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H. Miyashita, S. Shimmura, M. Kubota, K. Tsubota; Effect of Hypoxia Inducible Factor 2 Alpha on the Proliferation of Human Corneal Epithelial Cells in vitro. Invest. Ophthalmol. Vis. Sci. 2009;50(13):4608.
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In our previous study, hypoxic culture (under 2% O2) simulated the proliferation and inhibited the differentiation of human corneal limbal epithelial cells (HLEC). Hypoxia inducible factor (HIF) alpha are stabilized under hypoxia, dimerized with HIF1b, and acted as a transcriptional factor. Ubiquitous expressed HIF1a inhibits the proliferation and tissue-specific HIF2a stimulates the proliferation. In this study, we investigated the influence of HIF on corneal epithelial cells by using siRNA knockdown technology.
HLEC and SV40-transformed human corneal epithelial cell line (HCE-T) were cultured in serum-free low calcium medium (defined KSFM, Invitrogen) and serum-containing medium (supplemented hormonal epithelial medium), respectively. Cells were cultured under 21 % O2 or 2 % O2 by using multi-gas incubator. RT-PCR analysis was performed to determine HIF expressed in corneal epithelial cells. To confirm the effect of each HIF, 5000 cells were seeded in 96 well plate, and transfected with 5 nM of siRNA against each HIF (Qiagen). siRNA not homologous to mammalian gene was used as negative control (Qiagen). The effect of siRNA on target mRNA was analyzed by real time RT-PCR, and the effect on proliferation was measured by using nucleic acid-binding fluorescence dye (CyQuant NF, Invitrogen).
HLEC and HCE-T expressed HIF1a, HIF1b, and HIF2a, but not HIF3a mRNA. siRNA against human HIF1a and HIF2a decreased the target mRNA level in HCE-T by 19% and 21%, respectively. HIF1a siRNA tended to increase the proliferation of HLEC. In contrast, HIF2a siRNA significantly decreased the proliferation of HLEC in compared to control siRNA (n=3, p<0.05, Student t test). HCE-T proliferation was also inhibited by HIF2a siRNA.
HIF2a is expressed in human corneal epithelial cells and may stimulate the proliferation in vitro.
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