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Peipei Qi, Thanh V Hoang, Brad Wagner, Lin Liu, Aswati Subramanian, Panagiotis A. Tsonis, Chun Liang, Michael L Robinson; Expression and functional studies of miRNAs in lens development. Invest. Ophthalmol. Vis. Sci. 2017;58(8):1718.
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© ARVO (1962-2015); The Authors (2016-present)
This study aimed to provide a comprehensive comparison of miRNA expression between newborn mouse lens epithelial cells and lens fiber cells using small RNA-Seq. The role of several highly expressed candidate miRNAs (miR-1, miR-184 and miR-26) was then characterized in mice where these miRNAs were systematically deleted.
Total RNAs were extracted from newborn lens epithelial and fiber cell fractions and small RNAs were isolated by size-selection for 50bp-single-ended sequencing. Differential expression of miRNA was then analyzed using DESeq package. Knockout mice for miR-184 and for each of the three members of miR-26 family were generated using CRISPR/Cas9 genome editing via zygote microinjection. Histology staining was performed for gross morphological analysis on lenses of all knockout mice. Additionally, a series of miR-184 and miR-26 target genes were predicted using a combination of several bioinformatics tools. RT-qPCR assays were carried out to analyze the effects of miRNA deletion on the expression of these genes.
Our miRNA-Seq data analysis showed that the most abundant miRNAs in the lens include: miR-1-3p, miR-184-5p, miR-26a-5p, miR-204-5p, miR-211-5p and members of the let-7 family. Despite of high expression of miR-1-3p exclusively in lens fibers, no obvious morphological defects appeared in newborn single or double miR-1 (miR-1-1 and miR-1-2) knockout lenses. Mice deficient in miR-184 displayed no obvious morphological defects in the lens up to one year of age, but loss of miR-184 led to dysregulated gene expression. Deleting one or two of three miR-26 family members (miR-26a1, miR-26a2 and miR26b) failed to result in any obvious eye defects. However, loss of all three (6 alleles) of miR-26 family members caused severe nuclear cataract in mice over 6 weeks of age. Cataract-associated gene expression analysis showed that lenses lacking miR-26 exhibited increased Pten and EphA2 expression, but substantially reduced expression of Slc4a4 and Hsp25 relative to the control lenses.
This miRNA-Seq analysis provided a comprehensive view of both the relative abundance and differential expression of miRNAs from lens epithelial cells and lens fiber cells. Although no single miRNA gene deletion tested resulted in lens abnormalities, deletion of all six miRNA-26 alleles results in abnormal postnatal lens development and/or homeostasis.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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