The RNA N6-methyladenosine demethylase FTO regulates ATG5 to inhibit malignant progression of uveal melanoma.

PURPOSE: This research aimed to identify the function of fat mass- and obesity-associated protein (FTO), an eraser of N6-methyladenosine (m6A), and explore its possible mechanisms in uveal melanoma (UVM). METHODS: We performed quantitative real-time PCR (qPCR), Western blotting and gene correlation analysis with GEPIA2 to assess FTO expression and identify its potential targets in UVM. CCK-8, colony formation, cell cycle, cell apoptosis, wound healing and Transwell invasion assays were utilized to assess cell viability, cell cycle distribution, apoptosis, migration and invasion. Western blotting, qPCR and methylated RNA immunoprecipitation-qPCR (MeRIP-qPCR) were carried out to explore the underlying mechanism of FTO in 2 UVM cell lines. RESULTS: FTO, a key m6A demethylase, was found to be upregulated in human UVM tissues compared with normal choroid tissues. Knockdown of FTO in Mel270 and OMM2.3 cells significantly promoted proliferation and migration and suppressed apoptosis. Mechanistically, knockdown of FTO decreased the expression of ATG5, an autophagy-related gene, leading to attenuation of autophagosome formation, thereby inhibiting autophagy. Upon FTO knockdown, increased levels of methylated ATG5 and decreased ATG5 stability were detected. Furthermore, ATG5 dramatically alleviated FTO downregulation-induced tumor growth and metastasis. CONCLUSIONS: Our research highlights the importance of the m6A demethylase FTO in UVM by demonstrating that it direct regulates ATG5-induced autophagy in an m6A-dependent manner. These findings suggest that FTO may serve as a potential therapeutic target for UVM.

Metabolomic comparison followed by cross-validation of enzyme-linked immunosorbent assay to reveal potential biomarkers of diabetic retinopathy in Chinese with type 2 diabetes.

Purpose: To identify the biomarkers in the critical period of development in diabetic retinopathy (DR) in Chinese with type 2 diabetes using targeted and untargeted metabolomics, and to explore the feasibility of their clinical application. Methods: This case-control study described the differential metabolites between 83 Chinese type 2 diabetes mellitus (T2DM) samples with disease duration ≥ 10 years and 27 controls matched cases. Targeted metabolomics using high-resolution mass spectrometry with liquid chromatography was performed on plasma samples of subjects. The results were compared to our previous untargeted metabolomics study and ELISA was performed to validate the mutual differential metabolites of targeted and untargeted metabolomics on plasma. Multiple linear regression analyses were performed to adjust for the significance of different metabolites between groups. Result: Mean age of the subjects was 66.3 years and mean T2DM duration was 16.5 years. By cross-validating with results from previous untargeted metabolomic assays, we found that L-Citrulline (Cit), indoleacetic acid (IAA), 1-methylhistidine (1-MH), phosphatidylcholines (PCs), hexanoylcarnitine, chenodeoxycholic acid (CDCA) and eicosapentaenoic acid (EPA) were the most distinctive metabolites biomarkers to distinguish the severity of DR for two different metabolomic approaches in our study. We mainly found that samples in the DR stage showed lower serum level of Cit and higher serum level of IAA compared with samples in the T2DM stage, while during the progression of diabetic retinopathy, the serum levels of CDCA and EPA in PDR stage were significantly lower than NPDR stage. Among them, 4 differential key metabolites including Cit, IAA, CDCA and EPA were confirmed with ELISA. Conclusion: This is the first study to compare the results of targeted and untargeted metabolomics via liquid chromatography-mass spectrometry to find the serum biomarkers which could reflect the metabolic variations among different stages of DR in Chinese. The progression of DR in Chinese at different critical stages was related to the serum levels of specific differential metabolites, of which there is a significant correlation between DR progression and increased IAA and decreased Cit, hexanoylcarnitine, CDCA, and EPA. However, larger studies are needed to confirm our results. Based on this study, it could be inferred that the accuracy of targeted metabolomics for metabolite expression in serum is to some extent higher than that of untargeted metabolomics.

Evaluation of segmental scleral buckling surgery for stage 4A retinopathy of prematurity in China.

