Titolo della tesi: The role of miRNA expression in the pathogenesis of early stages of Retinopathy of Prematurity: Pilot Study
Introduction
Retinopathy of prematurity (ROP) remains a major cause of preventable vision impairment in premature infants, especially those born with lower gestational ages and reduced birth weights. Although advancements in neonatal care have improved survival, the complex pathophysiology of ROP—largely driven by abnormal retinal vascularization in response to oxygen level fluctuations—remains partially understood. Angiogenic agents, including VEGF, IGF-I, and TNF-α, are recognized as contributors to ROP development, but understanding of the underlying regulatory mechanisms is limited. Recent research has shown that microRNAs (miRNAs), short non-coding RNAs involved in post-transcriptional gene regulation, play a crucial role in cellular processes such as angiogenesis, apoptosis, and immune response, which are relevant to ROP. This study aims to investigate the expression patterns of selected miRNAs in infants with early-stage, untreated ROP, providing insights into miRNA involvement in early ROP stages.
Materials and Methods
Study Population
A prospective study was conducted at University Hospital Policlinico Umberto I, enrolling infants born before 32 weeks of gestation and weighing less than 1500 grams between January 2023 and September 2024. ROP screening followed American Academy of Ophthalmology guidelines, with stages assessed using the International Classification of ROP (ICROP) criteria. Blood samples were drawn at birth, with 2mL collected in EDTA-containing tubes to prevent hemolysis. After centrifugation and plasma separation, samples were stored at -80°C for RNA extraction.
miRNA Analysis
Based on prior studies in rat models and limited human studies, miRNAs miR-210, miR-21, miR-27b, miR-214, and miR-128a were selected for analysis. RNA extraction, reverse transcription, and quantitative PCR (qPCR) were conducted to quantify miRNA expression levels. Fold regulation and fold change were calculated using the GeneGlobe Data Analysis Center (Qiagen).
Statistical Analysis
Data analysis was performed using SPSS (version 27). Continuous variables were assessed for normality using the Shapiro–Wilk test and analyzed using unpaired t-tests or Mann-Whitney tests as appropriate. Correlations between variables were analyzed using Pearson or Spearman’s rank correlation tests. Significance was set at p<0.05.
Results
Out of 20 samples collected, 12 met the study criteria, with 5 samples from infants with ROP and 7 from controls. ROP patients had significantly lower gestational age (mean 27.5 weeks) and birth weight (mean 958 g) compared to controls (mean 30.1 weeks and 1363 g, respectively; p<0.05). Significant miRNA expression differences were observed: miR-210, miR-21, and miR-214 were notably downregulated in ROP cases, whereas miR-128a was upregulated. Additionally, miR-210 levels correlated positively with oxygen exposure, suggesting a possible link between prolonged oxygen exposure and miRNA expression patterns. No significant differences were noted in blood parameters between the two groups.
Discussion
This pilot study is the first clinical investigation to explore miRNA expression profiles in early-stage, untreated ROP cases. The observed downregulation of miR-210, miR-21, and miR-214 and upregulation of miR-128a in ROP patients suggest distinct miRNA involvement in early disease processes. The reduction in miR-210, a hypoxia-responsive miRNA, may indicate a shift from hypoxic to angiogenic signaling in early ROP stages, whereas miR-21 downregulation may imply insufficient anti-apoptotic activity. miR-128a upregulation could represent an early adaptive response to hypoxia or oxidative stress. These findings highlight the potential of miRNA biomarkers in identifying infants at risk for ROP.
Limitations include the small sample size and a focus on five specific miRNAs, restricting generalizability. Larger cohort studies are necessary to validate these findings and explore additional miRNAs in ROP pathogenesis.
Conclusion
This study underscores differential miRNA expression in early, untreated ROP cases, pointing to the regulatory role of miRNAs in early-stage disease progression. miR-210, miR-21, miR-214, miR-128a, and miR-27b may serve as promising biomarkers for ROP, providing preliminary evidence for the potential of miRNA-based diagnostic and prognostic tools in managing ROP. Future research with larger cohorts will be essential to confirm these findings and explore the therapeutic implications of targeting miRNA pathways in early ROP intervention.