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INTRODUCTION: The therapeutic effect of different doses of the traditional aqueous extract of dried leaves of yerba mate (Ilex paraguariensis A. St.-Hil.) was investigated in an experimental cataract model in chicken embryos. METHODS AND RESULTS: LC-MS/MS analysis allowed the identification and quantification of 53 metabolites. In the hydrocortisone-induced cataract model, lenses were examined morphologically after treatment and parameters related to oxidative stress (total antioxidant/oxidant status (TAS/TOS), glutathione (GSH), and malondialdehyde (MDA)) were evaluated. Antiproliferative cell nuclear antigen (PCNA) and caspase-3 H-scores were determined and crystallin alpha A (CRYAA) gene expression in the lenses was measured by RT-PCR. The degree of cataract decreased in all treatment groups. While there was no significant difference in TAS levels compared to the negative control, TOS, GSH, and MDA levels were dose-dependently regulated. Treatment groups other than the high-dose group regulated the decrease in PCNA and the increase in caspase-3. CRYAA gene expression increased significantly only at the lowest dose. CONCLUSION: YM, which is becoming increasingly popular as a traditional tea, showed a therapeutic effect on hydrocortisone-induced cataracts in chicken embryos at relatively low doses.
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Purpose: To investigated the biological effects of E156K-mutated αA-crystallin (CRYAA) in human lens epithelial cells (HLECs). Methods: FLAG-tagged, human, full-length, wild-type (WT), or E156K-mutated CRYAA was expressed in HLECs under CRYAA knockdown. CRYAA expression was determined by quantitative reverse transcription polymerase chain reaction and western blotting (WB). Rhodamine cytoskeleton staining was used to observe the changes in cell morphology following transfection with WT or E156K-mutated CRYAA plasmids. WB was performed to assess the expression of markers related to epithelial-mesenchymal transition (EMT) and migration. Results: Rhodamine cytoskeleton staining revealed changes in the morphology of cells transfected with E156K-mutated CRYAA and opposite responses occurred after treatment with a ß-catenin inhibitor. Cells transfected with E156K-mutated CRYAA expressed remarkably higher levels of the mesenchymal biomarkers N-cadherin and vimentin but decreased levels of the epithelial biomarker E-cadherin, whereas opposite trends were observed in cells treated with the ß-catenin inhibitor, ICG001. The migratory capability of E156K-mutated CRYAA cells was significantly greater than that of WT cells (P < 0.001). This effect was accompanied by significantly increased expression levels of phosphorylated (p)-focal adhesion kinase (FAK) and p-Src. These changes were decreased significantly by treatment with FAK and Src inhibitors. Conclusion: E156K-mutated CRYAA induced EMT, in which the HLECs lost cell polarity, and acquired a mesenchymal phenotype with greater migratory capability. These biological effects may be associated with activation of the Wnt/ß-Catenin and FAK/Src signaling pathways.
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Hereditary cataracts are characterized by significant clinical and genetic heterogeneity, which can pose challenges for early DNA diagnosis. To comprehensively address this problem, it is essential to investigate the epidemiology of the disease, perform population studies to determine the spectrum and frequencies of mutations in the responsible genes, and examine clinical and genetic correlations. Based on modern concepts, non-syndromic hereditary cataracts are predominantly caused by genetic disease forms associated with mutations in crystallin and connexin genes. Therefore, a comprehensive approach to studying hereditary cataracts is necessary for early diagnosis and improved treatment outcomes. The crystallin (CRYAA, CRYAB, CRYGC, CRYGD, and CRYBA1) and connexin (GJA8, GJA3) genes were analyzed in 45 unrelated families from the Volga-Ural Region (VUR) with hereditary congenital cataracts. Pathogenic and probably pathogenic nucleotide variants were identified in ten unrelated families, nine of which had cataracts in an autosomal dominant pattern of inheritance. Two previously undescribed likely pathogenic missense variants were identified in the CRYAA gene: c.253C > T (p.L85F) in one family and c.291C > G (p.H97Q) in two families. The known mutation c.272_274delGAG (p.G91del) was found in the CRYBA1 gene in one family, while no pathogenic variants were found in the CRYAB, CRYGC, or CRYGD genes in the examined patients. In the GJA8 gene, the known mutation c.68G > C (p.R23T) was found in two families, and previously undescribed variants were identified in two other families: a c.133_142del deletion (p.W45Sfs*72) and a missense variant, c.179G > A (p.G60D). In one patient with a recessive form of cataract, two compound-heterozygous variants were identified-a previously undescribed likely pathogenic missense variant, c.143A > G (p.E48G), and a known variant with uncertain pathogenetic significance, c.741T > G (p.I24M). Additionally, a previously undescribed deletion, c.del1126_1139 (p.D376Qfs*69), was identified in the GJA3 gene in one family. In all families where mutations were identified, cataracts were diagnosed either immediately after birth or during the first year of life. The clinical presentation of the cataracts varied depending on the type of lens opacity, resulting in various clinical forms. This information emphasizes the importance of early diagnosis and genetic testing for hereditary congenital cataracts to guide appropriate management and improve outcomes.
