Tissue specific comprehensive gene analysis in a murine lens-induced myopia model
Ontology highlight
ABSTRACT: To identify to identify target tissues and molecules involved with refractive myopic shift and axial length elongation in a murine lens-induced myopia model, we performed comprehensive analysis by microRNA array. Negative 30diptor (-30D) lens was fixed on right eye (-30D) of C57BL/6J mice (3weeks old, N=3) for 3 weeks, the refraction and the axial length were measured using a refractometer and a SD-OCT system in all eyes. Eye balls were enucleated and separated to cornea, iris, lens, retina, choroid and sclera. Total RNA was extracted from individual ocular components. MicroRNA expression analysis was carried out using Agilent Mouse miRNA Microarray (8×60K) miRBase21.0 (Agilent). Expression ratio calculation and miRNA varying expression were extracted by GeneSpring GX 14.5 (Agilent). After 3weeks of lens fixing, a refractive change and an axial length elongation change were observed (Normal vs -30D: 0.95 ± 1.85D vs -18.42 ± 3.98D, 0.155 mm ± 0.015mm vs 0.273 ± 0.009 mm), respectively. MiRNA expression changes that induced only by -30D lens fixing was confirmed in each part of the eyeball. By expression ratio calculation and miRNA varying expression analysis, upregulated miRNA (56 in cornea, 13 in iris, 6 in lens, 0 in retina, 29 in choroid and 30 in sclera) and downregulated miRNA (7 in cornea, 28 in iris, 17 in lens, 9 in retina, 7 in choroid and 40 in sclera) were observed. Overlapping miRNAs were also found while each eye tissues. In this study, miRNA varying expression were observed in each ocular part of the murine lens-induced myopia model. These miRNAs dysregulation may be functionally involved with the refractive myopia shift and the axial length elongation.
Project description:The normal gene expression profiles of the tissues in the eye are a valuable resource for considering genes likely to be involved with disease processes. This is based on the assumption that transcript abundances in healthy tissue are correlated to the continued health of that tissue. Expression values were compared with publically available EST and RNA-sequencing resources. The estimated gene and exon level abundances are available online on the Ocular Tissue Database. Ten different tissues were obtained from 6 different individuals and RNA was pooled. The tissues included: retina, optic nerve head (ONH), optic nerve (ON), ciliary body (CB), trabecular meshwork (TM), sclera, lens, cornea, choroid/RPE and iris.
Project description:Development of myopia is associated with large-scale changes in ocular tissue gene expression. Although differential expression of coding genes underlying development of myopia has been a subject of intense investigation, the role of non-coding genes such as microRNAs in the development of myopia is largely unknown. In this study, we explored myopia-associated miRNA expression profiles in the retina and sclera of C57Bl/6J mice with experimentally induced myopia using microarray technology. We found a total of 53 differentially expressed miRNAs in the retina and no differences in miRNA expression in the sclera of C57BL/6J mice after 10 days of visual form deprivation, which induced -6.93 ± 2.44 D (p < 0.000001, n = 12) of myopia. We also identified their putative mRNA targets among mRNAs found to be differentially expressed in myopic retina and potential signaling pathways involved in the development of form-deprivation myopia using miRNA-mRNA interaction network analysis. Analysis of myopia-associated signaling pathways revealed that myopic response to visual form deprivation in the retina is regulated by a small number of highly integrated signaling pathways. Our findings highlight substantial involvement of miRNAs in the regulation of refractive eye development, and in the development of myopia through the retinal gene regulation.
Project description:We sequenced mRNA from 34 retina/RPE/choroid samples taken from the right eyes of male chicks across a time-course of normal development or refractive error induction (defocus-induced myopia and hyperopia).
Project description:Following laboratory and clinical findings implicating circadian biology in the pathogenesis of myopia (nearsightedness), we examined gene expression in two crucial tissues controlling post-natal refractive development, the retina and choroid. Inducing unilateral visual form deprivation myopia in young chicks, a widely studied and validated model, we isolated retinal and choroidal tissues every 4 hours over a single day from myopic and contralateral control eyes during a period time when myopia progresses rapidly.
