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Background The aim of this study is to identify the genetic defects in a Chinese family with fundus albipunctatus. Methods Complete ophthalmic examinations, including slit-lamp biomicroscopy, dilated indirect ophthalmoscopy, fundus photography, autofluorescence, swept source optical coherence tomography (SS-OCT) and full-field electroretinography (ffERG) were performed. Genomic DNA was extracted from blood samples and whole genome sequencing was performed. Variants were validated with Sanger sequencing. Results Six members in this Chinese family, including three affected individuals and three controls, were recruited in this study. The ophthalmic examination of three recruited patients was consistent with fundus albipunctatus. Three variants, a novel frameshift deletion c.39delA [p.(Val14CysfsX47] and a haplotype of two rare missense variants, c.683G > A [p.(Arg228Gln)] along with c.710A > G [p.(Tyr237Cys], within the retinal dehydrogenase 5 (RDH5) gene were found to segregate with fundus albipunctatus in this family in an autosomal recessive matter. Conclusion We identified novel compound heterozygous variants in RDH5 responsible for fundus albipunctatus in a large Chinese family. The results of our study further broaden the genetic defects of RDH5 associated with fundus albipunctatus.

Retinal cell damage caused by diabetes leads to retinal microvascular injury. Roundabout 4 (ROBO4) is involved in angiogenesis, which varies with the development of diabetic retinopathy (DR). Here, we explored the transcriptional regulation and microRNA‐mediated modulation of ROBO4 expression and related retinal cell function in DR. A streptozotocin‐induced type I diabetic animal model was established to detect the expression of hypoxia inducible factor‐1α (HIF‐1α), specificity protein 1 (SP1) and ROBO4. Retinal pigment epithelium (RPE) cells were cultured under hyperglycaemia or hypoxia and used for mechanistic analysis. Furthermore, roles of miR‐125b‐5p and miR‐146a‐5p were evaluated, and their targets were identified using luciferase assays. The cell functions were evaluated by MTS assays, permeability analysis and migration assays. The development of DR increased the levels of HIF‐1α, SP1 and ROBO4 both in the DR model and in hyperglycaemic/hypoxic RPE cells. They were co‐expressed and up‐regulated in diabetic retinas and in RPE cells under hyperglycaemia/hypoxia. Knockdown of HIF‐1α significantly inhibited SP1 and ROBO4, whereas SP1 down‐regulation abolished ROBO4 expression in RPE cells under hyperglycaemia/hypoxia. miR‐125b‐5p and miR‐146a‐5p were down‐regulated by hyperglycaemia and/or hypoxia. Up‐regulation of miRNAs reversed these changes and resulted in recovery of target gene expression. Moreover, luciferase assays confirmed miR‐125b‐5p targeted SP1 and ROBO4, and miR‐146a‐5p targeted HIF‐1α and ROBO4 directly. The decreased cell viability, enhanced permeability, and increased cell migration under DR conditions were mitigated by knockdown of HIF‐1α/SP1/ROBO4 or up‐regulation of miR‐125b‐5p/miR‐146a‐5p. In general, our results identified a novel mechanism that miR‐125b‐5p/miR‐146a‐5p targeting HIF‐1α/SP1‐dependent ROBO4 expression could retard DR progression.

Purpose Vascular endothelial growth factor (VEGF) plays implicated roles in diabetic retinopathy (DR). The role of roundabout 4 (Robo 4) in angiogenesis and vasculogenesis is controversial; however, the interdependent relationship between these two factors has not been studied in DR. This study determined the colocalization of VEGF and Robo4 in fibrovascular membranes (FVM) from patients with proliferative diabetic retinopathy (PDR). MicroRNA (miRNA)-mediated modulation of VEGF and Robo4 was explored in diabetic rats and ARPE-19 tissue culture cells under hyperglycemia. Methods VEGF and Robo4 co-expression in the FVM was analyzed using immunofluorescence. VEGF and Robo4 levels were determined in diabetic retinas and ARPE-19 tissue culture cells under high glucose using western blotting and RT-qPCR. MicroRNA agomir was intraocularly injected to increase miR-15a expression and downregulate VEGF and Robo4 levels in diabetic retinas. Results VEGF and Robo4 colocalization in FVM vessels was observed. Increased VEGF levels were consistent in diabetic retinas and ARPE-19 tissue culture cells cultured under hyperglycemia. Robo4 decreased in ARPE-19 tissue culture cells exposed to hyperglycemia for 72 h, whereas it increased in diabetic rat retinas. Several miRNAs were differentially expressed during DR progression. Furthermore, miR-15a agomir injection inhibited high levels of VEGF and Robo4 in diabetic retinas. Conclusions VEGF and Robo4 were co-expressed in FVMs from PDR patients. In the early stages of DR, VEGF was upregulated and contributed to DR development, whereas, in the late stage of DR, VEGF and Robo4 worked together to aggravate DR progression. However, miR-15a could downregulate VEGF and Robo4 to ameliorate DR development.

