Motivation The analysis of longitudinal datasets and construction of gene regulatory networks provide a valuable means to disentangle the complexity of microRNA-mRNA interactions. However, there are no computational tools that can integrate, conduct functional analysis and generate detailed networks from longitudinal microRNA-mRNA datasets. Results We present TimiRGeN, an R package that uses time point based differential expression results to identify miRNA-mRNA interactions influencing signalling pathways of interest. miRNA-mRNA interactions can be visualised in R or exported to PathVisio or Cytoscape. The output can be used for hypothesis generation and directing in vitro or further in silico work such as gene regulatory network construction. Availability and implementation TimiRGeN is available for download on Bioconductor (https://bioconductor.org/packages/TimiRGeN) and requires R v4.0.2 or newer and BiocManager v3.12 or newer. Supplementary information Supplementary data is available at Bioinformatics online.

Changes 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, SOX9 and glycosaminoglycan levels. Potential miR-199a/b-5p targets were then identified using RNAseq combined with bioinformatic analysis to identify FZD6, ITGA3 and CAV1 as highly likely candidates. Through knockdown experiments we indicated a strong antagonistic relationship between miR-199a/b-5p and FZD6, ITGA3 and CAV1. Luciferase assays using FZD6 and ITGA3 3'UTRs luciferase assays indicated both mRNAs to be targets of miR-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 any system, and it highlights that many other miRNAs may be involved in the regulation of this system and was used to predict experimental gaps not covered by experimental work.

microRNAs (miRNAs) are non-coding RNAs which modulate the expression of other RNA molecules. One miRNA can target many transcripts, allowing each miRNA to play key roles in many biological pathways. miR-324 is a miRNA previously implicated in bone and cartilage maintenance, defects of which result in common age-related diseases, such as osteoporosis or osteoarthritis (OA). In global miR-324-null mice cartilage damage was increased in both surgically and ageing-induced OA, despite minimal changes to the cartilage transcriptome, with few predicted miR-324 targets dysregulated. However, micro-computed tomography and histology demonstrated that global miR-324-null the mice had an increase in bone mineral density, trabecular thickness and cortical thickness, with many parameters increasing with age. The bone marrow of miR-324-null mice also had reduced lipid content while and in vivo TRAP staining revealed a decrease in osteoclasts, with histomorphometry demonstrating an increased rate of bone formation in miR-324-null mice. Ex vivo assays revealed that the high bone mass phenotype of the miR-324-null mice resulted from increased osteoblast activity and decreased osteoclastogenesis. RNA-seq and qRT-PCR followed by miR-324 target prediction and validation in osteoblasts, osteoclasts and bone marrow macrophages identified the osteoclast fusion regulator Pin1 as a miR-324 target in the osteoclast lineage and the master osteogenic regulator Runx2 as a target of miR-324-5p in osteoblasts, the in vitro overexpression of which recapitulated the increased osteogenesis and decreased adipogenesis phenotype observed in vivo. These data point to important roles of miR-324 in skeletal biology with altered bone homeostasis in miR-324-null mice potentially causal for the increased cartilage damage observed during OA and ageing. Elucidation of pathways regulated by miR-324 offer promise for the treatment of bone diseases such as osteoporosis.