Molecular Vision最新文献

Purpose: In our earlier study, we identified hsa-miR-150-5p as a highly expressed miRNA in enriched corneal epithelial stem cells (CESCs). In this study, we aimed to understand the molecular regulatory function of hsa-miR-150-5p in association with the maintenance of stemness in CESCs.

Methods: The target mRNAs of hsa-miR-150-5p were predicted and subjected to pathway analysis to identify targets for functional studies. Primary cultured limbal epithelial cells were transfected with hsa-miR-150-5p mimic, inhibitor, or scrambled sequence using Lipofectamine 3000. The transfected cells were analyzed to determine (i) their colony-forming potential; (ii) the expression levels of stem cell (SC) markers/transcription factors (ABCG2, NANOG, OCT4, KLF4, and ΔNp63), the differentiation marker (Cx43), and the hsa-miR-150-5p predicted targets (JARID2, INHBA, AKT3, and CTNNB1) by qPCR; and (iii) the expression levels of ABCG2, p63α, Cx43, JARID2, AKT3, p-AKT3, β-catenin, and active β-catenin by immunofluorescence staining and/or western blotting.

Results: The ectopic expression level of hsa-miR-150-5p increased the colony-forming potential (8.29% ± 0.47%, p < 0.001) with the ability to form holoclone-like colonies compared with the control (1.8% ± 0.47%). The mimic-treated cells had higher expression levels of the SC markers but reduced expression levels of Cx43 and the targets of hsa-miR-150-5p that are involved in the Wnt-β-catenin signaling pathway. The expression levels of β-catenin and active β-catenin in the inhibitor-transfected cells were higher than those in the control cells, and the localized nuclear expression indicated the activation of Wnt signaling.

Conclusions: Our results indicate a regulatory role for hsa-miR-150-5p in the maintenance of CESCs by inhibiting the Wnt signaling pathway.

Retinoblastoma (Rb) is a rare childhood intraocular malignancy with an incidence rate of approximately 9000 children per year worldwide. The management of Rb is inherently complex and depends on several factors. The orders of priorities in the treatment of Rb are saving life, globe salvage and vision salvage. Rarity and the young age at diagnosis impede conducting randomized clinical trials (RCTs) for new therapeutic options, and therefore pre-RCTs studies are needed. This review provides an overview of advances in Rb treatment options, focusing on the emergence of new small molecules to treat Rb. Articles related to the management and treatments of Rb were searched in different databases. Several studies and animal models discussing recent advances in the treatment of Rb were included to have a better grasp of the biological mechanisms of Rb. Over the years, the principles of management and treatment of Rb have changed significantly. Innovations in targeted therapies and molecular biology have led to improved patient and ocular survival. However, there is still a need for further evaluation of the long-term effects of these new treatments.

Purpose: To evaluate the effect of diquafosol tetrasodium on the expression of secretory and membrane-associated mucins in multi-layered cultures of primary human conjunctival epithelial cells (HCEC) using intracellular extracellular signal regulated kinase (ERK) signaling.

Methods: HCECs were treated with hyperosmotic stress (400 mOsm/l) for 24 h after air-liquid interface cell culture followed by treatment with diquafosol. HCECs were stimulated for 1 h with or without PD98059, an ERK inhibitor, then treated with diquafosol for 6 h and 24 h. Mucin 1 (MUC1), mucin 16 (MUC16), and MUC5AC mRNA and protein expression levels were analyzed, and cell viability was detected using an MTT assay. Western blot analysis was used to examine p44/42 MAPK (Erk1/2) and phosphorylated p44/42 MAPK (Erk1/2) expression.

Results: Hyperosmotic stressed HCECs demonstrated increased MUC5AC secretion and gene expression when treated with diquafosol. MUC1 mRNA levels increased significantly at 24 h (p<0.01), and expression of MUC16 mRNA levels increased at 6 h and were maintained until 24 h (p<0.05).There was no significant difference in cell viability compared to the control group. Immunostaining results for MUC1, MUC16, and MUC5AC in diquafosol tetrasodium-treated HCECs at 24 h showed more positive cells than in the control group. Phosphorylation of p44/42 MAPK (Erk1/2) signaling molecules significantly increased from 5 min to 60 min (p<0.05). The effects of diquafosol on mucin expressions in hyperosmotic stressed HCECs were significantly inhibited by PD98059, an ERK inhibitor, at 6 h and 24 h.

