Prediction and Verification of miRNA Expression in Human and Rat Retinas (original) (raw)
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Prediction of microRNAs affecting mRNA expression during retinal development
BMC Developmental Biology, 2010
Background: MicroRNAs (miRNAs) are small RNA molecules (~22 nucleotides) which have been shown to play an important role both in development and in maintenance of adult tissue. Conditional inactivation of miRNAs in the eye causes loss of visual function and progressive retinal degeneration. In addition to inhibiting translation, miRNAs can mediate degradation of targeted mRNAs. We have previously shown that candidate miRNAs affecting transcript levels in a tissue can be deduced from mRNA microarray expression profiles. The purpose of this study was to predict miRNAs which affect mRNA levels in developing and adult retinal tissue and to confirm their expression. Results: Microarray expression data from ciliary epithelial retinal stem cells (CE-RSCs), developing and adult mouse retina were generated or downloaded from public repositories. Analysis of gene expression profiles detected the effects of multiple miRNAs in CE-RSCs and retina. The expression of 20 selected miRNAs was confirmed by RT-PCR and the cellular distribution of representative candidates analyzed by in situ hybridization. The expression levels of miRNAs correlated with the significance of their predicted effects upon mRNA expression. Highly expressed miRNAs included miR-124, miR-125a, miR-125b, miR-204 and miR-9. Over-expression of three miRNAs with significant predicted effects upon global mRNA levels resulted in a decrease in mRNA expression of five out of six individual predicted target genes assayed.
MicroRNA Profile of the Developing Mouse Retina
Investigative Ophthalmology & Visual Science, 2010
PURPOSE. MicroRNAs (miRNAs) are short, noncoding transcripts that negatively regulate gene expression. They are implicated in diverse cellular processes. The purpose of this study was to obtain a global expression profile of miRNAs in the developing retina and identify differences in miRNA expression between adult rod and cone photoreceptors. METHODS. Locked nucleic acid (LNA) microarrays were used to investigate the miRNA transcriptome of the developing mouse retina and brain. Real-time PCR was used to validate the array findings. Laser capture microdissection was used to determine the miRNA spatial pattern of expression. RESULTS. One hundred thirty-eight miRNAs were expressed at at least one of the investigated time points. Several miRNAs showed significant changes in expression between embryonic day 15 and adult age in both retina and brain. Cluster analysis identified subgroups of miRNAs showing defined expression profiles. Globally, correlation of expression was higher, with increasing sequence similarity of the mature miRNAs. The miRNAs with identical seed sequences exhibited highly correlated expression profiles. The co-expression of selected host gene and intronic miRNA pairs was confirmed in adult retina. In some cases, expression profiles of miRNAs showed weak correlation with those of their host transcripts, suggesting posttranscriptional regulation of miRNAs during development. In addition, the miRNA transcriptome of rod-and cone-dominant retinas showed only minor differences, and no miRNAs specific for either cell-type were identified. CONCLUSIONS. Global expression profiling revealed dozens of miRNAs with significant expression changes in the developing retina. Precise patterns of expression of miRNAs suggest their specific roles in development. (Invest Ophthalmol Vis Sci.
Journal of Biological Chemistry, 2007
Although microRNAs (miRNAs) provide a newly recognized level of regulation of gene expression, the miRNA transcriptome of the retina and the contributions of miRNAs to retinal development and function are largely unknown. To begin to understand the functions of miRNAs in retina, we compared miRNA expression profiles in adult mouse retina, brain, and heart by microarray analysis. Our results show that at least 78 miRNAs are expressed in adult mouse retina, 21 of which are potentially retina-specific. Among these, we identified a polycistronic, sensory organ-specific paralogous miRNA cluster that includes miR-96, miR-182, and miR-183 on mouse chromosome 6qA3 with conservation of synteny to human chromosome 7q32.2. In situ hybridization showed that members of this cluster are expressed in photoreceptors, retinal bipolar and amacrine cells. Consistent with their genomic organization, these miRNAs have a similar expression pattern during development with abundance increasing postnatally and peaking in adult retina. Target prediction and in vitro functional studies showed that MITF, a transcription factor required for the establishment and maintenance of retinal pigmented epithelium, is a direct target of miR-96 and miR-182. Additionally, to identify miRNAs potentially involved in circadian rhythm regulation of the retina, we performed miRNA expression profiling with retinal RNA harvested at noon (Zeitgeber time 5) and midnight (Zeitgeber time 17) and identified a subgroup of 12 miRNAs, including members of the miR-183/96/182 cluster with diurnal variation in expression pattern. Our results suggest that miR-96 and miR-182 are involved in circadian rhythm regulation, perhaps by modulating the expression of adenylyl cyclase VI (ADCY6).
