Angiogenic and Anti-Angiogenic Factors in human RPE

目的：microRNAs（miRNAs）调节转录组的重要部分。现在已经鉴定了超过400种脊椎动物miRNA，并且已经预测它们的靶标覆盖约30%的基因组。作为了解miRNA在人眼中功能的第一步，在三种眼组织中分析了miRNA表达。 方法：采用mirVana miRNA提取试剂盒提取总RNA，并将其分为小片段和大片段（<，> 200 nt）。采用RT-PCR方法检测人胚胎RPE（hfRPE，n=1）和16（n=3）、20（n=3）周人胚胎眼（WG）的天然视网膜、RPE和脉络膜中的miRNAs。来自Applied Biosystems（ABI）的Q RT-PCR测定覆盖排列在两个96孔板中的157个miRNA。在96孔板上用整个组的miRNA特异性引物逆转录每个RNA样品，并使用SDS 7900（ABI）在384孔板上一式三份地运行Q PCR。使用2-Ct方法计算相对定量。P<0.05（t检验）被认为是显著差异。 结果如下：对于整个组，将培养的hfRPE细胞测定两次，并且90%的miRNA在技术重复之间具有小于两倍的差异，表明miRNA的Q PCR测定是可再现的。检测的157种miRNA中约90%在视网膜、RPE和脉络膜中检测到，尽管丰度不同。8种miRNAs在视网膜中的表达比RPE高3至114倍，而31种miRNAs在视网膜中的表达比RPE低2至737倍。43种miRNAs在RPE中的表达水平比脉络膜中高2至754倍，25种miRNAs在RPE中比脉络膜中低3至62倍。24种miRNA在脉络膜中的表达水平比在视网膜中高2至855倍，而另外24种miRNA在脉络膜中的表达水平比在视网膜中低2至1148倍。在任何给定的组织中，miRNA表达在16和20 WG之间没有显著变化。如果给定组织中的miRNA在16和20 WG时的表达水平显著高于其他两个组织，则认为该组织中的miRNA相对富集。5种miRNAs在视网膜中的表达量是在视网膜色素上皮或脉络膜中的20 ~ 1148倍。与视网膜或脉络膜相比，六种miRNA在RPE中富集10至754倍。与视网膜或RPE相比，9种miRNA在脉络膜中富集3至1042倍。 结论：miRNAs在人胚胎视网膜、视网膜色素上皮和脉络膜中广泛表达，并具有高度的组织特异性。一小部分miRNAs在视网膜、RPE和脉络膜中相对富集。目前尚不清楚富集的miRNAs是否对这些组织的发育和功能至关重要。

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.