Abstract
Purpose: :
In our previous study, hypoxic culture (under 2% O2) stimulated the proliferation and inhibited the differentiation of human corneal limbal epithelial cells (HLEC). Hypoxia inducible factor alpha (HIF1A) are stabilized under hypoxia, dimerized with HIF1 beta (HIF1B)/ARNT, and acted as a transcriptional factor. Ubiquitous expressed HIF1a inhibits the proliferation and tissue-specific HIF2 alpha (HIF2A)/EPAS stimulates the proliferation. In this study, we investigated the influence of HIF on corneal epithelial cells by using siRNA knockdown.
Methods: :
HLEC and SV40-transformed human corneal epithelial cell line (HCE-T) were cultured in serum-free low calcium medium 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, five thousand cells were seeded in 96 well plate, and transfected with 5 nM of siRNA against each HIF. siRNA not homologous to mammalian gene was used as negative control. 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.
Results: :
HLEC and HCE-T expressed HIF1A, HIF1B, and HIF2A, but not HIF3A/IPAS mRNA. siRNA against human HIF1A and HIF2A decreased the target mRNA level in HCE-T by 19% and 21%, respectively. HIF1A siRNA did not affect on 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.
Conclusions: :
HIF2A is expressed in human corneal epithelial cells and may stimulate the proliferation in vitro.
Keywords: cornea: epithelium • hypoxia • proliferation