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AIM: To investigate the expression of αA-crystallin (CRYAA) in age-related cataract (ARC) models and its role in lens epithelial cells (LECs). METHODS: We used Flow cytometry to detect the apoptosis and cell cycle in HLEB3 cells and Real-time fluorescence quantitative polymerase chain reaction to detect the expression of CRYAA mRNA in HLEB3 and in rabbit lens. The expression of CRYAA in HLEB3 cells and rabbit lenses as well as the proteins related to apoptosis and autophagy in transfected cells were detected by western blotting. The lens structure in rabbits was investigated using hematoxylin-eosin staining. Protein thermostability assay was performed to detect the thermal stability of rabbit lens proteins. CCK- 8 assay was used to detect the viability of transfected cells, and the transfection was recorded by fluorescence photography. RESULTS: Hydrogen peroxide can promote apoptosis and arrest the cell cycle in HLEB3 cells, and naphthalene can cause cataract formation and damage the structure of the lens in rabbits. Both ARC models can reduce the expression of CRYAA. The expression of CRYAA silencing increased apoptosis and autophagy in HLEB3 cells.
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Catarata , Cristalinas , Cristalino , Animais , Coelhos , Apoptose , Catarata/genética , Catarata/metabolismo , Cristalinas/genética , Células Epiteliais/metabolismo , Cristalino/metabolismo , Proteínas/metabolismo , HumanosRESUMO
Deprivation of one sense can be followed by enhanced development of other senses via cross-modal plasticity mechanisms. To study the effect of whisker tactile deprivation on vision during the early stages of development, we clipped the bilateral whiskers of young mice and found that their vision was impaired but later recovered to normal levels. Our results demonstrate that inhibition of the PI3K/AKT/ERK signaling pathway caused short-term visual impairment during early development, while high expression levels of Crystallin Alpha A (CRYAA) and Gap Junction Protein Alpha 8 (GJA8) in the retina led to the recovery of developmental visual acuity. Interestingly, analysis of single-cell sequencing results from human embryonic retinas at 9-19 gestational weeks (GW) revealed that CRYAA and GJA8 display stage-specific peak expression during human embryonic retinal development, suggesting potential functions in visual development. Our data show that high expression levels of CRYAA and GJA8 in the retina after whisker deprivation rescue impaired visual development, which may provide a foundation for further research on the mechanisms of cross-modal plasticity and in particular, offer new insights into the mechanisms underlying tactile-visual cross-modal development.