Aim. To describe epidemiologic and clinical characteristics and prognostic factors influencing visual outcome after intraocular foreign bodies (IOFBs) injury. Methods. Medical records of 370 patients (373 eyes) with IOFBs were reviewed to identify the factors influencing visual acuity by univariate and multivariate analyses. Results. The majority of patients (97.0%) were men, with a mean age of 38.1 years. The most common cause of ocular injury was hammering (52.6%); magnetic IOFBs occurred in 84.7% of these cases. Factors associated with poor visual outcome (defined as P=0.046); worse presenting visual acuity (P<0.001); complications of retinal breaks (P=0.006) and endophthalmitis (P=0.032); vitrectomy (P=0.035); and intraocular C3F8 gas tamponade (P=0.038). Excellent visual outcome (defined as ≥0.5 logMAR) was associated with age P=0.003); better presenting visual acuity (PVA) (P<0.001); wound length P=0.005); absence of vitreous hemorrhage (P=0.026) and retinal breaks (P<0.001); nonvitrectomy surgery (P=0.043); and use of balanced saline (P=0.029). Conclusions. Multiple prognostic factors were identified that may predict visual outcome and globe survival after IOFBs injury. Age, initial presenting visual acuity, wound length, complications (vitreous hemorrhage, retinal breaks, and endophthalmitis), surgical approach, and intraocular tamponade were significant predictors of visual outcome.

Empagliflozin (EMPA) is the first sodium-glucose co-transporter 2 inhibitor to significantly reduce cardiovascular and kidney complications in type 2 diabetes mellitus. Given this, we speculate that EMPA may have the potential to intervene in diabetic retinopathy (DR), which is another diabetes-specific microvascular complication. Db/db mice were treated with EMPA for different periods to observe the retinas and related mechanisms. EMPA effectively balanced body weight and blood glucose levels, mitigated ocular edema and microaneurysm in db/db mice. EMPA significantly inhibited oxidative stress, apoptosis and recovered tight junction in diabetic retinas. MS/MS analyses showed that EMPA suppressed aberrant branched-chain amino acid (BCAAs) accumulation in db/db retinas, which led to the inhibition of the mammalian target of rapamycin activation, downregulation of inflammation, and angiogenic factors, including TNF-ɑ, IL-6, VCAM-1, and VEGF induced by diabetes. Furthermore, branched-chain α-keto acids (BCKAs), which are catabolites of BCAAs, were increased in diabetic retinas and decreased with EMPA application. Moreover, branched-chain ketoacid dehydrogenase kinase (BCKDK) was enhanced, BCKDHA and BCKDHB were decreased in diabetic retinas. This could be reversed by EMPA treatment, thus promoting BCAAs catabolism to decrease BCAAs and BCKAs accumulation in diabetic retinas. The high levels of BCAAs in the plasma and enhanced L-type amino acid transporter 1 (LAT1) were responsible for the high levels of BCAAs in diabetic retinas, which could be inhibited by EMPA. Overall, EMPA could ameliorate DR manifestations. The normalization of BCAAs catabolism and intake may play a role in this process. This study supports EMPA as a protective drug against DR.