Project description:Introduction: In human eyes, ocular enlargement/ growth, reflects active scleral extracellular matrix remodeling. miRNAs are small non-coding RNAs that regulate gene expression by base pairing with target sequences, and serve as nodes of signaling networks. We hypothesized that the sclera, like most tissues, expresses miRNAs, some of which modulate genes regulating ocular growth. In this study, the scleral miRNA expression profile of rapidly growing human fetal eyes was compared with that of stable adult donor eyes using high-throughput microarray and quantitative PCR analyses. Results: Human sclera expressed several miRNAs. Microarray comparison of adult and fetal samples revealed many to be differentially expressed (p<0.01, min p= 6.5x10^11), with increased expression of collagen specific mir-214, let-7c, let-7e, mir-103, mir-107, and mir-98 in fetal sclera subsequently confirmed (1.5 to 4 fold changes, p<0.01). For both adult and fetal samples, no significant differences in miRNA expression profiles of sclera from posterior and peripheral ocular regions were observed. Conclusion: This is the first study to catalogue miRNA expression in human sclera. The sclera expresses several miRNAs, some of which show age-related differential regulation, higher in rapidly growing fetal eyes, consistent with a role in ocular growth regulation. These findings may be useful for linking scleral miRNA expression with potential manipulation in disorders such as scleral ectasia/ axial myopia.
Project description:The iris is a fine structure that controls the amount of light that enters the eye. The ciliary body controls the shape of the lens and produces aqueous humor. The retinal pigment epithelium and choroid (RPE/choroid) are essential in supporting the retina and absorbing light energy that enters the eye. Proteins were extracted from iris, ciliary body, and RPE/choroid tissues of eyes from five individuals and fractionated using SDS-PAGE. After in-gel digestion, peptides were analyzed using LC-MS/MS on an Orbitrap Elite mass spectrometer. In iris, ciliary body, and RPE/choroid, we identified 2,959, 2,867, and 2,755 non-redundant proteins with protein false positive rate <1%. There were 43 unambiguous protein isoforms identified in iris, ciliary body, and RPE/choroid. Four “missing proteins” were found in ciliary body. The MS proteome database of the human iris, ciliary body, and RPE/choroid may serve as a valuable resource for future investigations of the eye in health and disease. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD001424.
Project description:Juvenile primates develop myopia when their visual experience is degraded by lid fusion. In response to the abnormal visual input, retinal neural networks cause an excessive growth of the postequatorial segment of the eye, but the mechanism underlying this axial elongation is unknown. By combining analysis of gene expression, injection of the thymidine analog 5-bromo-2'-deoxyuridine and immunocytochemistry, we show that the retinal periphery in both juvenile rhesus macaques and green monkeys harbors a population of mitotically active neuroprogenitor cells that proliferate when the visual experience is altered by lid fusion. Furthermore, the number of dividing cells is highly correlated with the axial elongation of the eye and the resulting myopic refractive error. Thus, the retina undergoes active growth during the postnatal development of the primate eye. This growth is modulated by the visual input and accelerates considerably when the eye develops axial myopia. Keywords: disease state analysis
Project description:The study aim was to determine microRNA (miRNA) expression profiles of ocular tissues in form-deprivation induced myopia (FDM) mice. Form-deprivation myopia was induced in C57BL/6Jmice over the right eye; the contralateral left eyes were used as controls. Whole genome microRNA expression profiles in myopic whole eye, retina, and sclera were determined using the Agilent mouse miRNA microarray. The normalized microarray data were performed ANOVA test to identify differences in miRNA expression between myopic and control eyes. The differential expression for selected miRNAs was validated by quantitative real time PCR (qRT-PCR).
Project description:The iris is a fine structure that controls the amount of light that enters the eye. The ciliary body controls the shape of the lens and produces aqueous humor. The retinal pigment epithelium and choroid (RPE/choroid) are essential in supporting the retina and absorbing light energy that enters the eye. Proteins were extracted from iris, ciliary body, and RPE/choroid tissues of eyes from five individuals and fractionated using SDS-PAGE. After in-gel digestion, peptides were analyzed using LC-MS/MS on an Orbitrap Elite mass spectrometer. In iris, ciliary body, and RPE/choroid, we identified 2,959, 2,867, and 2,755 non-redundant proteins with protein false positive rate <1%. There were 43 unambiguous protein isoforms identified in iris, ciliary body, and RPE/choroid. Four “missing proteins” were found in ciliary body. The MS proteome database of the human iris, ciliary body, and RPE/choroid may serve as a valuable resource for future investigations of the eye in health and disease. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD001424.
Project description:Because refractive development is governed largely by the retina, we analyzed the retinal transcriptome in chicks wearing a spectacle lens, a well-established means to induce refractive errors, to identify gene expression alterations and to develop novel mechanistic hypotheses about refractive development. One-week-old white Leghorn chicks wore a unilateral spectacle lens of + or –15 diopters for 6 hours or 3 days (n=6 for each condition). With total RNA from the retina/retinal pigment epithelium (RPE), Affymetrix Chicken GeneChips were used to compare gene expression levels between lens-wearing and contralateral control eyes. Normalized microarray signal intensities were evaluated by an analysis of variance approach. Selected differentially expressed genes were validated by qPCR in biologically independent samples.