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BACKGROUND: The mutations in the αA-crystallin (CRYAA) gene may contribute to the development of age-related cataract (ARC). In this study, we searched for single nucleotide polymorphisms (SNP) in exons of CRYAA and investigated the associations between the identified SNPs and the subtypes of ARC. MATERIALS AND METHODS: Peripheral venous blood was collected for the extraction of genomic DNA. Three exons of CRYAA were sequenced to detect SNPs. The frequency distributions of alleles and genotypes were compared between the ARC and control groups. RESULTS: There were 618 patients with various subtypes of ARC (nuclear cataract [NC], cortical cataract [CC], posterior subcapsular cataract [PSC]). The control group comprised 236 patients. The incidence of early-onset cataract was significantly greater in PSC patients (P = .002 for NC; P = .036 for CC). One SNP was detected in exon 3 of CRYAA (rs76740365 G>A). When the distribution of rs76740365 was compared among the ARC subtypes, only the difference between the PSC group and the control group was statistically significant (allele frequency: P = .000057, OR 2.945; genotype distribution frequency: P = .000458). The heterozygote genotype (GA) carried a significantly greater risk than the homozygous wild-type genotype (GG) by 1.742 times for all types of cataracts and 2.369 times for the PSC subtype. CONCLUSIONS: The SNP rs76740365 G>A in exon 3 of the CRYAA gene is associated with greater susceptibility of ARC, particularly the PSC subtype. Individuals carrying the SNP rs76740365 G>A may be more likely to develop PSC at a younger age than other subtypes.
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Catarata , Cristalinas , Humanos , Polimorfismo de Nucleotídeo Único , Cristalinas/genética , Catarata/genética , Éxons/genéticaRESUMO
AIM: Polymorphisms in alpha A crystallin (CRYAA) gene have been implicated in susceptibility to cataracts, but some published studies have reported inconclusive results. Our study aimed to conduct a meta-analysis investigating the association between polymorphisms in CRYAA and susceptibility to cataracts. METHODS: The PubMed, Excerpta Medica Database, Cochrane Library and China National Knowledge Infrastructure were searched for all articles published up to 20 March 2019 that reported cataracts and three polymorphisms (rs3761381, rs13053109, and rs7278468) of CRYAA. Afterwards, statistical analysis was performed for available articles. RESULTS: Four articles published between 2014 and 2017 were included, involving 869 cases and 1,950 controls. There was no statistical evidence of an association between cataract risk and CRYAA gene polymorphisms rs13053109 (p > .05) and rs3761382 (p > .05). Significant decreased cataract risks were observed for different gene models of rs7278468 polymorphism: for G vs T, OR = 0.6640; 95% CI, 0.5361-0.7736, p < .001; for GG vs TT, OR = 0.3864; 95% CI, 0.2379-0.6278, p < .001; for GG vs TT+GT, OR = 0.4492; 95% CI, 0.2829-0.7134, p = .001; for GG+GT vs TT, OR = 0.6645; 95% CI, 0.5058-0.8729, p = .003; for GT vs TT, OR = 0.7508; 95% CI, 0.5639-0.9996, p = .050. CONCLUSION: Our meta-analysis indicated that rs3761382 and rs13053109 polymorphisms of CRYAA may not be associated with susceptibility to cataracts. Individuals carrying mutant genotype of rs7278468 polymorphism are associated with a significantly decreased cataract risk. ABBREVIATIONS: CC: Congenital cataract; ARC: Age-related cataract; SNPs: single nucleotide polymorphisms; NOS: Newcastle-Ottawa Scale; HWE: Hardy-Weinberg equilibrium; OR: odds ratio; CI: confidence interval; qPCR: quantitative polymerase chain reaction; NO: nuclear opalescence; NC: nuclear color.
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Catarata , Cristalinas , Catarata/genética , Cristalinas/genética , Predisposição Genética para Doença , Genótipo , Humanos , Polimorfismo de Nucleotídeo ÚnicoRESUMO
BACKGROUND: Mutations in CRYAA, which encodes the α-crystallin protein, are associated with a spectrum of congenital cataract-microcornea syndromes. RESULTS: In this study, we performed clinical examination and subsequent genetic analysis in two unrelated sporadic cases of different geographical origins presenting with a complex phenotype of ocular malformation. Both cases manifested bilateral microphthalmia and severe anterior segment dysgenesis, primarily characterized by congenital aphakia, microcornea, and iris hypoplasia/aniridia. NGS-based analysis revealed two novel single nucleotide variants occurring de novo and affecting the translation termination codon of the CRYAA gene, c.520T > C and c.521A > C. Both variants are predicted to elongate the C-terminal protein domain by one-third of the original length. CONCLUSIONS: Our report not only expands the mutational spectrum of CRYAA but also identifies the genetic cause of the unusual ocular phenotype described in this report.
