Allele-specific influence of seed polymorphism rs2168518 in hsa-miR-4513 on expression of neovascularization-associated genes

Abstract

Purpose : MicroRNAs (miRNAs) are small post-transcriptional regulators offering promising molecules as biomarkers or targets for innovative approaches to treat complex diseases such as age-related macular degeneration (AMD). Hsa-miR-4513 appears an excellent candidate as it contains a seed polymorphism (rs2168518) which is specifically associated with the neovascular (NV) form of AMD. So far, little is known about the biological mechanisms underlying this association. We therefore aimed to identify allele-specific target genes of hsa-miR-4513 to clarify its contribution to NV.

Methods : We performed high-throughput RNA sequencing (RNA-Seq) in a miRNA overexpression model in human umbilical vein endothelial cells transfected with hsa-miR-4513, separated by the rs2168518 alleles. Promising allele-specific target genes were independently verified by quantitative reverse transcription PCR (qRT-PCR) and further validated via protein expression. Allele-specific miRNA binding was analyzed with a luciferase reporter assay. Further, publicly available databases were utilized to link target genes to phenotypes previously described to be associated with hsa-miR-4513.

Results : Overall, we identified 23 allele-specific target genes of hsa-miR-4513 by RNA-Seq and independently replicated 19 of these via qRT-PCR. Western Blot analysis and luciferase reporter assays conducted for five to six exemplary genes further confirmed the allele-specific regulation of these genes by hsa-miR-4513. Remarkably, multiple allele-specific target genes of hsa-miR-4513 are linked to various phenotypes while two genes, namely CDKN2A and TBC1D5, have been reported before to be related to eye phenotypes.

Conclusions : Our study revealed multiple promising allele-specific target genes of hsa-miR-4513, which offer the unique opportunity to elucidate the association of this miRNA with NV. Especially CDKN2A and TBC1D5 are highlighted as interesting allele-specific target genes and provide a basis for further follow-up studies.

This is a 2021 ARVO Annual Meeting abstract.

 

Flow of study design to identify and validate allele-specific target genes of hsa-miR-4513.