IntroductionVitrectomy combined with internal limiting membrane (ILM) peeling, flap or tamponade is widely used in the treatment of macular diseases, such as macular hole (MH) and high myopia macular hole retinal detachment (HMMHRD). However, movement of the ILM to a suitable position to prevent displacement is a difficult operation. Improving visual function after surgery remains controversial. Compared with ILM, the thicker and more flexible lens capsule is easy to obtain and operate. Previous studies have confirmed the effectiveness of lens capsule flap in the treatment of MH. This study aims to evaluate the efficacy and safety of vitrectomy combined with lens capsule flap transplantation in the treatment of HMMHRD.Methods and analysisThis single-centre, single-blind, prospective, randomised clinical trial will include 54 patients with HMMHRD who will first undergo phacoemulsification and intraocular lens implantation and then vitrectomy combined with lens capsule flap transplantation (experimental group) or ILM tamponade (control group). Study participants will be randomly allocated in a 1:1 ratio to experimental and control groups. Follow-up will be conducted 1, 3 and 7 days and 1, 3 and 6 months after surgery in both groups. Necessary examinations will be performed at each follow-up visit. Measurement outcomes include postoperative situation of macular hole closure, best-corrected visual acuity, macular retinal function and macular retinal sensitivity. The primary outcome is type I closure rate of MH 6 months after operation. Intergroup comparisons of the proportions of patients with type I closure of MH will be performed with Fisher’s exact test.Ethics and disseminationFull ethics approval for this study was obtained from the Ethics Committee of Shanghai General Hospital, Shanghai Jiaotong University, Shanghai, China. The outcomes of the trial will be disseminated through peer-reviewed journals and at scientific conferences.Trial registration numberChiCTR2200057836.

Background: The aim of this study is to identify the genetic defect in a Chinese family with congenital aniridia combined with cataract and nystagmus.Methods: Complete ophthalmic examinations, including slit-lamp biomicroscopy, dilatedindirect ophthalmoscopy, anterior segment photography, and anterior segment optical coherence tomography (OCT) were performed. Blood samples were collected from all family members and genomic DNA was extracted. Genome sequencing was performed in all family members and Sanger sequencing was used to verify variant breakpoints.Results: All the thirteen members in this Chinese family, including seven patients and six normal people, were recruited in this study. The ophthalmic examination of affected patients in this family was consistent with congenital aniridia combined with cataract and nystagmus. A novel heterozygous deletion (NC_000011.10:g.31802307_31806556del) containing the 5’ region of PAX6 gene was detected that segregated with the disease. Conclusion: We detected a novel deletion in PAX6 responsible for congenital aniridia in the affected individuals of this Chinese family. The novel 4.25kb deletion in PAX6 gene of our study would further broaden the genetic defects of PAX6 associated with congenital aniridia.

Autophagy is a self-degradative pathway involving intracellular substance degradation and recycling. Recently, this process has attracted a great deal of attention for its fundamental effect on physiological processes in cells, tissues, and the maintenance of organismal homeostasis. Dysregulation of autophagy occurs in some diseases, including immune disease, cancer, and neurodegenerative conditions. Diabetic retinopathy (DR), as a serious microvascular complication of diabetes, is the main cause of visual loss in working-age adults worldwide. The pathogenic mechanisms of DR are thought to be associated with accumulation of oxidative stress, retinal cell apoptosis, inflammatory response, endoplasmic reticulum (ER) stress, and nutrient starvation. These factors are closely related to the regulation of autophagy under pathological conditions. Increasing evidence has demonstrated the potential role of autophagy in the progression of DR through different pathways. However, to date this role is not understood, and whether the altered level of autophagy flux protects DR, or instead aggravates the progression, needs to be explored. In this review, we explore the alterations and functions of autophagy in different retinal cells and tissues under DR conditions, and explain the mechanisms involved in DR progression. We aim to provide a basis on which DR associated stress-modulated autophagy may be understood, and to suggest novel targets for future therapeutic intervention in DR.

Background The aim of this study is to identify the genetic defect in a Chinese family with congenital aniridia combined with cataract and nystagmus. Methods Complete ophthalmic examinations, including slit-lamp biomicroscopy, dilated indirect ophthalmoscopy, anterior segment photography, and anterior segment optical coherence tomography (OCT) were performed. Blood samples were collected from all family members and genomic DNA was extracted. Genome sequencing was performed in all family members and Sanger sequencing was used to verify variant breakpoints. Results All the thirteen members in this Chinese family, including seven patients and six normal people, were recruited in this study. The ophthalmic examination of affected patients in this family was consistent with congenital aniridia combined with cataract and nystagmus. A novel heterozygous deletion (NC_000011.10:g.31802307_31806556del) containing the 5′ region of PAX6 gene was detected that segregated with the disease. Conclusion We detected a novel deletion in PAX6 responsible for congenital aniridia in the affected individuals of this Chinese family. The novel 4.25 kb deletion in PAX6 gene of our study would further broaden the genetic defects of PAX6 associated with congenital aniridia.

