Determination of miRNA in tear extracellular vesicles significantly associated with treatment-requiring retinopathy of prematurity: a pilot study

Abstract

Retinopathy of prematurity (ROP) develops in some premature infants and may be characterized by permanent severe retinal damage necessitating early detection and prompt treatment. The purpose of this study was to investigate whether specific miRNAs in tear extracellular vesicles (EVs) are associated with the development of ROP requiring treatment. Tear samples were collected from 47 infants, including 33 with ROP and 14 without ROP; of the ROP group, 18 infants required treatment. The miRNAs expressed in EVs were profiled by real-time PCR array. An exploratory analysis of differential miRNA expression using tear EV samples from 35 infants was performed. Network analysis conducted for the miRNAs for ROP requiring treatment revealed critical networks of miRNAs linked to IGF1R and VEGF. A machine learning study utilizing the random forest model identified 13 miRNAs with high importance score for the treatment-requiring ROP eyes. After adjustments for birth weight, miR-520a-5p was identified as a candidate marker. Network analysis confirmed a significant association of miR-520a-5p with the VEGF-centered network. When the Gradient boosting decision tree was applied, miR-520a-5p discriminated treatment-requiring ROP with accuracy of 77.8% and an area under the curve (AUC) of 0.889. For the validation phase, 12 unanalyzed infants were examined for the diagnostic accuracy of miR-520a-5p, and the findings confirmed a high diagnostic accuracy of 91.7% and AUC of 0.875 for treatment-required ROP eyes. Notably, miR-520a-5p expression was strongly influenced by infant immaturity, as reflected by gestational age. These findings provide new insights into ROP pathophysiology and suggest that tear-derived miRNAs, particularly those in EVs, may serve as potential biomarkers and inform future therapeutic strategies.

Keywords: Exosome; Extracellular vesicle; Retinopathy of prematurity; Tear; miRNA.

Fig. 1

Diagram of study design. For the initial screening of miRNAs, tear extracellular vesicles from 35 infants were analyzed using a PCR array. Candidate miRNAs identified in this phase were then tested for validation in an independent cohort of patients.

Fig. 2

Expression of the surface marker on extracellular vesicles (EVs) in tears. The EVs in tears were examined for surface expression of CD9, CD147, and EpCAM using the Exoscreen Assay.

Fig. 3

Differential expression of 202 miRNA in extracellular vesicles in the tears of ROP infants. Fourteen treatment-requiring ROP eyes, 11 non-treatment-requiring ROP eyes, 10 eyes without ROP were analyzed as identification phase data set. The heat map is shown with clustering analysis. Intensity scale indicates Log10 expression values.

Fig. 4

Network analysis of miRNA in extracellular vesicles in tear of treatment-requiring ROP infants. The miRNA in the extracellular vesicles of tear were analyzed using the Ingenuity pathway analysis. Network (P = 10⁻²⁶) associated with angiogenesis are shown. The network was associated with VEGF, ERK, NFκB, and p38 MAPK. miR-520a-5p at the top node of the network was connected downstream of the miR-515 family, illustrating its association within this angiogenic regulatory network. Image was created using Ingenuity pathway analysis software.

Fig. 5

Importance score to discriminate treatment-requiring ROP infants. Importance score was analyzed using random forest and Boruta programs using 14 treatment-requiring ROP, 11 non-treatment-requiring ROP infants, and 10 infants without ROP as identification phase data set.