Identification of novel RUNX1-associated downstream targets by transcriptomics analysis.

Abstract

Purpose : The transcription factor RUNX1 has been implicated in angiogenic and fibrotic complications associated with vision threatening diseases including proliferative diabetic retinopathy, age-related macular degeneration, retinopathy of prematurity, and proliferative vitreoretinopathy. These diseases are known to encompass pathways such as cell proliferation and migration, epithelial to mesenchymal transition, and TNFα-signaling where RUNX1 has been implicated as a canonical mediator. Here we sought to identify and validate novel downstream effectors of RUNX1 using bulk transcriptomics analysis of primary human microvascular retinal endothelial cells (HMRECs) to generate relevant new avenues of research and establish potential therapeutic targets.

Methods : HMRECs were subjected to either RUNX1 nucleofection or siRNA knockdown to identify relevant anticorrelated genes. Samples (n = 6 per group) were then analyzed by bulk RNA sequencing following library generation and mRNA enrichment. Sequencing results were input into gene ontology databases to identify relevant pathways associated with manipulations in RUNX1 expression. Top differentially expressed genes were compared between the library sequencing results and the top DEGs from each pathway to identify targets for validation. Validation by qPCR of the top differentially expressed genes from this analysis was performed and changes in expression of select proteins were assessed by Western blotting.

Results : Bioinformatics analysis yielded 2052 significantly differentially expressed anticorrelated genes between cells subjected to RUNX1 overexpression or knockdown. 26 of the genes (including GPX7, CCL2, CDKN1A, and OPTN) were found to play key roles in RUNX1-associated pathways (including epithelial mesenchymal transition, TNFα-signaling, E2 transcription factor targets, cell cycle) and thus were selected for validation. qPCR results showed directional alignment of gene expression between groups when RUNX1 was either overexpressed or knocked down. This was successfully confirmed by Western blot analysis.

Conclusions : These results support the validity of the 2052 anticorrelated targets within the original sequencing library which could offer potential novel avenues of research or therapeutic targets associated with RUNX1 signaling mechanisms that have never been previously explored.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.