Conclusions: ERK signaling may regulate the expression levels of MUC1, MUC16, and MUC5AC induced by diquafosol in hyperosmotic stressed HCECs.

Purpose: Retinitis pigmentosa (RP) is a group of highly heterogenetic inherited retinal degeneration diseases. Molecular genetic diagnosis of RP is quite challenging because of the complicated disease-causing mutation spectrum. The aim of this study was to explore the mutation spectrum in Chinese RP patients using next-generation sequencing technology and to explore the genotype-phenotype relationship.

Method: In this study, a cost-effective strategy using whole-exome sequencing (WES) was employed to address the genetic diagnosis of 28 RP families in China. One to two patients and zero to two healthy relatives were sequenced in each family. All mutations in WES data that passed through the filtering procedure were searched in relation to 662 gene defects that can cause vision-associated phenotypes (including 89 RP genes in the RetNet Database). All patients visiting the outpatient department received comprehensive ophthalmic examinations.

Result: Twenty-five putative pathogenic mutations of 12 genes were detected by WES and were all confirmed by Sanger sequencing in 20 (20/28, 71.4%) families, including the 12 following genes: USH2A, CYP4V2, PRPF31, RHO, RP1, CNGA1, CNGB1, EYS, PRPF3, RP2, RPGR, and TOPORS. Three families were rediagnosed as having Bietti crystalline dystrophy (BCD). USH2A (4/20, 20%) and CYP4V2 (3/20, 15%) were found to be the most frequent mutated genes. Seven novel mutations were identified in this research, including mutations in USH2A1, USH2A2, PRPF31, RP2, TOPORS, CNGB1, and RPGR. Phenotype and genotype relationships in the 12 RP genes were analyzed, which revealed later disease onset and more severe visual function defects in CYP4V2.

Conclusion: Twenty-five putative pathogenic mutations of 12 genes were detected by WES, and these were all confirmed by Sanger sequencing in 20 (20/28, 71.4%) families, including seven novel mutations. USH2A and CYP4V2 were found to be the most frequent genes in this research. Phenotype and genotype relationships were revealed, and the mutation spectrum of RP in Chinese populations was expanded in this research, which may benefit future cutting-edge therapies.

Purpose: Orbital venous malformation (OVM), the most common type of vascular malformation in adults, has a great impact on both visual and cosmetic factors. Circular RNAs (circRNAs) play important roles in various ophthalmological diseases; however, little is known about their function in the pathogenesis of OVM.

Methods: We obtained differentially expressed circRNAs, mRNAs, and miRNAs based on RNA sequencing of four OVM tissues and four normal orbital vascular tissues. The circRNA-mRNA coexpression network and circRNA-miRNA-mRNA and competing endogenous RNA (ceRNA) networks were constructed using miRanda software. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed to identify the up- and downregulated mRNAs in the circRNA-miRNA-mRNA ceRNA network.

Results: Overall, we identified 45 upregulated and 144 downregulated circRNAs, as well as 2,175 upregulated and 1,274 downregulated mRNAs and 156 upregulated and 168 downregulated miRNAs in OVM samples compared with normal orbital vascular tissues. The expression changes of mRNAs and circRNAs detected by quantitative real-time PCR (qRT-PCR) were in line with the RNA-seq results. Then, a ceRNA regulatory network was constructed with these differentially expressed circRNAs, mRNAs, and miRNAs. GO functional analysis revealed that most related biological processes involved extracellular matrix organization, positive regulation of actin nucleation, and so on, which were thought to be involved in the evolution of OVM. KEGG pathway analysis of upregulated mRNAs showed that mucin-type O-glycan biosynthesis, glycosaminoglycan degradation, and the PI3K (Gene ID: 5290; OMIM: 613089)-AKT (Gene ID: 207; OMIM: 114500) signaling pathway were all enriched in OVM samples.

Conclusions: Our study provides novel insight into the regulatory mechanism of circRNAs, miRNAs, and mRNAs in the pathogenesis of OVM.

Purpose: To describe the clinical phenotype and genetic basis of non-syndromic retinitis pigmentosa (RP) in one family and two sporadic cases with biallelic mutations in the transcription factor neural retina leucine zipper (NRL).

