Purchase this article with an account.
Kwaku Osei, Champion Deivanayagam, Jason J Nichols; Hyperosmolar stress upregulates Glycoprotein 340 mRNA expression in human corneal epithelial cells. Invest. Ophthalmol. Vis. Sci. 2020;61(7):137.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Glycoprotein 340 (Gp340), encoded by the Deleted in Malignant Brain Tumor 1 (DMBT1) gene, is a normal component of tears and ocular surface tissues which is dysregulated in dry eye. To understand the potential role of Gp340 in dry eye, this study aimed to investigate the expression of DMBT1 in human corneal epithelial cells (HCECs).
SV40-transformed HCECs (Riken Cells, Ibaraki, Japan) were seeded at 3 × 105 cells/flask and grown to 70-80% confluency in supplemented DMEM/F12 media. The media was replaced with fresh basal media and the cells were incubated for 12 hours. The basal media was removed and the cells were exposed to a hyperosmolar media (500 mOsmol/kg) for 15 minutes, 1 hour, and 2 hours (n = 3 per exposure time). Cells were then harvested and stored at -80 °C until mRNA expression analysis. Cells exposed to 295 mOsmol/kg basal media for 2 hours served as the control. Total RNA isolated using RNeasy Mini Kit (Qiagen, Valencia, CA) was used to generate cDNA. Gp340 mRNA expression was analyzed by real-time PCR using Taqman assays for DMBT1 (Hs01069306_m1, Life Technologies) and 18s RNA (Hs99999901_s1, Life Technologies). The real-time PCR results were validated through qualitative (gel-based) RT-PCR. Real-time DMBT1 expressions were normalized to both 18SRNA and the control treatment, and presented as fold changes. For the qualitative RT-PCR, the intensities of all mRNA bands were quantified by densitometry and DMBT1 expression levels were normalized to 18S RNA. The difference in DMBT1 expression between each hyperosmolar stress exposure time and the control was analyzed using Mann-Whitney U test.
In response to HCEC exposure to hyperosmolar media (500 mOsmol/kg) for 15 minutes, 1 hour, and 2 hours; Gp340 mRNA overexpressed 2, 6.5, and 8-fold, respectively from real-time PCR (Figure 1). Analysis of qualitative RT-PCR data (Figure 2) further confirmed the proportional higher Gp340 mRNA expression in hyperosmolar-stressed cells relative to the control (Mann Whitney U test, P ≤ 0.05).
Hyperosmolar stress results in overexpression of Gp340 mRNA. It is, however, unknown if there is a corresponding upregulation of protein expression. Future studies will determine Gp340 protein expression in hyperosmolar stress and explore its potential role in dry eye pathophysiology.
This is a 2020 ARVO Annual Meeting abstract.
Real-time RT-PCR for Gp340 mRNA expression
Qualitative RT-PCR for Gp340 mRNA expression
This PDF is available to Subscribers Only