Purpose To quantify the levels of three inflammatory cytokines in the aqueous humour of patients with prior acute primary angle closure (APAC) and investigate their correlation with surgical outcomes of trabeculectomy. Methods In this prospective cohort study, aqueous humour samples were collected from 44 prior APAC eyes. Analyte concentrations of monocyte chemoattractant protein-1 (MCP-1), vascular endothelial growth factor (VEGF) and interleukin-6 (IL-6) were measured using multiplexed immunoassay kits. Intraocular pressure was measured using Goldmann application tonometry. Results Forty-four prior APAC eyes were followed up for 24 months after trabeculectomy and divided into success and failure groups according to surgical outcomes. Monocyte chemoattractant protein-1 (MCP-1) levels in the aqueous humour were significantly higher in the failure group (p = 0.0118). Univariate and multivariate analyses showed that MCP-1 level was a significant risk factor for trabeculectomy outcomes (univariate analysis: p = 0.016, odds ratio = 14.538; multivariate analysis: p = 0.023, odds ratio = 13.718). When prior APAC eyes were divided according to MCP-1 levels, the overall success rate was significantly higher in eyes with low MCP-1 levels than eyes with high MCP-1 levels (p = 0.0249). Conclusion In prior APAC patients, the MCP-1 level in the aqueous humour predicts trabeculectomy results. Therefore, modulation of MCP-1 expression may have potential clinical applications after filtration surgery.

Roundabout4 (Robo4) is a gene that is expressed specifically in vasculature and is involved in the angiogenesis and integrity of blood vessels. The expression level of Robo4 increases gradually along with the development of diabetic retinopathy (DR). In this study, we explored the mechanism of transcriptional regulation of Robo4 in retinal endothelial cells, and investigated the effects of this regulation on cellular functions under hyperglycemic conditions. Human retinal endothelial cells (HREC) exposed to hyperglycemia were used to detect the expression levels of specificity protein 1 (SP1) and Robo4 by RT-qPCR and western blotting. Small interfering RNA (SiRNA) transfection technology was used to analyze the regulatory relationship between SP1 and Robo4. The effect of transcription factor SP1 on Robo4 promoter activity and the location of SP1 binding sites were investigated using chromatin immunoprecipitation (ChIP) and luciferase assay. Cell migration, monolayer permeability and tube formation assays were performed to demonstrate the role of SP1/Robo4 in regulating HREC functions in hyperglycemic conditions. The results showed that hyperglycemia upregulated the mRNA and protein levels of SP1 and Robo4 in HREC. Depletion of SP1 by siRNA transfection inhibited the hyperglycemia induced overexpression of Robo4. ChIP combined with luciferase assay showed that under hyperglycemic conditions, SP1 significantly increased the transcriptional level of Robo4 via an additional SP1 binding site at − 1912/− 1908 in the Robo4 promoter. Repressing the SP1/Robo4 pathway effectively mitigated the abnormity in HREC migration, permeability and angiogenesis induced by hyperglycemia. All these findings indicate that hyperglycemia-induced upregulation of Robo4 is mediated by enhanced transcription of SP1. The SP1/Robo4 signaling pathway can regulate the migratory ability, monolayer permeability and angiogenesis of HREC under hyperglycemic conditions, suggesting that it may play an important role in microvascular dysfunction during DR.

BACKGROUND Diabetic retinopathy is a primary contributor of visual impairment in adult diabetes mellitus patients. Diabetic retinopathy causes breakdown of blood retinal barrier (BRB), and leads to diabetic macular edema. Previous studies have demonstrated angiopoietin-like protein 4 (ANGPTL4) as an effective diabetic retinopathy therapeutic target, however, its role in maintaining the outer BRB in diabetic retinopathy has yet not elucidated. MATERIAL AND METHODS We established an in vivo diabetic rat model with the use of streptozotocin injections and cultured ARPE-19 cells under (hypoxia, 1%) condition. We first investigated the expression of hypoxia induced factor-1alpha (HIF-1alpha) and ANGPTL4 in vivo and subsequently studied the transcriptional regulation and underlying molecular mechanisms in ARPE-19 cells under oxygen-deprived situations. RESULTS The expression of HIF-1alpha and ANGPTL4 was increased with diabetic retinopathy progression both in vivo and in vitro. Depletion of HIF-1alpha by siRNA inhibited hypoxia-induced ANGPTL4 expression. Repressing the HIF-1alpha/ANGPTL4 signaling effectively alleviated the migration and cellular permeability induced by hypoxia in ARPE-19 cells. Depletion of ANGPTL4 by siRNA significantly alleviated signal transducer and activator of transcription 3 (STAT3) activity in vitro, thereby attenuating the decrease of tight junction proteins occludin and zona occludens-1 (ZO-1) under hypoxia in ARPE-19 cells. CONCLUSIONS Our results suggest that ANGPTL4 partially modulates STAT3 and could serve as an effective diabetic retinopathy treatment strategy.

