant FIH-1 to increase FIH-1 levels. Antago-31 raised FIH-1 levels and significantly reduced glycogen stores in HCEKs compared to irrelevant-antago treatment. Similarly, HCEKs retrovirally transduced with a miR-31-resistant FIH-1 had markedly reduced glycogen levels compared with empty vector controls. In addition, we observed no change in a HIF-1? reporter or known genes downstream of HIF-1? indicating that the action of FIH-1 and miR-31 on glycogen is HIF-1?-independent. An enzyme-dead FIH-1 mutation failed to restore glycogen stores, indicating that FIH-1 negatively regulates glycogen in a hydroxylase-independent manner. FIH-1 overexpression in HCEKs decreased AKT signaling, activated GSK-3?, and inactivated glycogen synthase. Treatment of FIH-1-transduced HCEKs with either a myristolated Akt or a GSK-3? inhibitor restored glycogen stores, confirming the direct involvement of Akt/GSK-3? signaling. Silencing FIH-1 in HCEKs reversed the observed changes in Akt-signaling. Glycogen regulation in a HIF-1?-independent manner is a novel function for FIH-1 and provides new insight into how the corneal epithelium regulates its energy requirements.

SUBMITTER: Peng H

PROVIDER: S-EPMC3405266 | biostudies-other | 2012 Aug

REPOSITORIES: biostudies-other

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MicroRNA-31 targets FIH-1 to positively regulate corneal epithelial glycogen metabolism.

Peng Han H Hamanaka Robert B RB Katsnelson Julia J Hao Liang-Liang LL Yang Wending W Chandel Navdeep S NS Lavker Robert M RM

FASEB journal : official publication of the Federation of American Societies for Experimental Biology 20120424 8

Corneal epithelium relies on abundant glycogen stores as its primary energy source. MicroRNA-31 (miR-31), a corneal epithelial-preferred miRNA, negatively regulates factor inhibiting hypoxia-inducible factor-1 (FIH-1). Since HIF-1α is involved in anaerobic energy production, we investigated the role that miR-31 and FIH-1 play in regulating corneal epithelial glycogen. We used antagomirs (antago) to reduce the level of miR-31 in primary human corneal epithelial keratinocytes (HCEKs), and a miR-31 ...[more]

PMID: 22532441

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Project description:BACKGROUND:Epigenetic factors, such as microRNAs, are important regulators in the self-renewal and differentiation of stem cells and progenies. Here we investigated the microRNAs expressed in human limbal-peripheral corneal (LPC) epithelia containing corneal epithelial progenitor cells (CEPCs) and early transit amplifying cells, and their role in corneal epithelium. METHODOLOGY/PRINCIPAL FINDINGS:Human LPC epithelia was extracted for small RNAs or dissociated for CEPC culture. By Agilent Human microRNA Microarray V2 platform and GeneSpring GX11.0 analysis, we found differential expression of 18 microRNAs against central corneal (CC) epithelia, which were devoid of CEPCs. Among them, miR-184 was up-regulated in CC epithelia, similar to reported finding. Cluster miR-143/145 was expressed strongly in LPC but weakly in CC epithelia (P?=?0.0004, Mann-Whitney U-test). This was validated by quantitative polymerase chain reaction (qPCR). Locked nucleic acid-based in situ hybridization on corneal rim cryosections showed miR-143/145 presence localized to the parabasal cells of limbal epithelium but negligible in basal and superficial epithelia. With holoclone forming ability, CEPCs transfected with lentiviral plasmid containing mature miR-145 sequence gave rise to defective epithelium in organotypic culture and had increased cytokeratin-3/12 and connexin-43 expressions and decreased ABCG2 and p63 compared with cells transfected with scrambled sequences. Global gene expression was analyzed using Agilent Whole Human Genome Oligo Microarray and GeneSpring GX11.0. With a 5-fold difference compared to cells with scrambled sequences, miR-145 up-regulated 324 genes (containing genes for immune response) and down-regulated 277 genes (containing genes for epithelial development and stem cell maintenance). As validated by qPCR and luciferase reporter assay, our results showed miR-145 suppressed integrin ?8 (ITGB8) expression in both human corneal epithelial cells and primary CEPCs. CONCLUSION/SIGNIFICANCE:We found expression of miR-143/145 cluster in human corneal epithelium. Our results also showed that miR-145 regulated the corneal epithelium formation and maintenance of epithelial integrity, via ITGB8 targeting.

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MicroRNA-31 targets FIH-1 to positively regulate corneal epithelial glycogen metabolism.

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MicroRNA-31 targets FIH-1 to positively regulate corneal epithelial glycogen metabolism.

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