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

MicroRNA（miRNA）是一组短的（19-25个核苷酸）非编码RNA，其能够通过与靶mRNA的3个非翻译区（3UTR）碱基配对来下调基因表达。 miRNAs在许多物种中高度保守，表明它们的功能重要性。从hfRPE中鉴定出几种miRNAs，它们的表达与其邻近组织（包括视网膜和脉络膜）相比相对较高。这些hfRPE富集miRNA是miR-184、miR-187、miR-200、miR-221/222、miR-204、miR-211，并且发现其中几种对于hfRPE的总组织抗性是关键的，表明它们在RPE屏障功能中的重要性。 为了进行这项研究，我们通过基因靶向方法产生了miR-204敲除（KO）小鼠系。 将miR-204基因完全切除，并通过同源重组用新霉素抗性基因表达盒替换。 位于neo基因侧翼的一对Lox-p位点，因此，靶向等位基因内的neo基因可以通过Cre-Loxp机制消除。通过Southern杂交和尾DNA PCR验证敲除小鼠中miR-204基因的缺失。 通过北方杂交在通常富含miR-204的组织（例如眼和脑）中确定miR-204表达的丧失。 敲除小鼠是可行的，没有严重的发育缺陷。然而，成年眼的OCT分析显示，在2/3的敲除小鼠中，视网膜中的血管不规则，并且从透镜上皮突出额外的肿块。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显示与对照相比在视杆光感受器中减弱的a波振幅响应（n = 3）。将需要额外的功能研究（OCT，ERG）来表征视网膜缺陷，未来的研究将确定miR-204 KO小鼠眼睛中可能的结构改变，并鉴定负责生理变化的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.