Analysis of microRNA expression in rat retina at the early stages of retinopathy development

Full Text

Age-related macular degeneration (AMD), a neurodegenerative eye disease, is the primary cause of blindness globally, especially affecting the elderly population. MicroRNAs, a type of single-stranded, non-coding RNAs of about 22 nucleotides in length, primarily regulate gene expression negatively. Alterations in the miRNA profile during AMD’s development could potentially aid in early detection and progression monitoring of the disease. However, no data are available on the microRNA expression patterns in the retina during the early stages of AMD. Investigating the molecular mechanisms that underlie age-related retinal degeneration at an early stage, prior to the manifestation of any symptoms, could provide insights into the molecular events that initiate the irreversible stage. In the case of OXYS rats with accelerated aging, dystrophic changes in the RPE, neuroretinal thinning, and impaired choroidal microcirculation — the primary indicators of the dry form of AMD — spontaneously occur by the age of 3 months [1]. A comparative analysis was conducted in this study on microRNA expression in the retinas of OXYS rats at two different stages of retinopathy — 20 days (preclinical) and 3 months (manifestation) — as well as in control Wistar rats. Small RNA-seq sequencing was performed on the DNBSEQ platform.

According to differential expression analysis (DESeq2, q-value <0.05), at 20 days of age, 2 microRNAs exhibited altered expression in OXYS rats compared to Wistar rats. Similarly, at 3 months of age, the expression of 16 microRNAs in OXYS rats was altered compared to Wistars, with 7 miRNAs showing increased levels and 9 miRNAs showing decreased levels. Analysis of age-related changes in miRNA expression demonstrated that in the OXYS rat retina between 20 days and 3 months, the levels of 134 miRNAs altered: 59 miRNAs increased, and 75 miRNAs decreased. Over the course of 20 days to 3 months, 94 miRNAs underwent alterations in the retinas of Wistar rats. Among these, the level of 48 miRNAs increased while the level of 46 miRNAs decreased. The RNAhybrid, miRanda, and TargetScan programs were used to search for target genes of differentially presented miRNAs among different groups. Following this, gene ontologies were studied. In comparison to Wistar rats, “adhesion contacts” and “synaptic vesicle cycle” categories are noticeably populated with target genes of miRNA DE at 20 days in OXYS rats. MiRNA target genes with lower levels at 3 months in OXYS rats compared to Wistar rats are significantly enriched in categories such as vascular branching, extracellular matrix organization, adhesion, and protein deubiquitination. Target genes with increased miRNA levels at 3 months in OXYS rats, compared to Wistar rats, are significantly represented in various signaling pathways, such as mTOR, MAPK, VEGF, and thyroid hormone. Target genes, which serve as common targets for at least 10 microRNAs (miRNAs), were chosen for analysis of the targetome subject to regulation by miRNA molecules exhibiting modified expression. Targetomes containing 400 to 600 genes were obtained for miRNAs that increased or decreased levels in both OXYS and Wistar rats between 20 days and 3 months. Notably, these targetomes were enriched in categories such as axonogenesis, retinal layer formation, visual learning, focal adhesion, and endocytosis. Profiling of 84 miRNAs of the retina of OXYS and Wistar rats at the age of 20 days and 3 months was carried out using PCR panels (Qiagen) to verify the results of small RNA-seq. Our analysis revealed highly convergent results between sequencing and PCR panels. Thus, 16 miRNAs showing altered expression in OXYS retina may serve as potential biomarkers and new targets for studying AMD pathogenesis.

ADDITIONAL INFORMATION

Funding sources. The study was supported by the Russian Science Foundation, grant No. 21-15-00047.

Authors' contribution. All authors made a substantial contribution to the conception of the work, acquisition, analysis, interpretation of data for the work, drafting and revising the work, and final approval of the version to be published and agree to be accountable for all aspects of the work.

Competing interests. The authors declare that they have no competing interests.

About the authors

O. S. Kozhevnikova

Institute of Cytology and Genetics of Siberian Branch of the Russian Academy of Sciences

Author for correspondence.
Email: oidopova@bionet.nsc.ru
Russian Federation, Novosibirsk

N. G. Kolosova

Institute of Cytology and Genetics of Siberian Branch of the Russian Academy of Sciences

Email: oidopova@bionet.nsc.ru
Russian Federation, Novosibirsk

References

Supplementary files