Inhibitor of growth 4 (ING4), a potential tumor suppressor, is implicated in cell migration and angiogenesis. However, its effects on diabetic retinopathy (DR) have not been elucidated. In this study, we aimed to evaluate ING4 expression in normal and diabetic rats and clarify its effects on hypoxia-induced dysfunction in human retinal pigment epithelial (ARPE-19) cells. A Type 1 diabetic model was generated by injecting rats intraperitoneally with streptozotocin and then killed them 4, 8, or 12 weeks later. ING4 expression in retinal tissue was detected using western blot analysis, reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR), and immunohistochemistry assays. After transfection with an ING4 overexpression lentiviral vector or small interfering RNA (siRNA), ARPE-19 migration under hypoxia was tested using wound healing and transwell assays. The angiogenic effect of conditioned medium (CM) from ARPE-19 cells was examined by assessing human retinal endothelial cell (HREC) capillary tube formation. Additionally, western blot analysis and RT-qPCR were performed to investigate the signaling pathways in which ING4, specificity protein 1 (Sp1), matrix metalloproteinase 2 (MMP-2), MMP-9, and vascular endothelial growth factor A (VEGF-A) were involved. Here, we found that ING4 expression was significantly reduced in the diabetic rats' retinal tissue. Silencing ING4 aggravated hypoxia-induced ARPE-19 cell migration. CM collected from ING4 siRNA-transfected ARPE-19 cells under hypoxia promoted HREC angiogenesis. These effects were reversed by ING4 overexpression. Furthermore, ING4 suppressed MMP-2, MMP-9, and VEGF-A expression in an Sp1-dependent manner in hypoxia-conditioned ARPE-19 cells. Overall, our results provide valuable mechanistic insights into the protective effects of ING4 on hypoxia-induced migration and angiogenesis regulation in ARPE-19 cells. Restoring ING4 may be a novel strategy for treating DR.

Diabetic retinopathy (DR) is the leading cause of blindness in working-age adults across the world. The pathogenesis of DR is multifactorial and the molecular mechanisms are still not fully understood. Accumulating evidence has demonstrated that noncoding RNAs (ncRNAs) may be aberrantly expressed and may play vital roles in the development of DR. Amongst ncRNAs, miRNAs and long ncRNAs (lncRNAs) are known for their regulatory functions. Here, we summarize the functions and mechanisms of known aberrantly expressed miRNAs and lncRNAs in DR. Additionally, a novel lncRNA–mRNA–miRNA network is included in this review. We highlight original studies that provide detailed data about the mechanisms of miRNAs and lncRNAs, their applications as diagnostic or prognostic biomarkers, and their potential therapeutic targets. In conclusion, this review will help us gain a better understanding of the molecular mechanisms by which miRNAs and lncRNAs perform their functions in DR, and provide general strategies and directions for future research.

Rationale: Neuromyelitis optica spectrum disorders (NMOSD) is considered as an immune-mediated disorder in the central nervous system (CNS). Numerous autoimmune diseases are frequently complicated with NMOSD and distinct clinical characteristics are noted in NMOSD patients with other autoimmune diseases. However, to our best knowledge, co-occurrence of NMOSD and thrombopenic purpura is rarely identified. Patient concerns: We presented a rare case of a 72-year-old female with 6-year history of thrombopenic purpura, and 1-month history of blurred vision as well as chest zonethesia. Anti-aquaporin-4 (AQP4) antibodies was positive in the serum of the patient. Diagnoses: With the addition of laboratory findings, iconography findings and physical examination results, the diagnosis of NMOSD was established according to the most recent diagnostic criteria. Interventions and outcomes: With the treatment of intravenous immunoglobulin (IVIg), the patient felt better at discharge without changing of expanded disability status scale (EDSS) score. Lessons: The case indicates that NMOSD could co-occur with thrombopenic purpura. The disturbance of immune system balance may explain this overlap. Further studies are warranted to reveal the mechanism and to explore whether patients with NMOSD with and without thrombopenic purpura have distinct clinical feature, drug responsiveness or prognosis.

