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William Samuel, Olga Postnikova, Cynthia Jaworski, Todd Duncan, T. Michael Redmond; Long Non-coding RNA LINC00276 is involved in the differentiation of human retinal pigment epithelial cells. Invest. Ophthalmol. Vis. Sci. 2020;61(7):1534.
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© ARVO (1962-2015); The Authors (2016-present)
ARPE-19, a widely used retinal pigment epithelial (RPE) cell line, is subject to loss of RPE-like phenotype after multiple passages. We have shown that appropriate differentiation of ARPE-19 fosters a phenotype and gene expression profile similar to native RPE. Along with microRNAs (miRNAs) that are known to influence RPE cell differentiation, a number of long non-coding RNAs (lncRNAs) were also identified by RNA-Seq analysis. In particular, the expression of LINC00276 was highly increased. In this study, we investigated the function of LINC00276 in the differentiation of ARPE-19 cells.
Total RNA extracted from ARPE-19 cells cultured in DMEM containing 1% FBS and 1 mM sodium pyruvate for 4-day or 4-month were used for analyzing the expression of RPE-specific mRNAs, miRNAs, and lncRNAs by RT-PCR, and proteins by western blotting. ARPE-19 cells transfected with antisense LINC00276 GapmeR or overexpression constructs were analyzed for the expression of mRNA and miRNA by RT-PCR. Dual-luciferase assays were performed using LINC00276 luciferase constructs with or without miRNA binding sites. Biotinylated miRNA mimics were used for RNA pull-down to identify their targets.
ARPE-19 cells cultured for 4 months exhibited native RPE phenotype along with expression of genes, proteins, and miRNAs preferentially expressed in RPE. RT-PCR analysis showed a >200-fold increase in LINC00276 expression in the differentiated cells. LINC00276 silencing decreased the expression of MITF, TRPM1, TRPM3, and miR-204/211 as well as other RPE-specific genes, while LINC00276 overexpression increased their expression. Addition of miR-211 mimic increased luciferase activity of the LINC00276 luciferase construct containing a miR-211 binding site whereas the miR-211 binding site deletion construct showed no luciferase activity. RNA pull-down using biotinylated miR-211 showed an increased LINC00276 expression by RT-PCR analysis.
ARPE-19 cells cultured for 4 months develop a phenotype characteristic of native RPE, and also express proteins, mRNAs, and miRNAs specific to RPE as well as a number of lncRNAs. Of these, alteration of LINC00276 expression modulates the expression of genes associated with RPE differentiation. Further, miRNAs that are involved in RPE differentiation regulate LINC00276 expression suggesting that LINC00276 may play a key role in regulating RPE characteristics.
This is a 2020 ARVO Annual Meeting abstract.
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