Systems analysis of miR-199a/b-5p and multiple miR-199a/b-5p targets during chondrogenesis

Abstract

AbstractChanges in chondrocyte gene expression can contribute to the development of osteoarthritis (OA), and so better understanding of the regulative processes during chondrogenesis can highlight potential drug targets for OA. microRNAs (miRNAs) have been a focus of chondrogenesis/ OA research and we have used a combined experimental, bioinformatic, and systems biology approach to explore multiple miRNA-mRNA interactions that regulate chondrogenesis. We identified paralogues miR-199a-5p and miR-199b-5p as pro-chondrogenic regulators of chondrogenesis through bioinformatic analysis. Experimental work demonstrated alteration of miR-199a-5p or miR-199b-5p expression led to significant inverse modulations of chondrogenic biomarkers:ACAN,COL2A1,SOX9and glycosaminoglycan levels. Potential miR-199a/b-5p targets were then identified using RNAseq combined with bioinformatic analysis to identifyFZD6, ITGA3andCAV1as highly likely candidates. Through knockdown experiments we indicated a strong antagonistic relationship between miR-199a/b-5p andFZD6,ITGA3andCAV1. Luciferase assays usingFZD6andITGA33’UTRs luciferase assays indicated both mRNAs to be targets ofmiR-199a-5p.The experimental work was used to generate and parameterize a multi-miRNA 14-day chondrogenesis kinetic model to be used as a repository for the experimental work and as a resource for further investigation of this system. This is the first multi-miRNA model of a chondrogenesis-based system, and it highlights complex relationships between regulatory miRNAs, and predicted indirect miRNA-miRNA interactions, via modulation of miR-140-5p by knockdown of miR-199a/b-5p.