miRNA profiling of developing rat retina in the first three postnatal weeks
The morphogenesis of the mammalian retina depends on the precise control of gene expression during development. Small non-coding RNAs, including microRNAs play profound roles in various physiological and pathological processes via their regulation of gene expression. A systematic analysis of the expression profile of small non-coding RNAs in developing Wistar rat retinal tissues was executed using IonTorrent PGM next-generation sequencing technique in order to reveal the crucial players in the early postnatal retinogenesis. Our analysis reveals extensive regulatory potential of microRNAs during retinal development. We found a group of microRNAs that show constant high abundance (rno-mir-19, rno-mir-101; rno-mir-181, rno-mir-183, rno-mir-124 and let-7) during the development process. Others are present only in the early stages (such as rno-mir-20a, rno-mir-206, rno-mir-133, rno-mir-466, rno-mir-1247, rno-mir-3582), or at later stages (rno-mir-29, rno-mir-96, rno-mir-125, rno-mir-344 ...
Analysis of mir-9 Expression Pattern in Rat Retina during Postnatal Development
International Journal of Molecular Sciences, 2021
It is well established that miR-9 contributes to retinal neurogenesis. However, little is known about its presence and effects in the postnatal period. To expand our knowledge, miRNA-small RNA sequencing and in situ hybridization supported by RT-qPCR measurement were carried out. Mir-9 expression showed two peaks in the first three postnatal weeks in Wistar rats. The first peak was detected at postnatal Day 3 (P3) and the second at P10, then the expression gradually decreased until P21. Furthermore, we performed in silico prediction and established that miR-9 targets OneCut2 or synaptotagmin-17. Another two microRNAs (mir-135, mir-218) were found from databases which also target these proteins. They showed a similar tendency to mir-9; their lowest expression was at P7 and afterwards, they showed increase. We revealed that miR-9 is localized mainly in the inner retina. Labeling was observed in ganglion and amacrine cells. Additionally, horizontal cells were also marked. By dual miRNA...
Identification of miRNAs in a Model of Retinal Degenerations
Investigative ophthalmology & visual science, 2014
We investigated the expression profile of and identify all microRNAs (miRNAs) that potentially regulate inflammation in a light-induced model of focal retinal degeneration. Sprague Dawley (SD) rats aged 90 to 140 postnatal days were exposed to 1000 lux white fluorescent light for 24 hours. At 24 hours, and 3 and 7 days after exposure, the animals were euthanized and retinas processed for RNA. Expression of 750 miRNAs at 24 hours of exposure was assessed using low density array analysis. Significantly modulated miRNAs and their target mRNAs were used to assess the potential biological effects. Expression of seven miRNAs, potentially modulating inflammation, was investigated across a protracted time course after light exposure using quantitative PCR. Photoreceptor cell death was analyzed using TUNEL. Intense light exposure for 24 hours led to differential expression of a number of miRNAs, 37 of which were significantly modulated by 2-fold or more. Of those, 19 may potentially regulate...
