Stage-aware non-coding RNA panels emerge as diabetic retinopathy biomarker framework

A structured narrative review concludes that circulating microRNAs offer the strongest current evidence for stage-associated biomarker development in diabetic retinopathy, while long non-coding RNAs and circular RNAs remain exploratory components best interpreted within network-informed panels. The review assessed human studies measuring these non-coding RNAs, or ncRNAs, in blood and extracellular vesicles, and organized the evidence around three clinical questions: detecting early disease, stratifying risk within non-proliferative diabetic retinopathy, and monitoring the boundary between severe NPDR and early proliferative disease. It found that direct evidence for discrimination between clinically adjacent stages and for prediction of future progression remains scarce.

Diabetic retinopathy is a progressive microvascular complication whose prognosis and management change substantially as it advances from non-proliferative disease to pathological neovascularization in proliferative diabetic retinopathy. Fundus photography and optical coherence tomography effectively identify established structural abnormalities, but they offer limited information about early molecular changes or the likelihood of an imminent stage transition. Existing blood markers, including vascular endothelial growth factor, inflammatory mediators, oxidative-stress indicators and metabolic measures, often reflect systemic disease, vary substantially among individuals and do not reliably distinguish retinal stages. The authors therefore propose a framework in which molecular testing would complement, rather than replace, established imaging.

MicroRNAs are short ncRNAs, generally about 18–25 nucleotides long, that regulate messenger RNA stability and translation. Long non-coding RNAs, usually longer than 200 nucleotides, influence chromatin, transcription and post-transcriptional regulation, while circular RNAs have covalently closed structures that make them comparatively resistant to degradation. All three classes have been linked in experimental and clinical literature to endothelial dysfunction, inflammation, oxidative stress and angiogenesis, processes that are central to diabetic retinopathy. MicroRNAs are the most extensively investigated circulating class and currently provide the most reproducible stage-associated signals, although evidence for most individual candidates remains limited by small cohorts, cross-sectional designs and inconsistent methods.

Early-stage evidence is particularly sparse and requires careful qualification. MicroRNA-3976 was reported as increased in a serum extracellular-vesicle fraction in a discovery and internal validation study comparing 18 people with early NPDR and diabetes but no retinopathy. MicroRNA-23a-3p was lower in serum in a single cohort of 45 people, while microRNA-126-5p was lower in plasma in an RNA-sequencing discovery, quantitative reverse-transcription PCR validation and clinical cohort involving mild NPDR. MicroRNA-26a-5p was associated with retinal nerve fibre layer thinning in an early-disease correlation study. These candidates are “anchored” to explicit early-stage comparisons or retinal structural changes, but the review’s qualitative grading assigned them low evidence because of limited replication and validation.

More established signals appear at more advanced stages. MicroRNA-21, which rises across the non-proliferative-to-proliferative severity continuum, has produced some of the strongest reported discrimination results. One study reported an area under the receiver operating characteristic curve of 0.89, with 95% confidence interval 0.86–0.96, sensitivity of 82–86% and specificity of 84–95% for distinguishing PDR from NPDR, and the review rated its evidence as moderate because it involved three independent cohorts and multistage validation. A three-microRNA serum panel comprising microRNA-21, microRNA-181c and microRNA-1179 also achieved an AUC of 0.89 for PDR versus NPDR across two validation sets. These are cross-sectional diagnostic results, not evidence that baseline microRNA levels predict subsequent progression.

Severe NPDR is the pivotal clinical transition because it precedes overt neovascularization and is associated with substantial near-term risk. Natural-history evidence cited in the review indicates approximately a 50% one-year risk of PDR conversion for severe NPDR and more than 75% for very severe NPDR. MicroRNA-21 reportedly increases progressively from normal fundus through advancing NPDR to PDR, whereas microRNA-34a declines, a pattern the authors interpret as complementary evidence of increasing inflammatory and angiogenic signaling alongside depletion of cytoprotective reserves. Yet these gradients are largely derived from cross-sectional studies. Direct comparisons of mild versus moderate NPDR, moderate versus severe NPDR, and severe NPDR versus early PDR are uncommon, and few studies preselected these adjacent-stage contrasts as primary endpoints.

