Angiogenic and Anti-Angiogenic Factors in human RPE

目的：microRNAs(MiRNAs)调节转录组的很大一部分。现在已经鉴定了400多个脊椎动物miRNAs，它们的靶标预计覆盖了大约30%的基因组。作为了解miRNA在人眼中功能的第一步，我们对miRNA在三个眼组织中的表达进行了分析。 方法：用mirVana miRNA分离试剂盒提取总RNA，收集大、小RNA片段(~lt；、~gt；200nt)。用培养的人胎视网膜色素上皮(hfRPE，n=1)和孕16周(n=3)和20周(n=3)人胎眼自然视网膜、RPE和脉络膜的小RNA片段进行定量RT-PCR检测miRNAs。来自应用生物系统公司(ABI)的Q RT-PCR分析涵盖了排列在两个96孔板上的157个miRNAs。每个RNA样品在96孔板上用整个小组的miRNA特异性引物进行逆转录，并在384孔板上用SDS7900(ABI)进行Q-PCR。用2-CT法计算相对定量。P&lt；0.05(t检验)为显著差异。 结果：对培养的hfRPE细胞进行了两次检测，90%的miRNAs在两次技术重复之间的差异小于2倍，表明Q-PCR方法具有重复性。在被检测的157个miRNAs中，大约90%在视网膜、RPE和脉络膜中检测到，尽管丰度不同。8个miRNAs在视网膜中的表达比RPE高3~114倍，31个miRNAs在视网膜中的表达比RPE低2~737倍。43个miRNAs在RPE中的表达水平是脉络膜的2~754倍，25个miRNAs在RPE中的表达水平是脉络膜的3~62倍。24个miRNAs在脉络膜中的表达水平是视网膜的2~855倍，而另外24个miRNAs在脉络膜中的表达水平是视网膜的2~1148倍。在任何给定的组织中，miRNA的表达在16到20周之间没有显著变化。给定组织中的miRNA如果在16和20 WG时的表达水平显著高于其他两个组织，则被认为是相对丰富的。5种miRNAs在视网膜中的表达水平是RPE或脉络膜表达水平的20~1148倍。与视网膜或脉络膜相比，6个miRNAs的RPE含量增加了10到754倍。9个miRNAs在脉络膜中的丰度是视网膜或RPE的3到1042倍。 结论：miRNAs在人胎儿视网膜、视网膜色素上皮和脉络膜中广泛表达，具有高度的组织特异性。少数miRNAs在视网膜、RPE和脉络膜中相对丰富。这些富含的miRNAs对这些组织的发育和功能是否起关键作用还有待确定。

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.