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Catarata , Cristalinas , Anormalidades do Olho , Cristalinas/genética , Anormalidades do Olho/genética , Humanos , Mutação/genética , Nucleotídeos , Linhagem , FenótipoRESUMO
Creatine kinase (CK) is an energy storage enzyme that plays an important role in energy metabolism. CK/phosphocreatine functions as an energy buffer and links ATP production sites with ATP utilization sites. Several key mutations in the αA-crystallin (cryaa) and αB-crystallin (cryab) genes have been linked with autosomal-dominant, hereditary human cataracts. The cryaa-R49C mutation was identified in a four-generation Caucasian family. We previously identified an increase in the quantity of CK complexed with α-crystallin in the lenses of knock-in mice expressing the cryaa-R49C mutation using proteomic analyses. Increased levels of CK in postnatal cataractous lenses may indicate increased ATP requirements during early cataract development. To gain a further understanding of the relationship between CK and α-crystallin, we investigated whether α-crystallin interacts with and forms complexes with CK, in vitro. Isothermal titration calorimetry (ITC) showed that each CK dimer bound to 28 α-crystallin subunits, with a Kd of 3.3 × 10-7 M, and that the interaction between α-crystallin and CK was endothermic, thermodynamically favorable, and entropy-driven. High-salt concentrations did not affect the interaction between CK and α-crystallin, suggesting that the interaction between CK and α-crystallin is primarily hydrophobic. Gel permeation chromatography (GPC) detected water-soluble α-crystallin and CK complexes, as determined by increased light scattering after complex formation. In addition, CK and α-crystallin formed partially-water-insoluble, high-molecular-mass complexes. Enzyme-linked immunosorbent assay (ELISA)-based enzymatic activity analyses of lens homogenates showed a 17-fold increase in CK activity in the postnatal lenses of cryaa-R49C knock-in mice. These studies indicate that the interaction between α-crystallin and CK is functionally important and that increased CK levels may be necessary to meet the increased ATP demands of ATP-dependent functions in cataractous lenses.
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Previous research has shown that CXCR5-/- mice develop retinal degeneration (RD) with age, a characteristic related to age macular degeneration (AMD). RD in these mice is not well-understood, and in this study, we sought to characterize further the RD phenotype and to gain mechanistic insights into the function of CXCR5 in the retina. CXCR5-/- and WT control mice were used. Fundus images demonstrated a significant (p < 0.001) increase of hypo-pigmented spots in the retina of aged CXCR5-/- mice compared with WT control mice. PAS staining indicated localization of deposits in the sub-retinal pigment epithelia (RPE) layer. AMD-associated proteins Cryab, amyloid beta, and C3d were detected within the RPE/sub-RPE tissues by immunofluorescence (IF). In addition, western blot analysis of COX-2, Arg1, and VEGF-a revealed an increase in the signaling of these molecules within the RPE/choroid complex. Transmission electron microscopy (TEM) indicated a drusen-like structure of sub-RPE deposits with an accumulation of vacuolated cellular debris. Loss of photoreceptors was detected by peanut lectin staining and was corroborated by a reduction in MAP2 signaling. Loss of blood-retinal barrier integrity was demonstrated by a reduction of ZO-1 expression. Inflammatory cells were detected in the sub-RPE space, with an increase in IBA-1 positive microglia cells on the surface of the RPE. Mass spectrometry analysis of CXCR5-/- mouse RPE/choroid proteins extracts, separated by SDS-page and incubated with autologous serum, identified autoantibodies against AMD-associated proteins: Cryaa, Cryab, and Anxa2. In vitro evaluations in BV-2 cell culture indicated a significant increase in production of Arg-1 (p < 0.001) and COX-2 (p < 0.01) in the presence of anti-CXCR5 antibody when compared with Igg-treated control BV-2 cells stimulated with IL-4 and TNFα/IFNγ, respectively. Anti-CXCR5 antibody treatment without stimulating agents did not affect Arg-1 and COX-2 expression; this suggests that CXCR5 may have a regulatory role in microglia cells activation. These results indicate that with age, CXCR5-/- mice develop RD characterized by microglia dysfunction, increased production of CXCL13 in the RPE progressive photoreceptor, neuronal loss, and sub-RPE deposition of cellular debris, resulting in the production of immunogenic proteins and autoimmune-mediated RD.
