Molecular Characterization of Two Human Lens Epithelial Cell Lines: HLE-B3 And SRA01/O4
Date
2016-05
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
University of Delaware
Abstract
The ocular lens is a transparent tissue that focuses light on the retina, allowing high-resolution vision. The loss of lens transparency causes an eye defect termed cataract, which is the leading cause of blindness worldwide. Half of the U.S. population above age 80 is affected by cataracts, for which surgical treatment is the principal therapeutic intervention. In 2014, over 3.6 million cataract surgeries were performed in the United States, and over 20 million surgeries were performed globally. Age-related cataracts are caused by a variety of stress conditions including elevated oxidative stress, increased sugar (diabetes), smoking or environmental insults such as exposure to ultraviolet (UV) radiation. Thus, characterization of biological and environmental factors that affect lens transparency is the first critical step toward the development of new therapies for cataracts. Recent discoveries have revealed a surprising link between proteins that are components of cytoplasmic RNA granules (RGs) and mammalian cataract. RGs like Processing Bodies (PBs) and Stress Granules (SGs) represent specialized cytoplasmic sites for regulation of RNA in the control of gene expression. PBs, the constitutive class of RGs, are RNA-protein complexes that silence or channel mRNA to decay, and therefore function to regulate the cellular proteome. SGs form as an early reaction to cellular stress, specifically allowing the translation of proteins that function in homeostasis. To investigate RG function in lens cells, we molecularly characterized two human lens epithelial cell lines (LECs), SRA01/04 and HLE-B3. These previously uncharacterized cell lines can serve as valuable in vitro tools for investigating the intricate molecular basis for human cataractogenesis. It is important in cell culture studies to validate the origin and identity of the cell line. Therefore, our first step was to confirm that the cell lines were of human origin. Analyzing genome-specific Short Tandem Repeats by PCR, we authenticated that the LEC lines are indeed human-derived. We next analyzed gene expression in these LECs by high throughput Illumina microarray analysis to investigate the extent of their retention of lens-like character. The microarray data demonstrates that both LECs retain expression of genes that are expressed and/or enriched in normal lens epithelial cells. Using a bioinformatics tool “iSyTE” developed to predict genes implicated in lens biology and cataract, we further validated the expression of cataract associated genes in these LECs using RT-PCR. Furthermore, we find that both LECs support formation of RGs, and exhibit formation of SGs under various stress conditions such as chemical and UV-stress. Thus, these studies present the first cellular models for investigating the molecular biology of UV-radiation exposure in lens epithelial cells, and therefore represent a new resource for study of factors that are linked to age-associated cataract in humans.
Description
Keywords
cell lines, molecular characterization, biology, biological sciences