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William Samuel, Todd Duncan, Olga Postnikova, Cynthia Jaworski, R Krishnan Kutty, T. Michael Redmond; Long Non-coding RNA LINC00276 is involved in the differentiation of human retinal pigment epithelial cells. Invest. Ophthalmol. Vis. Sci. 2018;59(9):5382. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
The retinal pigment epithelial (RPE) cell line ARPE-19 is widely used and provides a dependable alternative to native RPE, but is subject to loss of RPE-like phenotype after multiple passages. Recently, we have shown that appropriate differentiation of ARPE-19 fosters a phenotype and gene expression profile similar to native RPE. While microRNAs (miRNAs) have been shown to influence RPE cell differentiation, the role of long non-coding RNAs (lncRNAs) in RPE differentiation remains poorly understood. In this study, we investigated the function of LINC00276 in the differentiation of ARPE-19 cells.
Total RNA extracted from 4-day or 4-month cultured ARPE-19 cells were used for RNA-Seq analysis. The reads from RNA-Seq were aligned to the human genome version hg19 to identify lncRNAs that are differentially expressed. Expression of lncRNAs, RPE-specific mRNAs and miRNAs were assessed by RT-PCR, and proteins by western blotting. LINC00276 expression in ARPE-19 cells was modulated by transfecting them with antisense LINC00276 GapmeR or overexpression plasmid constructs.
ARPE-19 cells cultured for 4 months differentiated into a classic native RPE phenotype with dark pigmentation along with expression of genes/proteins preferentially expressed in RPE such as RPE65, RDH5, RDH10, and miR-204/211. RNA-Seq analysis provided a comprehensive view of the relative abundance and differential expression of lncRNAs in the differentiated ARPE-19 cells. Of the 403 lncRNAs with detectable signals, 82 lncRNAs were upregulated, and 37 were down-regulated with a fold change of ≥ 2.5 in cells grown for 4 months compared to cells grown for 4 days. Further analysis by RT-PCR showed that the expression of LINC00276 was increased >200-fold in 4-month cells compared to 4-day cells. In addition, LINC00276 silencing decreased the expression of MITF, TRPM1, TRPM3, and miR-204/211, while LINC00276 overexpression increased their expression.
A number of lncRNAs were differentially expressed during development of RPE characteristics in ARPE-19 cells cultured for 4 months. Of these, LINC00276 is increased ≥ 200-fold in differentiated ARPE-19 compared to undifferentiated cells. We found that alteration of LINC00276 expression modulates the expression of genes associated with RPE differentiation, suggesting that LINC00276 may play a key role in regulating acquisition of RPE characteristics.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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