Methods: Exome sequencing was performed in one affected family member. Microsatellite genotyping was used for haplotype analysis. PCR and Sanger sequencing were used to confirm mutations in and screen other family members where they were available. The SMART tool for domain prediction helped us build the protein schematic diagram.

Results: For family MM1 of Pakistani origin, whole-exome sequencing and microsatellite genotyping revealed homozygosity on chromosome 14 and identified a homozygous stop-loss mutation in NRL, NM_006177.5: c.713G>T, p.*238Lext57, which is predicted to add an extra 57 amino acids to the normal protein chain. The variant segregated with disease symptoms in the family. For case RP-3051 of Spanish ancestry, clinical exome sequencing focusing on the morbid genome highlighted a homozygous nonsense mutation in NRL, c.238C>T, p.Gln80*, as the most likely disease candidate. For case RP-1553 of Romanian ethnicity, targeted-exome sequencing of 73 RP/LCA genes identified a homozygous nonsense mutation in NRL, c.544C>T, p.Gln182*. The variants were either rare or absent in the gnomAD database.

Conclusions: NRL mutations predominantly cause dominant retinal disease, but there have been five published reports of mutations causing recessive disease. Here, we present three further examples of recessive RP due to NRL mutations. The phenotypes observed are consistent with those in the previous reports, and the observed mutation types and distribution further confirm distinct patterns for variants in NRL causing recessive and dominant diseases.

Purpose: Positron emission tomography (PET) is widely used in high-precision imaging, which may provide a simple and noninvasive method for the detection of pathology and therapeutic effects. [¹⁸F]-DPA-714 is a second-generation translocator protein (TSPO) positron emission tomography radiotracer that shows great promise in a model of neuroinflammation. In this study, [¹⁸F]-DPA-714 micro-PET imaging was used to evaluate retinal inflammation in mice exposed to blue light, a well-established model of age-related macular degeneration (AMD) for molecular mechanism research and drug screening.

Methods: C57BL/6J melanized mice were subjected to 10,000, 15,000, and 20,000 lux blue light for 5 days (8 h/day) to develop the retinal injury model, and the structure and function of the retina were assessed using hematoxylin-eosin (HE) staining, electroretinography (ERG), and terminal-deoxynucleotidyl transferase (TdT)-mediated nick-end labeling (TUNEL) immunostaining. Then, [¹⁸F]-DPA-714 was injected approximately 100 μCi through each tail vein, and static imaging was performed 1 h after injection. Finally, the mice eyeballs were collected for biodistribution and immune analysis.

Results: The blue light exposure significantly destroyed the structure and function of the retina, and the uptake of [¹⁸F]-DPA-714 in the retinas of the mice exposed to blue light were the most significantly upregulated, which was consistent with the biodistribution data. In addition, the immunohistochemical, western blot, and immunofluorescence data showed an increase in microglial TSPO expression.

Conclusions: [¹⁸F]-DPA-714 micro-PET imaging might be a good method for evaluating early inflammatory status during retinal pathology.

Purpose: To clarify the effect of a previously identified single nucleotide polymorphism (SNP; rs76740365 G>A) in the exon-3 of the alpha A-crystallin (CRYAA) gene on the properties of CRYAA and to investigate its function in human lens epithelial cells (HLECs).

Methods: The human recombinant wild-type and mutant CRYAA (E156K) were constructed, and the molecular weight was measured by mass spectrometry. The structural changes induced by E156K mutation were analyzed by UV circular dichroism spectra and intrinsic tryptophan fluorescence and were predicted using Schrödinger software. The chaperone-like ability of wild-type and E156K mutant CRYAA was invested against the heat-induced aggregation of βL-crystallin and the DTT-induced aggregation of insulin. HLECs expressing wild-type and mutated CRYAA were subjected to quantitative PCR (qPCR) and western blot. Cell apoptosis was determined using flow cytometry analysis, and the expression of apoptosis-related proteins were determined using western blot.

Results: The mass spectrometric detection revealed that E156K mutation had no significant effect on the apparent molecular mass of the CRYAA oligomeric complex. Evaluation of the structures of the CRYAA indicated that E156K mutation did not significantly affect the secondary structures, while causing perturbations of the tertiary structure. The mutant CRYAA displayed an increase in chaperone-like activity, which might be related to the increase of the surface hydrophobicity. We also predicted that E156K mutation would induce a change from negatively charged surface to positively charged, which was the possible reason for the disturbance to the surface hydrophobicity. Transfection studies of HLECs revealed that the E156K mutant induced anti-apoptotic function in HLECs, which was possibly associated with the activation of the p-AKT signal pathway and downregulation of Casepase3.

