Angiogenic and Anti-Angiogenic Factors in human RPE

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Purpose: MicroRNAs (miRNAs) regulate a significant portion of the transcriptome. Over 400 vertebrate miRNAs have now been identified and their targets have been predicted to cover approximately 30% of the genome. As a first step in understanding the function of miRNA in the human eye, miRNA expression was profiled in three ocular tissues. Methods: Total RNA was isolated using mirVana miRNA isolation kit and collected in small and large RNA fractions (<, >200nt). Small RNA fractions from cultured human fetal RPE (hfRPE, n=1) and native retina, RPE and choroid of human fetal eyes at 16 (n=3) and 20 (n=3) weeks of gestation (WG) were used in quantitative RT-PCR to detect miRNAs. The Q RT-PCR assays from Applied Biosystems (ABI) covers 157 miRNAs arrayed in two 96-well plates. Each RNA sample was reverse transcribed with miRNA specific primers for the entire panel on a 96 well plate and Q PCR was run in triplicates on a 384 well plate using SDS 7900 (ABI). Relative quantitation is calculated using the 2-Ct method. P<0.05 (t-test) is considered as significant difference. Results: Cultured hfRPE cells were assayed twice for the entire panel and 90% of miRNAs had less than two fold difference between the technical repeats, indicating the Q PCR assays for miRNAs are reproducible. Approximately 90% of 157 miRNAs examined were detected in retina, RPE, and choroid albeit in variable abundance. Eight miRNAs were expressed 3 to 114 fold higher in retina than in RPE while 31 miRNAs were 2 to 737 fold lower in retina than in RPE. Expression level for 43 miRNAs were 2 to 754 fold higher in RPE than in choroid and 25 miRNAs were 3 to 62 fold lower in RPE than in choroid. 24 miRNAs were expressed at levels that are 2 to 855 fold higher in choroid than in retina and while levels of another 24 miRNAs were 2 to 1148 fold lower in choroid than in retina. In any given tissue, miRNA expression did not change significantly between 16 and 20 WG. A miRNA in a given tissue is considered relatively enriched if it is expressed at significantly higher levels than in two others tissues at both 16 and 20 WG. The expression levels of five miRNAs enriched in retina were 20 to 1148 fold higher than that in RPE or choroid. Six miRNAs were enriched in RPE by a factor of 10 to 754 fold compared to retina or choroid. Nine miRNAs were enriched in choroid by a factor of 3 to 1042 fold compared to retina or RPE. Conclusions: miRNAs are extensively expressed in human fetal retina, RPE, and choroid with highly tissue-specific profiles. A small set of miRNAs was relatively enriched in retina, RPE and choroid. It remains to be determined if the enriched miRNAs are critical for the development and functions of these tissues.