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Autoimunidade/imunologia , Modelos Animais de Doenças , Degeneração Macular/imunologia , Receptores CXCR5/imunologia , Degeneração Retiniana/imunologia , Peptídeos beta-Amiloides/imunologia , Peptídeos beta-Amiloides/metabolismo , Animais , Autoimunidade/genética , Proteínas de Ligação ao Cálcio/imunologia , Proteínas de Ligação ao Cálcio/metabolismo , Linhagem Celular , Ciclo-Oxigenase 2/imunologia , Ciclo-Oxigenase 2/metabolismo , Imunofluorescência , Degeneração Macular/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas dos Microfilamentos/imunologia , Proteínas dos Microfilamentos/metabolismo , Microglia/citologia , Microglia/imunologia , Microglia/metabolismo , Microscopia Eletrônica de Transmissão , Receptores CXCR5/deficiência , Receptores CXCR5/genética , Degeneração Retiniana/genética , Epitélio Pigmentado da Retina/imunologia , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/ultraestrutura , Fator A de Crescimento do Endotélio Vascular/imunologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Proteína da Zônula de Oclusão-1/imunologia , Proteína da Zônula de Oclusão-1/metabolismo , Cadeia B de alfa-Cristalina/imunologia , Cadeia B de alfa-Cristalina/metabolismoRESUMO
BACKGROUND: Corneal neovascularization (angiogenesis and lymphangiogenesis) compromises corneal transparency and transplant survival, however, the molecular mechanisms of corneal host epithelial and stromal cells in neovascularization have not yet been fully elucidated. Furthermore, the contribution and mechanism of corneal host endothelial cells involved in neovascularization are largely unexplored. METHODS: Liquid chromatography-mass spectrometry, immunoblotting, and ELISA were used to screen and identify potential neovascularization-related factors in human full-thickness vascularized corneal tissues. Lipopolysaccharide was used to induce inflammation in three kinds of corneal host cells in vitro, including corneal epithelial, stromal, and endothelial cells. Fungus was used to establish an animal model of corneal neovascularization in vivo. Tube formation and spheroid sprouting assays were used to evaluate the contribution of three kinds of corneal host cells to the degree of neovascularization under various stimuli. Matrix metalloproteinase (MMP)-2, alpha-crystallin A chain (CRYAA), galectin-8, Bcl-2, neuropilin-2, MMP-9 plasmids, and recombinant human fibronectin were used to identify the key proteins of corneal host cells involved in corneal inflammatory neovascularization. FINDINGS: All three kinds of corneal host cells influenced corneal neovascularization to varying degrees. MMP-9 in human corneal epithelial cells, MMP-2, and CRYAA in human corneal stromal cells, and MMP-2 and galectin-8 in human corneal endothelial cells are potential key proteins that participate in corneal inflammatory neovascularization. INTERPRETATION: Our data indicated that both the effects of key proteins and corneal host cells involved should be considered for the treatment of corneal inflammatory neovascularization.