Aims: To describe the long-term effect of scleral buckling (SB) surgery for stage 4A retinopathy of prematurity (ROP). Methods: A retrospective chart review was conducted for patients with a diagnosis of stage 4A ROP who underwent SB between October 2010 and October 2021. Basic data were collected from patient charts, including gender, birth weight, gestational age at birth, disease stage, presence of plus disease, preoperative treatment [laser photocoagulation, intravitreal anti-vascular endothelial growth factor (VEGF) agent therapy, or a combination of both] and complications (vitreous hemorrhages), postmenstrual age at surgery, intraoperative combined treatment, and total length of follow-up. Retinal attachment status after surgery, postoperative complications (glaucoma, cataract), date and type of subsequent retinal surgeries (if performed), and refractive status 1 year after surgery were evaluated. The follow-up time after the first procedure was over 1 year. Results: Six-two eyes from forty-eight patients met the inclusion criteria for this study. The initial reattachment rate was 93.5% (58/62 eyes), and the final reattachment rate was 100% after two procedures at the end of follow-up. The incidence of cataracts was 3.2% (2/62), with no eye subsequently needing lensectomy surgery. None of the patients developed glaucoma during the follow-up time. The average spherical equivalent refraction value for patients was -3.00 ± 2.51 D (-7.60 D to +2.75 D) 1 year after surgery. Conclusion: SB, especially segmental buckling, which induces less myopia and does not require buckle removal, has the potential to provide a significant positive impact in the treatment of stage 4A ROP.

Serum Untargeted Metabolomics Reveal Potential Biomarkers of Progression of Diabetic Retinopathy in Asians.

Purpose: To reveal molecular mechanisms of diabetic retinopathy (DR) in Asians and facilitate the identification of new therapeutic targets through untargeted metabolomics. To determine the differences in serum metabolites and metabolic pathways between different stages of diabetic retinopathy in patients with type 2 diabetic mellitus (T2DM) and proliferative DR (PDR) and non-proliferative DR (NPDR) and identify differential metabolites between T2DM and DR (NPDR and PDR) patients. Methods: This prospective observational registration study described the differential metabolites between 45 T2DM patients and 15 control cases with no significant differences in clinical characteristics. Their biospecimens and clinical information were collected and recorded in their medical reports. DR phenotypes of the subjects were verified by retina specialists. Serum metabolites were analyzed using high-resolution mass spectrometry with liquid chromatography. Untargeted metabolomics was performed on serum samples from 15 T2DM patients, 15 non-proliferative diabetic retinopathy patients, 15 proliferative diabetic retinopathy patients, and 15 diabetic controls. Discriminatory metabolic features were identified through partial least squares discriminant analysis (PLS-DA), hierarchical clustering analysis (HCA), and generalized linear regression models. Result: Through untargeted metabolomics, 931 features (523 in positive and 408 in negative modes) with 102 common metabolites highly relevant to the presence of DR were detected. In the adjusted analysis, 67 metabolic features differed significantly between T2DM and NPDR patients. Pathway analysis revealed alterations in metabolisms of amino acids and fatty acids. Glutamate, phosphatidylcholine, and 13-hydroperoxyoctadeca-9,11-dienoic acid (13-PHODE) were key contributors to these pathway differences. A total of 171 features distinguished PDR patients from T2DM patients, and pathway analysis revealed alterations in amino acid metabolism, fatty acid metabolism, nitrogen metabolism, and tricarboxylic acid cycle. Aspartate, glutamate, glutamine, ornithine, N-acetyl-l-glutamate, N-acetyl-l-aspartate, citrate, succinate, N-(L-arginino)succinate, 2-oxoglutarate, 13-hydroperoxyoctadeca-9,11-dienoic acid, methionine, lysine, threonine, phenylalanine, N(pi)-methyl-l-histidine, phosphatidylcholine, and linoleate were major contributors to the pathway differences. Between NPDR patients and PDR patients, there were 79 significant differential metabolites. Enrichment pathway analysis showed changes in amino acid metabolism, fatty acid metabolism, pantothenate, and CoA biosynthesis. Aspartate, glutamine, N-acetyl-l-glutamate, N-acetyl-l-aspartate, pantothenate, dihomo-gamma-linolenate, docosahexaenoic acid, and icosapentaenoic acid were key factors for the differences of these pathways. Conclusion: This study demonstrated that the pathways of arginine biosynthesis metabolism, linoleic acid metabolism, alanine, aspartate, and glutamate metabolism, as well as d-glutamine and d-glutamate metabolism, were dysregulated in DR patients of the Asian population. Increased levels of glutamate, aspartate, glutamine, N-acetyl-l-glutamate, and N-acetyl-l-aspartate and decreased levels of dihomo-gamma-linolenate, docosahexaenoic, and icosapentaenoic were considered as the metabolic profile that could distinguish PDR from NPDR in Asians. Phosphatidylcholine and 13-PHODE were identified as two major novel metabolite markers in advanced stages of DR in our study.