Downregulated LncRNA-ANCR promotes osteoblast differentiation by targeting EZH2 and regulating Runx2 expression

Abstract

Long noncoding RNAs (lncRNAs) are key regulators of diverse biological processes such as transcriptional regulation, cell growth and differentiation. Previous studies have demonstrated that the lncRNA-ANCR (anti-differentiation ncRNA) is required to maintain the undifferentiated cell state within the epidermis. However, little is known about whether ANCR regulates osteoblast differentiation. In this study, we found that the ANCR expression level is significantly decreased during hFOB1.19 cell differentiation. ANCR-siRNA blocks the expression of endogenous ANCR, resulting in osteoblast differentiation, whereas ANCR overexpression is sufficient to inhibit osteoblast differentiation. We further demonstrated that ANCR is associated with enhancer of zeste homolog 2 (EZH2) and that this association results in the inhibition of both Runx2 expression and subsequent osteoblast differentiation. These data suggest that ANCR is an essential mediator of osteoblast differentiation, thus offering a new target for the development of therapeutic agents to treat bone diseases.

Introduction

Mesenchymal stem cells (MSCs) have the potential to differentiate into multiple connective tissue cell types, including osteoblasts, myoblasts, adipocytes, and chondrocytes [1], [2]. The differentiation of MSCs into specific lineage depends on the extracellular cues received by these cells. Bone morphogenetic protein (BMP)-2, parathyroid hormone (PTH), and Runt-related transcription factor 2 (Runx2) plays an important roles in the regulation of osteoblast differentiation [2], [3], [4]. The alteration or malfunction of this regulation results in pathological consequences [5].

Long noncoding RNAs (lncRNAs) have been proposed to be a key regulators of diverse biological processes, such as transcriptional regulation, cell growth, and differentiation [6], [7]. Ng et al., demonstrated that lncRNAs promote neuronal differentiation by associating with chromatin modifiers and transcription factors [6]. Some hESC-specific lncRNAs involved in pluripotency maintenance were shown to physically interact with SOX2 and SUZ12 [6]. Sunwoo et al., found that MEN ε/β lncRNAs are upregulated during muscle differentiation and are essential components of paraspeckles [8]. The knockdown of MEN ε/β results in the disruption of nuclear paraspeckles. In the epidermis, lncRNA-ANCR (anti-differentiation ncRNA) loss abolishes the normal blockade of differentiation in the progenitor-containing compartment. Thus, ANCR is required to maintain the undifferentiated cell state within the epidermis. However, little is known about the role of ANCR in the osteoblast differentiation of hMSCs.

EZH2 is highly expressed in embryonic stem cells (ESCs) and is involved in the differentiation of these cells into different cell lineages, including osteoblasts, hepatocytes and neurons [9], [10]. EZH2 catalyzes histone H3 trimethylation at lysine 27 (H3K27me3) in target gene promoters to silence gene expression [11]. Wei et al., demonstrated that the CDK1-dependent phosphorylation of EZH2 disrupts methyltransferase activity of EZH2 and suppresses the methylation of H3K27, promoting the differentiation of MSCs into osteoblasts. Yu et al., found that EZH2 regulates the neuronal differentiation of MSCs through PIP5K1C-dependent calcium signaling [9]. EZH2 also regulates muscle gene expression and skeletal muscle differentiation [12]. Recent studies have demonstrated that some lncRNAs physically interact with EZH2 to regulate gene expression. lncRNA-HOTAIR targets Polycomb Repressive Complex 2 (which contains the H3K27 methylase EZH2) to silence the HOXD gene [13]. Therefore, we speculated that ANCR may regulate the osteoblast differentiation of ESCs by associating with EZH2.

Based on these findings, we investigated the molecular mechanism by which ANCR regulates osteoblast differentiation. We found that ANCR is an important regulator of osteoblast differentiation. ANCR associates with EZH2, and this association results in the inhibition of both Runx2 expression and subsequent osteoblast differentiation.