The pathological mechanisms of diabetic retinopathy (DR), a leading cause of blindness in adults with diabetes mellitus, remain incompletely understood. Because microRNAs (miRNAs) represent effective DR therapeutic targets, we identified aberrantly expressed miRNAs associated with cellular dysfunction in early DR and detected their potential targets. We exposed human retinal endothelial cells (HRECs) and a cell line of retinal pigment epithelial (RPE) cells to high glucose (25 mmol/L, 1–7 days) to mimic DR progression and used streptozotocin-injected rats (4–8 weeks) for an in vivo diabetes model. HREC/RPE viability decreased after 24 h incubation and diminished further over 6 days, and Hoechst staining revealed hyperglycemia-induced HREC/RPE apoptosis. Although miR-124/-125b expression decreased with DR progression in vitro and in vivo, miR-135b/-199a levels decreased in retinal cells under hyperglycemia exposure, but increased in diabetic retinas. Moreover, miR-145/-146a expression decreased gradually in high-glucose-treated HRECs, but increased in hyperglycemia-exposed RPE cells and in diabetic rats. Our findings suggested that aberrant miRNA expression could be involved in hyperglycemia-induced retinal-cell dysfunction, and the identified miRNAs might vary in different retinal layers, with expression changes associated with DR development. Therefore, miRNA modulation and the targeting of miRNA effects on transcription factors could represent novel and effective DR-treatment strategies.

MicroRNA-124 (miR-124) has been shown to be downregulated in glioma; however, its biological functions in glioma are not yet fully understood. The aim of this study was to examine the Smad4‑dependent effects of miR‑124 on C6 glioma cell proliferation. In this study, the level of miR‑124 was found to be enhanced in C6 cells upon transfection with miR‑124 mimics, and the mechanisms of action of miR‑124 in C6 cells were investigated by reverse transcriptase-quantitative polymerase chain reaction, MTT assay, western blot analysis and luciferase reporter assays in vitro. The results revealed that miR‑124 expression was significantly lower in the C6 cells than in either normal rat brain tissue or astrocytes. Upon the overexpression of miR‑124, the proliferation of the C6 cells decreased and Smad4 expression was significantly suppressed. Smad4 was identified as a direct target of miR‑124 through luciferase reporter assays. Furthermore, miR‑124 was found to modulate signal transducer and activator of transcription 3 (Stat3) by downregulating Smad4 expression. Using small interfering RNA targeting Smad4 mRNA, we also confirmed that miR‑124 downregulated c‑Myc by modulating Smad4 expression. In addition, caspase‑3 expression was induced by miR‑124 overexpression, but not via Smad4 downregulation. On the whole, our results demonstrate that miR‑124 upregulation inhibits the growth of C6 glioma cells by targeting Smad4 directly. These findings may be clinically useful for the development of therapeutic strategies directed toward miR‑124 function in patients with glioma.

Retinal pigment epithelium (RPE) cell-based gene expression studies performed under hypoxia and/or hyperglycemia show huge potential for modeling cell responses in diabetic retinopathy, retinopathy of prematurity and other retinal diseases. However, normalization of gene expression on RPE cells under those conditions has commonly been done using either GAPDH or β-actin as reference genes without any validation of their expression stability. Therefore, we aimed to establish a suitable set of reference genes for studies on RPE cells cultured under both normal culturing glucose and atmospheric oxygen tension (normoxia, 21%), under a low oxygen tension (hypoxia, 1%), under a high glucose growth medium (25 mmol/l) and under the combination of the two changed conditions above for distinct time points taking together from 24h to 7 days. Quantitative real-time PCR (qRT-PCR) was applied on RNA obtained from a cell line, ARPE-19. Stability of 14 commonly used reference genes was assessed and ranked according to their stability values using the geNorm and NormFinder softwares with the aim to find the most stable expressed gene under all conditions. Our findings confirm that HPRT1, GUSB and PPIA are the most suitable reference genes for RPE cell gene expression experiments subjected to hypoxia and/or hyperglycemia. To emphasize the importance of selecting the most stably expressed reference genes for obtaining reliable results, mRNA expression levels of hypoxia induced factor-1α were analyzed vs the best reference genes, the worst ones and the most commonly used ones. These reference genes gave the most reliable normalization for comparative analyses of gene transcription under those conditions.