Identification of miR-1 as a micro RNA that supports late-stage differentiation of growth cartilage cells

Abstract

The process of endochondral ossification is strictly regulated by a variety of extracellular and intracellular factors. Recently, it has become recognized that specific miRNAs are involved in this process by regulating the expression of the relevant genes at the post-transcriptional level. In this present study we obtained the first evidence of the involvement of a specific micro RNA (miRNA) in the regulation of the chondrocyte phenotype during late stages of differentiation. By use of the microarray technique, miR-1 was identified as this miRNA, the expression of which was most repressed upon hypertrophic differentiation. Transfection of human chondrocytic HCS-2/8 cells and chicken normal chondrocytes with miR-1 led to repressed expression of aggrecan, the major cartilaginous proteoglycan gene. Therefore, miR-1 was found to be involved in the regulation of the chondrocytic phenotype and thus to play an important role in chondrocytes during the late stage of the differentiation process, maintaining the integrity of the cartilage tissue.

Introduction

Endochondral ossification is a central biological process involved in the development of long bones and related bone components. In multiple steps, mesenchymal cells are first condensed and begin to differentiate into chondrocytes. These chondrocytes form the cartilaginous anlage for each bone to be formed, proliferate for growth and extensively differentiate into hypertrophic chondrocytes. At the final step, these chondrocytes conduct mineralization, allowing the invasion of vascular endothelial cells and osteoblasts. Along with chondrocyte differentiation, a number of growth factors and cytokines are involved in this process of endochondral ossification under exact regulation by many intracellular regulators of gene expression [1]. It has now been suggested that the expression of such genes may be controlled not only by protein molecules, such as transcription factors, but also by micro RNAs (miRNAs) [1].

The miRNAs constitute a major group of small noncoding RNAs (ncRNAs) having a regulatory function. This regulation occurs post-transcriptionally in collaboration with protein counterparts forming RNA-induced silencing complexes (RISC). These complexes repress target gene expression by interfering with the translation or by accelerating the degradation of mRNA through direct binding to partially complementary sequences in the 3′-untranslated region (UTR) of the target mRNA. The miRNAs play important roles in the differentiation of numerous kinds of the cells including stem cells and in the development of a number of tissues including skeletal ones [2], [3], [4], [5]. In the cartilage tissue as well, certain miRNAs are specifically expressed; however, their distinct roles in chondrocyte differentiation are yet to be identified.

Recently, we found that miR-18a regulates the phenotype of human chondrocytic cells via CCN family protein 2/connective tissue growth factor (CCN2/CTGF) [6]. CCN2 is one of the classical members of the multifunctional CCN family and plays a central role in endochondral ossification. During this process, its gene expression is strictly regulated by intracellular molecules at both transcriptional and post-transcriptional levels in order to enable its stage-specific expression [7], [8], [9]. The expression of miR-18a is strongly repressed in chondrocytic cells, which consequently de-represses the mature chondrocytic phenotype by releasing the 3′-UTR-mediated translational interference of CCN2 mRNA. Nevertheless, the expression level of miR-18a does not change during the late differentiation after chondrogenesis [9]; thus involvement of miR-18a therein is unlikely. Therefore, we initiated the present study by comprehensively analyzing the expression profile of miRNAs in differentiating chondrocytes for the purpose of identifying miRNAs involved in the regulation of the chondrocytic phenotype during late stages of differentiation.