Genome-Wide MicroRNA and Gene Analysis of Mesenchymal Stem Cell Chondrogenesis Identifies an Essential Role and Multiple Targets for miR-140-5p.

Matt J Barter,David A Young,Maria Tselepi,Wang Hui,Daryl P Shanley,Steven Woods,Graham R Smith,Rodolfo Gómez,Ian M Clark

doi:10.1002/stem.2093

Abstract

microRNAs (miRNAs) are abundantly expressed in development where they are critical determinants of cell differentiation and phenotype. Accordingly miRNAs are essential for normal skeletal development and chondrogenesis in particular. However, the question of which miRNAs are specific to the chondrocyte phenotype has not been fully addressed. Using microarray analysis of miRNA expression during mesenchymal stem cell chondrogenic differentiation and detailed examination of the role of essential differentiation factors, such as SOX9, TGF‐β, and the cell condensation phase, we characterize the repertoire of specific miRNAs involved in chondrocyte development, highlighting in particular miR‐140 and miR‐455. Further with the use of mRNA microarray data we integrate miRNA expression and mRNA expression during chondrogenesis to underline the particular importance of miR‐140, especially the ‐5p strand. We provide a detailed identification and validation of direct targets of miR‐140‐5p in both chondrogenesis and adult chondrocytes with the use of microarray and 3′UTR analysis. This emphasizes the diverse array of targets and pathways regulated by miR‐140‐5p. We are also able to confirm previous experimentally identified targets but, additionally, identify a novel positive regulation of the Wnt signaling pathway by miR‐140‐5p. Wnt signaling has a complex role in chondrogenesis and skeletal development and these findings illustrate a previously unidentified role for miR‐140‐5p in regulation of Wnt signaling in these processes. Together these developments further highlight the role of miRNAs during chondrogenesis to improve our understanding of chondrocyte development and guide cartilage tissue engineering. Stem Cells 2015;33:3266–3280

Highlights

Cartilage is found at the end of bones in articulating joints where it provides both a structural role to resist compression and a lubricating function for low friction movement
mesenchymal stem cells (MSCs) were cultured in chondrogenic differentiation medium for 14 days in Transwell hanging cell culture inserts
This was accompanied by a failure of the cells to effectively upregulate gene expression of selected cartilage components to the level of nontargeting siRNA (Fig. 1G), together indicating that miRNAs are essential for the process of chondrogenic differentiation

Summary

Introduction

Cartilage is found at the end of bones in articulating joints where it provides both a structural role to resist compression and a lubricating function for low friction movement. Chondrocytes are solely capable of producing cartilage extracellular matrix (ECM) which is largely composed of collagen for structural integrity and proteoglycan for water adsorption. During development chondrocytes lay the scaffold for long bone formation by terminally differentiating and undergoing hypertrophy to allow resorption and ossification, characteristic of bone formation [1]. Chondrocytes develop from undifferentiated mesenchymal stem cells (MSCs) in limbs to form cartilage anlage. Cells aggregate to form high density condensations which provide cell-cell contacts required for initiating.

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Conclusion