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Córnea/citologia , Neovascularização da Córnea/etiologia , Neovascularização da Córnea/patologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Biomarcadores , Linhagem Celular , Cromatografia Líquida , Córnea/metabolismo , Neovascularização da Córnea/metabolismo , Meios de Cultivo Condicionados , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Ensaio de Imunoadsorção Enzimática , Células Epiteliais/metabolismo , Feminino , Expressão Gênica , Humanos , Masculino , Espectrometria de Massas , Pessoa de Meia-Idade , Proteoma , Proteômica , Ratos , Células Estromais/metabolismo , Adulto JovemRESUMO
BACKGROUND: Congenital cataract is the leading cause of blindness in children worldwide. Approximately half of all congenital cataracts have a genetic basis. Protein aggregation is the single most important factor in cataract formation. METHODS: A four-generation Chinese family diagnosed with autosomal dominant congenital cataracts and microphthalmia was recruited at the Shengjing Hospital of China Medical University. Genomic DNA was extracted from the peripheral blood of the participants. All coding exons and flanking regions of seven candidate genes (CRYAA, CRYBA4, CRYBB2, CRYGC, GJA8, MAF, and PITX3) were amplified and sequenced. Restriction fragment length polymorphism (RFLP) assays were performed to confirm the candidate causative variant, c.35G > T in the CRYAA gene. We constructed pcDNA3.1(+)-CRYAA expression plasmids containing either the wild-type or the R12L mutant alleles and respectively transfected them into HEK293T cells and into HeLa cells. Western blotting was performed to determine protein expression levels and protein solubility. Immunofluorescence was performed to determine protein sub-cellular localization. RESULTS: A heterozygous variant c.35G > T was identified in exon 1 of CRYAA, which resulted in a substitution of arginine to leucine at codon 12 (p.R12L). The nucleotide substitution c.35G > T was co-segregated with the disease phenotype in the family. The mutant R12L-CRYAA in HEK293T cells showed a significant increase in the expression level of the CRYAA protein compared with the wild-type cells. Moreover, a large amount of the mutant protein aggregated in the precipitate where the wild-type protein was not detected. Immunofluorescence studies showed that the overexpressed mutant CRYAA in HeLa cells formed large cytoplasmic aggregates and aggresomes. CONCLUSIONS: In summary, we described a case of human congenital cataract and microphthalmia caused by a novel mutation in the CRYAA gene, which substituted an arginine at position 12 in the N-terminal region of αA-crystallin. The molecular mechanisms that underlie the pathogenesis of human congenital cataract may be characterized by the prominent effects of the p.R12L mutation on αA-crystallin aggregation and solubility. Our study also expands the spectrum of known CRYAA mutations.
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Catarata/genética , Transtornos Cromossômicos/genética , Cristalinas/genética , Microftalmia/genética , Mutação de Sentido Incorreto , Agregação Patológica de Proteínas/genética , Adulto , Povo Asiático , Sequência de Bases , Catarata/diagnóstico , Catarata/etnologia , Catarata/patologia , Criança , China , Transtornos Cromossômicos/diagnóstico , Transtornos Cromossômicos/etnologia , Transtornos Cromossômicos/patologia , Éxons , Feminino , Expressão Gênica , Testes Genéticos , Células HEK293 , Células HeLa , Heterozigoto , Humanos , Masculino , Microftalmia/diagnóstico , Microftalmia/etnologia , Microftalmia/patologia , Linhagem , Polimorfismo de Fragmento de Restrição , Agregação Patológica de Proteínas/diagnóstico , Agregação Patológica de Proteínas/etnologia , Agregação Patológica de Proteínas/patologiaRESUMO
BACKGROUND/AIMS: Age-related cataract (ARC) remains the leading cause of visual impairment among the elderly population. Long non-coding RNAs (lncRNAs) have emerged as potential regulators in many ocular diseases. However, the role of lncRNAs in nuclear ARC, a subtype of ARC, requires further elucidation. METHODS: LncRNA sequencing was performed to identify differentially expressed lncRNAs between the capsules of transparent and nuclear ARC lenses. Expression validation was confirmed by qRT-PCR. MTT assay, Calcein-AM and propidium iodide double staining, Rhodamine 123 and Hoechst double staining, EdU and transwell assay were used to determine the role of H19 or miR-675 in the viability, apoptosis, proliferation and migration of primary cultured human lens epithelial cells (HLECs). Bioinformatics and luciferase reporter assays were used to identify the binding target of miR-675. RESULTS: Sixty-three lncRNAs are differentially expressed between the capsules of transparent and nuclear ARC lenses. One top abundantly expressed lncRNA, H19, is significantly up-regulated in the nuclear ARC lens capsules and positively associated with nuclear ARC grade. H19 knockdown accelerates apoptosis development and reduces the proliferation and migration of HLECs upon oxidative stress. H19 is the precursor of miR-675, and a reduction of H19 inhibits miR-675 expression. miR-675 regulates CRYAA expression by targeting the binding site within the 3'UTR. Moreover, miR-675 increases the proliferation and migration while decreasing the apoptosis of HLECs upon oxidative stress. CONCLUSION: H19 regulates HLECs function through miR-675-mediated CRYAA expression. This finding would provide a novel insight into the pathogenesis of nuclear ARC.
