Biological function microRNAs enriched in RPE: in vitro and in vivo models

MicroRNA(MiRNA)是一组短小的(19-25个核苷酸)非编码RNA，能够通过与靶mRNAs的3个非翻译区(3UTRs)进行碱基配对来下调基因的表达。MiRNAs在许多物种中高度保守，表明它们在功能上的重要性。从hfRPE中发现了几个miRNAs，它们的表达水平高于其邻近组织，包括视网膜和脉络膜。这些hfRPE富集型miRNAs是miR-184、miR-187、miR-200、miR-221/222、miR-204、miR-211，其中几个对hfRPE的总组织阻力至关重要，提示它们在RPE屏障功能中具有重要作用。 为了进行这项研究，我们通过基因打靶的方法培育了miR-204基因敲除(KO)小鼠系。通过同源重组，miR-204基因被完全去除，代之以新霉素抗性基因表达盒。位于neo基因两侧的一对Lox-p位点，因此，靶基因中的neo基因可以通过Cre-loxP机制消除。Southern杂交和尾部DNA聚合酶链式反应证实基因敲除小鼠中miR-204基因缺失。Northern杂交检测到miR-204在正常情况下富含miR-204的组织中表达缺失，如眼和脑。基因敲除的小鼠是可以存活的，并且没有严重的发育缺陷。然而，对成人眼的OCT分析显示，在2/3的基因敲除小鼠中，视网膜血管不规则，晶状体上皮细胞中有额外的肿块突出。它们在眼睛中的生理功能可能受到损害，因为从这些小鼠获得的ERG显示，与对照组(n=3)相比，视杆状光感受器的a波幅度反应减弱。将需要更多的功能研究(OCT，ERG)来表征视网膜缺陷，未来的研究将确定miR-204 KO小鼠眼睛可能的结构变化，并确定导致生理变化的miR-204靶基因。

MicroRNA (miRNA) is a group of short (19-25 nucleotide), non-coding RNAs that are capable of downregulateing gene expression by base pairing with the 3 untranslated regions (3UTRs) of target mRNAs. The miRNAs are highly conserved across many species, indicating their functional importance. Several miRNAs were identified from hfRPE that their express is relative high compared to its adjacent tissues, including retina and choroids. These hfRPE enriching miRNAs are miR-184, miR-187, miR-200, miR-221/222, miR-204, miR-211 and several of them were found to be critical to the total tissue resistance of the hfRPE, suggesting their importance in RPE barrier function. To carry out this study, we produced a miR-204 knockout (KO)mouse line by a gene-targeting approach. The miR-204 gene was completely ablated and replaced with a neomycin resistant gene expression cassette via homologous recombination. A pair of the Lox-p sites flanking a neo gene, thus, the neo gene that within the targeted allele can be eliminated by Cre-Loxp mechanism. Lack of miR-204 gene in the knockout mice was verified by Southern hybridization and PCR on tail DNA. Loss of miR-204 expression was determined in tissues normally enriching in miR-204, such as eye and brain, by Northern hybridization. The knockout mice are viable and dont have gross developmental defects. However, OCT analysis of adult eye showed an irregular vascularature in retina and an extra mass protruding from the lens epithelium in 2/3 of the knockout mice. Their physiological function in eye is likely compromised as ERG obtained from these mice showed an a wave amplitude response that was diminished in rod photoreceptors compared to control (n = 3). Additional functional studies (OCT, ERG) will be required to characterize the retinal defects and future studies will determine the possible structural alteration in eyes of miR-204 KO mice and identity the miR-204 target genes responsible for the physiological changes.