Conclusions: Taken together, our results for the first time showed that E156K mutation in CRYAA associated with ARC resulted in enhanced chaperone-like function by inducing its surface hydrophobicity, which was directly related to the activation of its anti-apoptotic function.

Purpose: This study was designed to dissect the role of long noncoding RNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) in retinoblastoma (RB) and its underlying mechanism.

Methods: Gain- and loss-of-function experiments were adopted to explore the effects of MALAT1 and microRNA (miR)-598-3p on the biologic behaviors of RB cells. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) was used to assess the expression of MALAT1 and miR-598-3p in Y79 and HXO-RB44 cells. The proliferation of RB cells was determined with the cell counting kit-8 (CCK-8) assay and 5-ethynyl-2'-deoxyuridine (EdU) staining. Flow cytometry was employed for the measurement of the apoptotic rate, western blotting for examination of the expression of apoptosis-related proteins (Bax and Bcl-2) and phosphoinositide 3-kinase/protein kinase-B (PI3K/AKT) pathway-related factors (PI3K, AKT, p-PI3K, and p-AKT), and the luciferase reporter assay for assessment of the interaction between MALAT1 and miR-598-3p.

Results: High expression of MALAT1 and low expression of miR-598-3p were noticed in Y79 and HXO-RB44 cells. MALAT1 upregulation or miR-598-3p downregulation facilitated RB cell proliferation and inhibited cell apoptosis, as evidenced by the increased proliferation rate and Bcl-2 expression, as well as diminished Bax expression and apoptotic rate, in the RB cells after transfection with pcDNA3.1-MALAT1 or miR-598-3p inhibitor. MALAT1 bound to and negatively regulated miR-598-3p. The PI3K/AKT pathway activation occurred with MALAT1 overexpression. MALAT1 promoted RB cell proliferation and repressed cell apoptosis by repressing miR-598-3p to activate the PI3K/AKT pathway.

Conclusions: MALAT1 repressed miR-598-3p to activate the PI3K/AKT pathway, thus facilitating cell proliferation and inhibiting cell apoptosis in RB.

Background: Familial exudative vitreoretinopathy (FEVR) is a rare inherited disorder marked by incomplete retinal vascularization associated with exudation, neovascularization, and tractional retinal detachment. FEVR is genetically heterogeneous and is caused by variants in six genes: FZD4, LRP5, NDP, TSPAN12, ZNF408, and CTNNB1. In addition, the phenotypic overlap between FEVR and other disorders has been reported in patients harboring variants in other genes, such as KIF11, ATOH7, and RCBTB1.

Purpose: To identify pathogenic variants in Vietnamese pediatric patients diagnosed with FEVR and to investigate the clinical findings in correlation with each causative gene.

Methods: A total of 20 probands underwent ocular examinations with fundoscopy (ophthalmoscopy) or fluorescein angiography. Genomic DNA was extracted from the peripheral blood of the probands and their family members. Multiplex ligation-dependent probe amplification (MLPA) was employed to detect copy number variants of FEVR-causing genes. Short variants were screened by whole-exome sequencing (WES) and then validated by Sanger sequencing.

Results: Fluorescein angiography showed retinal vascular anomalies in all patients. Other ocular abnormalities commonly found were strabismus, nystagmus, exudation, and retinal detachment. Genetic analysis identified 12 different variants in the FZD4, NDP, KIF11, and ATOH7 genes among 20 probands. Four variants were novel, including FZD4 c.169G>C, p.(G57R); NDP c.175-3A>G, splicing; KIF11 c.2146C>T, p.(Q716*) and c.2511_2515del, p.(N838Kfs*17). All patients with the KIF11 variant showed signs of microcephaly and intellectual disability. The patient with Norrie syndrome and their family members were found to have a deletion of exon 2 in the NDP gene.

Conclusions: This study sheds light on the genetic causes of ocular disorders with the clinical expression of FEVR in Vietnamese patients. WES was applied as a comprehensive tool to identify pathogenic variants in complex diseases, such as FEVR, and the detection rate of pathogenic mutations was up to 60%.