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T. Michael Redmond, Cynthia Jaworski, William Samuel, R Kutty, Todd Duncan, Toral Parikh; Analysis of microRNA expression changes following explanation of bovine RPE. Invest. Ophthalmol. Vis. Sci. 2013;54(15):283 - D0232.
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© ARVO (1962-2015); The Authors (2016-present)
An enduring challenge in studying retinal pigment epithelium (RPE) is the limited quantity of tissue available. Although cultured RPE cells are widely used, the phenotypes of cultured cells, with time, differ substantially from native tissue. For example, many cultured RPE cells do not express RPE65 protein, a distinguishing feature of RPE. To better understand this alteration in gene expression, in particular the loss of key visual cycle proteins, we asked whether the expression pattern of microRNAs (miRs) is altered after explantation, in concert with the changes in gene and protein expression.
RPE was harvested from freshly dissected bovine eyes and placed in tissue culture. RNA and protein were extracted by standard means from fresh tissue and from cells harvested after 4 or 8 weeks in culture. MiR expression was assessed using the miRCURY LNA miR Array (Exiqon Services). Quantified signals were normalized. Expression levels of selected miRs were validated by real time RT-PCR.
Principal component analysis indicates that samples cluster according to their time in culture. Hierarchical clustering reveals the specific miRs comprising each cluster. Nineteen miRs were significantly different (p-value less than 0.05 with Bonferroni multiple testing) between native tissue and 4-week cultures; 78 differences were found between native tissue and 8-week cultures. There were 28 microRNAs that differed between the 4 and 8 week culture samples. Several miRs show evidence of a transient change of expression at 4 weeks, returning to levels similar to that of native tissue upon more prolonged culture time. Interestingly, the “sensory cluster” of miR-96, -182, and -183 all had diminished expression with culture, as did mir-204 and -211, reported to be characteristic of RPE. Real time RT-PCR of selected miRs corroborated the expression profiles. Expression of miR-21 and -184 rose markedly with growth in culture; sensory cluster miR levels fell.
The pattern of microRNA expression changes significantly when bovine RPE cells are explanted and grown in culture, suggesting that this class of regulatory molecules may be important in producing the altered culture phenotype and/or in maintaining the characteristics that define a functional in situ RPE cell. Analysis of potential gene targets showing increase or decrease will help us discriminate between miRs regulating these opposing trends.
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