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Han Peng, Spiro Getsios, Robert M. Lavker; By Targeting Factor-inhibiting Hypoxia-inducible Factor 1 (FIH-1), microRNA-31 Promotes Corneal Epithelial Transit Amplifying (TA) Cell Differentiation. Invest. Ophthalmol. Vis. Sci. 2011;52(14):326.
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© ARVO (1962-2015); The Authors (2016-present)
Central to corneal epithelial homeostasis is an orderly progression of relatively undifferentiated stem cell progeny (TA cells) into more mature TA cells. Regulation of early TA cell differentiation is incompletely understood but involves the activation of Notch receptor signaling. We have shown that microRNAs (miRNAs) play key roles in regulating corneal epithelial survival and migration. Our previous studies demonstrated that miRNA-31 (miR-31) was preferentially expressed in corneal epithelium when compared with other stratified epithelia. To elucidate how this miRNA functions in corneal epithelium, we have focused on FIH-1, a putative target of miR-31 that also dampens Notch signaling.
We used luciferase reporter assays in HeLa cells to validate potential targets of miR-31 identified by bioinformatics. To confirm these findings, we conducted gain- and loss-of- function experiments with miR-31 in conjunction with Northern, Western and real time PCR analyses using primary cultures of human corneal epithelial keratinocytes (HCEKs). These studies were complemented by ectopic expression of FIH-1 in submerged and organotypic raft cultures of human epithelia.
Luciferase assays confirmed that FIH-1 is a direct target of miR-31. Suppression of miR-31 in HCEKs following treatment with an antagomir (Antago-31) increased FIH-1 protein with a coordinated decrease of the Notch1 receptor. Consistent with the decline in Notch1 transcriptional activity, the expression of Hey2 also decreased. When FIH-1 levels were increased in HCEKs by Antago-31 treatment or ectopic expression of FIH-1, calcium-induced differentiation was disrupted, as evidenced by the lack of induction of keratin 3, involucrin and mucin1. Accordingly, organotypic raft cultures generated from keratinocytes retrovirally transduced with FIH-1 gave rise to an epithelium that was morphologically altered when compared with controls. In contrast, decreasing levels of FIH-1, by gene silencing promoted HCEK differentiation in low calcium.
Collectively, these observations define a novel intrinsic regulatory network for TA cell differentiation where Notch signaling potential is attenuated through a miR-31/FIH-1 nexus.
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