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Catarata/patologia , RNA Longo não Codificante/metabolismo , Regiões 3' não Traduzidas , Idoso , Antagomirs/metabolismo , Apoptose , Sequência de Bases , Catarata/genética , Movimento Celular , Células Cultivadas , Cristalinas/genética , Cristalinas/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Humanos , Cristalino/citologia , MicroRNAs/antagonistas & inibidores , MicroRNAs/genética , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Estresse Oxidativo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Alinhamento de Sequência , Regulação para CimaRESUMO
Circular RNAs (circRNAs) are a novel class of non-coding RNAs generated from back splicing. Accumulating evidence has demonstrated their vital regulation in several biological processes and ocular diseases. However, the role of circRNAs in age-related cataract (ARC), the leading cause of visual impairment worldwide, is still unknown. CircRNA sequencing reveals that 101 circRNAs are differentially expressed between the capsules of transparent and ARC lenses, including 75 down-regulated circRNAs and 26 up-regulated circRNAs transcripts. Eight of 10 differentially expressed circRNAs are further verified by quantitative RT-PCRs. One highly conserved circRNA, circHIPK3, is significantly down-regulated in all cortical, nuclear and posterior subcapsular subtypes of ARC. The silencing of circHIPK3, but not HIPK3 mRNA, significantly accelerates apoptosis development upon oxidative stress and decreases cell viability and proliferation in primary cultured human lens epithelial cells (HLECs). The expression of α-SMA and vimentin was downregulated, while the expression of E-cadherin and ZO-1was upregulated, suggesting the repression of epithelial-mesenchymal transition after circHIPK3 knockdown. CircHIPK3 silencing increases miR-193a expression. miR-193a regulates CRYAA expression by targeting the binding site within the 3'UTR. Moreover, miR-193a decreases the viability and proliferation, and increases the apoptosis of HLECs upon oxidative stress. This study suggests that circRNAs are the potential regulators in cataractogenesis. CircHIPK3 regulates HLECs function through miR-193a-mediated CRYAA expression. This finding would provide a novel insight into the pathogenesis of ARC.
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Cristalinas/metabolismo , Células Epiteliais/citologia , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Cristalino/citologia , MicroRNAs/metabolismo , Proteínas Serina-Treonina Quinases/fisiologia , RNA/fisiologia , Apoptose , Catarata/etiologia , Proliferação de Células , Células Cultivadas , Humanos , Estresse Oxidativo , RNA CircularRESUMO
BACKGROUND: Age-related cataract (ARC) is the leading cause of visual impairment worldwide, and α-crystallin (CRYAA) is the predominant structural protein involved in the maintenance of lens clarity and refractive properties. We previously demonstrated that CRYAA genes undergo epigenetic repression in the lens epithelia in ARC. We further analyze the underlying mechanism in the current study. METHODS: The transcription factor binding sites of the CpG island of CRYAA promoter were predicted by TESS website. An electrophoretic mobility shift assay (EMSA) was used to analyze the impact of the methylation of CpG sites on transcription factors. Human lens epithelial B-3 (HLE B-3) Cells were treated with demethylation agent zebularine in the concentrations of 0 (PBS as control), 10 µM, 20 µM, 50 µM, 100 µM and 200 µM, respectively. After treatment in the above concentrations for 24 h, 48 h and 72 h, respectively, CRYAA mRNA expression levels were detected by Quantitative Real-Time RT-PCR. RESULTS: The methylation of the CpG site of the CRYAA promoter decreased the DNA-binding capacity of transcription factor Sp1. Zebularine increased CRYAA expression in HLE B-3 Cells in a dose- dependent and time- dependent pattern. CONCLUSIONS: The evidence presented suggests that the methylation of the CpG sites of the CRYAA promotor directly affect Sp1 binding, leading to down expression of CRYAA in human lens epithelial cells. Zebularine treatment could restore CRYAA expression in a dose- dependent and time- dependent pattern.
