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

RPE 中富集的生物学功能 microRNA：体外和体内模型

• 批准号: 7968404
• 负责人: Sheldon Miller
• 金额: $ 25.72万
• 依托单位: NATIONAL EYE INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7968404
• 关键词: 3' Untranslated Regions; Adult; Aging; Alleles; Autoimmune Diseases; Base Pairing; Biological Process; Brain; Choroid; DNA; Defect; Development; Down-Regulation; Epithelium; Eye; Functional RNA; Future; Gene Expression; Gene Targeting; Genes; Heart Diseases; Human; In Vitro; Knock-out; Knockout Mice; Malignant Neoplasms; MicroRNAs; Mus; Neomycin resistance gene; Nucleotides; Ocular Physiology; Physiological; Relative (related person); Resistance; Retina; Retinal Defect; Site; Structure of retinal pigment epithelium; Tail; Tissues; fetal; homologous recombination; in vivo Model; lens; northern hybridization; response; retinal rods; southern hybridization

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.

MicroRNA （miRNA）是一组短的（19-25个核苷酸）非编码rna，能够通过与目标mrna的3个非翻译区（3UTRs）进行碱基配对来下调基因表达。这些mirna在许多物种中高度保守，表明它们的功能重要性。从hfRPE中鉴定出几个mirna，它们的表达与邻近组织（包括视网膜和脉络膜）相比相对较高。这些富集hfRPE的mirna是miR-184、miR-187、miR-200、miR-221/222、miR-204、miR-211，其中有几个被发现对hfRPE的总组织抗性至关重要，这表明它们在RPE屏障功能中的重要性。