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

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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.