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Ilhas de CpG/genética , Cristalinas/genética , Metilação de DNA , Células Epiteliais/metabolismo , Cristalino/metabolismo , Regiões Promotoras Genéticas/genética , Fator de Transcrição Sp1/metabolismo , Células Cultivadas , Cristalinas/metabolismo , Citidina/análogos & derivados , Citidina/farmacologia , Células Epiteliais/efeitos dos fármacos , Humanos , Cristalino/efeitos dos fármacos , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo RealRESUMO
The fish Astyanax mexicanus presents, within the same species, populations of river-dwelling surface fish (SF) and blind cave-living fish. In cavefish (CF), the eyes develop almost normally during embryogenesis. But 40 h after fertilization, the lens enters apoptosis, triggering the progressive degeneration of the entire eye. Before apoptosis, the CF lens expresses early differentiation factors correctly. Here, we searched for possible late differentiation defects that would be causal in CF lens degeneration. We reasoned that crystallins, the major lens structural proteins, could be defective or misregulated. We surveyed the CF and SF transcriptomes and uncovered 14 Astyanax crystallins from the beta, gamma, lambda, mu, and zeta families. These proteins are less polymorphic and accumulate more fixed mutations, some at highly conserved positions, in CF than in SF, suggesting relaxed selection at these loci in CF. In situ hybridizations and qPCR show that crybb1c, crybgx, crygm5 are expressed at much lower levels or are not expressed in the CF lens. For the best crystallin candidates, we tested a potential causal role in CF lens apoptosis. Crybgx, crybb1c (not expressed in CF from very early on), and cryaa (previously shown to be faintly expressed in CF) failed to induce any defect when knocked-down in zebrafish embryos. However, the anti-apoptotic cryaa protected lens cells from apoptosis when reexpressed by transgenesis in CF, suggesting a cell-autonomous effect of cryaa on lens cell survival. Altogether, these data suggest that crystallin sequence evolution and expression defects may contribute to the loss of eyes in CF.
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Evolução Molecular , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Cristalino/metabolismo , Peixe-Zebra/metabolismo , alfa-Cristalinas/metabolismo , Animais , Apoptose/fisiologia , Diferenciação Celular/fisiologia , Sobrevivência Celular/fisiologia , Cristalino/anormalidades , Transcriptoma/fisiologia , Peixe-Zebra/embriologia , Peixe-Zebra/genéticaRESUMO
PURPOSE: To describe at molecular level a family with pulverulent congenital cataract associated with a CRYGC gene mutation. METHODS: One family with several affected members with pulverulent congenital cataract and 230 healthy controls were examined. Genomic DNA from leukocytes was isolated to analyze the CRYGA-D cluster, CX46, CX50 and MIP genes through high-resolution melting curve and DNA sequencing. RESULTS: DNA sequencing in the affected members revealed the c.143G>A mutation (p.R48H) in exon 2 of the CRYGC gene; 230 healthy controls and ten healthy relatives were also analyzed and none of them showed the c.143G>A mutation. No other polymorphisms or mutations were found to be present. CONCLUSION: In the present study, we described a family with pulverulent congenital cataract that segregated the c.143G>A mutation (p.R48H) in the CRYGC gene. A few mutations have been described in the CRYGC gene in autosomal dominant cataract, none of them with pulverulent cataract making clear the clinical heterogeneity of congenital cataract. This mutation has been associated with the phenotype of congenital cataract but also is considered an SNP in the NCBI data base. Our data and previous report suggest that p.R48H could be a disease-causing mutation and not an SNP.