Regulatory mechanisms of circular RNAs during human mesenchymal stem cell osteogenic differentiation

Abstract

Human osteogenic differentiation is a complex and well-orchestrated process which involves a plethora of molecular players and cellular processes. A growing number of studies have underlined that circular RNAs (circRNAs) play an important regulatory role during human osteogenic differentiation. CircRNAs are single-stranded, covalently closed non-coding RNA molecules that are acquiring increased attention as epigenetic regulators of gene expression. Given their intrinsic high conformational stability, abundance, and specificity, circRNAs can undertake various biological activities in order to regulate multiple cellular processes, including osteogenic differentiation. The most recent evidence indicates that circRNAs control human osteogenesis by preventing the inhibitory activity of miRNAs on their downstream target genes, using a competitive endogenous RNA mechanism. The aim of this review is to draw attention to the currently known regulatory mechanisms of circRNAs during human osteogenic differentiation. Specifically, we provide an understanding of recent advances in research conducted on various human mesenchymal stem cell types that underlined the importance of circRNAs in regulating osteogenesis. A comprehensive understanding of the underlying regulatory mechanisms of circRNA in osteogenesis will improve knowledge on the molecular processes of bone growth, resulting in the potential development of novel preclinical and clinical studies and the discovery of novel diagnostic and therapeutic tools for bone disorders.

Keywords: circRNA; circular RNA; crosstalk; mesenchymal stem cell; miRNA; microRNA; osteogenesis; osteogenic differentiation.

Figure 1

Circular RNAs (circRNAs) biogenesis mechanisms and functions. In the nucleus, a single molecule of pre-mRNA is subject to back-splicing by the canonical spliceosomal machinery. The generation of mature circRNA molecules, which generally contains two or three exons occurs through two different modalities. The first exploits the base complementarity between two circRNA flanked introns to create a secondary structure that makes possible back-splicing. In the second, specific RNA-binding proteins recognize/bind to specific regions of circRNAs' flanked introns thus leading to the back-splicing secondary structure formation. The final products of this process are various mature RNA molecules, including a mRNA, a circRNA and one or more intronic lariats, obtained from the removal of introns interposed between circRNA exons. Mature circRNAs are crucial molecules which regulate several cell mechanisms, such as: a) Regulation of transcription by enhancing of RNA polymerase II (RNA pol II) and Ten-eleven translocation protein (TET) promoter demethylation activities. b) Sponging and regulation of RNA-binding proteins half-life favoring proteasome mediated degradation. c) Translation of particular circRNA regions through a CAP-independent mechanism. b) Sponging-mediated negative modulation of microRNAs (miRNA). This figure was made by using the BioRender online tool (www.biorender.com).

Figure 2

Pathways involved in human mesenchymal stem cells (MSCs) osteogenic differentiation. Wnt/β-catenin, bone morphogenetic protein (BMP) and transforming growth factor-beta (TGF-β) cascades are the main signaling pathways leading to MSCs osteogenic differentiation. Wnt/β-catenin induces MSCs osteogenic differentiation through β-catenin translocation into the nucleus, leading to the expression of target genes, including RUNX2. TGF-β and BMP through the binding with their respective receptors lead to the activation of Smad-dependent and -independent cascades. In TGF-β Smad-dependent signaling, Smad2/3 is phosphorylated upon ligand-receptor binding and interacts with Smad4, leading to its migration into nucleus. Here, this complex induces RUNX2 expression interacting with CBP and P300 co-activators. Smad2/3 without Smad4 interaction, forms a complex with HDAC4/5, blocking RUNX2 expression. Unphosphorylated Smad2/3 is degraded by ubiquitination. In BMP Smad-dependent pathway, BMPs receptors of type I and II (BMPR-I and BMPR-II) are activated by their ligands and lead to Smad1/5/8 phosphorylation. These molecules form a complex with Smad4 and move into the nucleus, acting as transcriptional regulator of target genes, including RUNX2 and Osterix (OSX). Unphosphorylated Smad1/5/8 is degraded by ubiquitination. This Smad-dependent cascade also comprises Smad6/7 and Smurf1/2, which are negative regulators of this pathway. In Smad-independent cascade, TGF-β/BMP signaling pathways promote the maturation and proliferation of osteoblasts inducing DLX5, RUNX2 and OSX phosphorylation through a cascade that comprises TAK1-TAB complex, ERK and p38. This figure was made by using the BioRender online tool (www.biorender.com).

Figure 3

Regulatory mechanisms of circular RNAs (circRNAs) during Wnt/β-catenin and bone morphogenetic protein (BMP)-mediated human mesenchymal stem cell (MSC) osteogenic differentiation. CircRNAs are able to positively and/or negatively regulate MSCs osteogenesis by molecularly sponging numerous miRNAs, inhibiting, in turn, the miRNA-mediated regulatory activity on downstream osteogenic target genes. This regulatory activity can occur on different target genes implicated in (A) Wnt/β-catenin pathway, (B) bone morphogenetic protein (BMP) signaling pathway. § miRNAs previously reported as implicated in Wnt/β-catenin pathway (Zhang et al., 2017). # predicted mechanism. This figure was made by using the BioRender online tool (www.biorender.com).

Figure 4

Regulatory mechanisms of circular RNAs (circRNAs) during human mesenchymal stem cell (MSC) osteogenic differentiation mediated by both Wnt/β-catenin and bone morphogenetic protein (BMP) signaling pathways and by additional pathways involved in human osteogenesis. CircRNAs are able to positively and/or negatively regulate MSCs osteogenesis by molecular sponging numerous miRNAs, inhibiting, in turn, the miRNA-mediated regulatory activity on downstream osteogenic target genes. This regulatory activity can occur on different target genes implicated in (A) both Wnt/β-catenin and bone morphogenetic protein (BMP) signaling pathways, (B) additional pathways involved in osteogenesis. *Predicted interaction. These miRNAs have been reported to be implicated in Both Wnt/β-catenin and BMP pathways (Huang et al., 2010, Diomede et al., 2016). This figure was made by using the BioRender online tool (www.biorender.com).