MicroRNAs in the Neural Retina
International journal of genomics, 2014
The health and function of the visual system rely on a collaborative interaction between diverse classes of molecular regulators. One of these classes consists of transcription factors, which are known to bind to DNA and control the transcription activities of their target genes. For a long time, it was thought that the transcription factors were the only regulators of gene expression. More recently, however, a novel class of regulators emerged. This class consists of a large number of small noncoding endogenous RNAs, namely, miRNAs. The miRNAs compose an essential component of posttranscriptional gene regulation, since they ultimately control the fate of gene transcripts. The retina, as a part of the central nervous system, is a well-established model for unraveling the molecular mechanisms underlying neuronal and glial functions. Numerous recent efforts have been made towards identification of miRNAs and their inferred roles in the visual pathway. In this review, we summarize the ...
miRNA Profile in Three Different Normal Human Ocular Tissues by miRNA-Seq
Investigative Opthalmology & Visual Science, 2016
PURPOSE. Because microRNAs (miRNAs) have been associated with eye diseases, our study aims to profile ocular miRNA expression in normal human ciliary body (CB), cornea, and trabecular meshwork (TM) using miRNA-Seq to provide a foundation for better understanding of miRNA function and disease involvement in these tissues. METHODS. Total RNAs were extracted from seven normal human CB, seven cornea, and seven TM samples using mirVana total RNA isolation kit. miRNA-Seq was done with Illumina MiSeq. Bowtie software was used to trim and align generated sequence reads, and only exact matches to mature miRNAs from miRBase were included. The miRTarBase database was used to analyze miRNA target interactions, and the expression of five selected miRNAs was validated using droplet digital PCR (ddPCR). RESULTS. Using the miRNA extracted from 21 human samples, we found 378 miRNAs collectively expressed, of which the 11 most abundant miRNAs represented 80% of the total normalized reads. We also identified uniquely expressed miRNAs, of which five share 18 highly validated gene targets, and created a profile of miRNAs known to target genes associated with keratoconus and glaucoma. Using ddPCR, we validated the expression profile of five miRNAs from miRNA-Seq. CONCLUSIONS. For the first time, we profiled miRNA expression in three human ocular tissues using miRNA-Seq, identifying many miRNAs that had not been previously reported in ocular tissue. Defining the relative expression of miRNAs in nondiseased eye tissues could help uncover changes in miRNA expression that accompany diseases such as glaucoma and keratoconus.
microRNA regulatory circuits in a mouse model of inherited retinal degeneration
Scientific reports, 2016
miRNA dysregulation is a hallmark of many neurodegenerative disorders, including those involving the retina. Up-regulation of miR-1/133 and miR-142, and down-regulation of miR-183/96/182 has been described in the RHO-P347S mouse retina, a model for a common form of inherited blindness. High-throughput LC-MS/MS was employed to analyse the protein expression of predicted targets for these miRNAs in RHO-P347S mouse retinas; 133 potential target genes were identified. Pathway over-representation analysis suggests G-protein signaling/visual transduction, and synaptic transmission for miR-1, and transmembrane transport, cell-adhesion, signal transduction and apoptosis for miR-183/96/182 as regulated functions in retina. Validation of miRNA-target mRNA interactions for miR-1, miR-96/182 and miR-96 targeting Ctbp2, Rac1 and Slc6a9, respectively, was demonstrated in vitro. In vivo interaction of miR-183/96/182 and Rac1 mRNA in retina was confirmed using miR-CATCH. Additional miRNAs (includin...
Journal of Comparative Neurology, 2019
MiRNAs play important roles in post-transcriptional processes to regulate gene expression. MiRNAs control various biological processes, such as growth, development and differentiation. The continuous physiological function of photoreceptors and retinal pigment epithelium requires precise regulation to maintain their homeostasis and function; hence, these cells are highly susceptible to premature death in retinal degenerative disorders. MiRNAs are essential for the retinal cell maturation and function; the miR-183 cluster represents one of the most important regulatory factors for the photoreceptor cells. Various studies together with bioinformatics analyses have shown that many genes contributing to the differentiation pathway of photoreceptors are targets of the miR-183 cluster, and the miR-183 cluster dysregulation causes certain defects in the differentiation of the photoreceptors and other retinal neurons by influencing the expression of target genes. Misexpression of miR-183 cluster in the human retinal epithelial cells leads to the reprogramming and transformation of these cells to neuron-and photoreceptor-like cells, which are associated with the expression of neuron-and photoreceptor-specific markers in human retinal pigment epitheliums (hRPE) cells. The knockout of this cluster causes the destruction of the outer segment of the photoreceptors, which subsequently causes the cells to exhibit severe susceptibility to light and eventually degenerate. Hundreds of target genes in this family are