Long non-coding RNAs and circular RNAs occupy a different evidentiary position. The EV-associated PPT2-EGFL8 transcript was lower in PDR than in non-PDR and diabetes-without-retinopathy groups, while NR2F1-AS1 increased across healthy, diabetes, NPDR and PDR categories. MIAT, HOTTIP and SNHG16 were higher in PDR than NPDR, and the circular RNA hsa_circ_0001953 was elevated in PDR compared with NPDR and healthy controls. Serum EV-associated circMKLN1 also rose across control, diabetes, NPDR and PDR groups. Each finding came from a single small, cross-sectional study, however, with no longitudinal prediction or independent replication. The review accordingly treats these molecules as advanced-stage-associated or context-defining candidates rather than standalone diagnostic markers.

Extracellular vesicles may help stabilize circulating ncRNAs because their lipid membranes protect cargo from enzymatic degradation, and they can selectively package molecules involved in intercellular communication. This has led some researchers to describe vesicle-associated ncRNAs as potentially enriched for disease-relevant signals. The review warns, however, that vesicle association does not establish retinal origin. Blood–retinal barrier disruption is more pronounced in advanced disease and may partly explain why circulating signals are easier to detect in PDR. Isolation methods such as ultracentrifugation, polymer precipitation and size-exclusion chromatography also differ in yield, purity and enrichment, while particle size or a common marker alone cannot prove that a preparation consists of endosome-derived exosomes. Serum, plasma, total circulating RNA and EV-enriched fractions therefore cannot be treated as interchangeable.

The proposed solution is a hierarchy of purpose-built panels rather than a longer catalogue of isolated markers. MicroRNAs would provide core effector readouts, while lncRNAs and circRNAs could add regulatory context and vesicle compartments could improve analytical stability. A competing-endogenous-RNA framework offers a biological rationale for combining them: lncRNAs and circRNAs can buffer shared microRNAs, meaning that a transcript’s diagnostic behavior may depend on its network and disease context rather than its abundance alone. Such interactions are often inferred from correlations or computational predictions, so direct binding, perturbation and rescue evidence are needed before a predicted relationship can be treated as established molecular competition.

Clinical translation would depend on cohorts designed around the difficult transitions rather than broad DR-versus-no-DR comparisons. The review calls for longitudinal, severity-stratified and imaging-integrated studies with prespecified endpoints, independent multicenter validation, adjustment for age, renal function, glycemic control, systemic inflammation, diabetes duration, medication use and relevant comorbidities, and transparent controls for hemolysis, vesicle characterization and RNA normalization. Machine learning and multi-omics may help refine biologically grounded panels, but they cannot replace mechanistic or prospective validation. Until adjacent-stage discrimination and incremental value beyond imaging are demonstrated, circulating ncRNA panels remain promising but unproven decision-support tools rather than clinically established tests for diabetic retinopathy.

Subject of Research: Stage-specific circulating and extracellular vesicle-associated non-coding RNAs as potential biomarkers for diabetic retinopathy

Article Title: Circulating and extracellular vesicle-associated non-coding RNAs in diabetic retinopathy: a stage-aware framework for network-informed biomarker panels

Article References: Yu, J.-W., Choi, M.-J., Lee, D. C., & Park, J.-H. (2026). Circulating and extracellular vesicle-associated non-coding RNAs in diabetic retinopathy: a stage-aware framework for network-informed biomarker panels. Frontiers in Endocrinology, 17, Article 1970838. https://doi.org/10.3389/fendo.2026.1970838

Image Credits: AI Generated

DOI: 10.3389/fendo.2026.1970838

Keywords: diabetic retinopathy, non-coding RNAs, microRNAs, long non-coding RNAs, circular RNAs, extracellular vesicles, biomarker panels, risk stratification, liquid biopsy, Circulating, extracellular, vesicle-associated