Biochemical and Biophysical Research Communications
Identification of miR-1 as a micro RNA that supports late-stage differentiation of growth cartilage cells
Research highlights
► Expression of miR-1 is most decreased upon hypertrophy of chondrocytes. ► Introduction of miR-1 represses gene expression for cartilage ECM synthesis. ► ECM synthesis by hypertrophic chondrocytes is supported by miR-1 down-regulation.
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.
Section snippets
Cell culture
Human chondrocytic HCS-2/8 cells [10], [11] were cultured in Dulbecco’s modified Eagle’s minimum essential medium (D-MEM) containing 10% fetal bovine serum (FBS). Primary chicken chondrocytes were isolated from the upper 1/3 portion (upper sternum: US cells) and lower 1/3 portion (lower sternum: LS cells) of the sternal cartilage of chicken embryos, as described previously [12], [13]. The cells were resuspended in high-glucose D-MEM supplemented with 10% FBS, and then plated at a density of 1.0 ×
Screening for chicken miRNAs down-regulated upon late differentiation of chicken sternum chondrocytes
First, in order to find specific miRNAs involved in the late stage of chondrocyte differentiation during endochondral ossification, we performed microarray analysis. Small miRNAs were extracted and purified from chicken LS and US chondrocytes representing proliferating and hypertrophic chondrocytes, respectively, which representation was confirmed by the comparable level of type II collagen gene expression and US cell-specific expression of the type X collagen gene (Fig. 1A). Subsequently, the
Discussion
In the present study, we specified several miRNAs that were down-regulated upon hypertrophic differentiation of chicken sternum chondrocytes. After a series of analyses in silico and in vitro, we found one of these miRNAs, miR-1, was capable of regulating the synthesis of aggrecan, a cartilage-specific proteoglycan. By transfecting cells with a synthetic miR-1 duplex, we found that the expression of aggrecan was repressed in chicken hypertrophic US chondrocytes and human HCS-2/8 cells. However,
Acknowledgments
This study was supported by Grants-in-aid for Scientific Research (S) [to M.T.], and C [to S.K.] and the Support Program for Improving Graduate School Education (C014) [to K.S] from the Japan Society for the Promotion of Science and Ministry of Education, Culture, Sports, Science and Technology–Japan, respectively. We thank Drs. Eriko Aoyama and Takako Hattori for their helpful suggestions.
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