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1. Introduction

Osteoporosis and osteoporotic fractures are associated with high medical costs and can lead to poor quality of life. Osteoporotic fractures can happen from minor trauma, such as slipping or falling. Worldwide, osteoporotic vertebral fracture (OVF) is the most frequent type of osteoporotic fracture [1,2]^[1][2][3][4]. OVF increases in incidence with age and women and is associated with an increased risk of death ^[2]. Osteoporosis and OVF are influenced by a multifactorial environment, including genetic factors [3–5]^[5][6][7]. Osteoporosis is generally thought to be caused by a reduction in the number of osteoblasts along with preferential differentiation to fat cells in the aged skeleton [6,7]^[3][4]. This may decrease the quantity and functionality of osteoblasts and increase bone marrow fat in aged bones [6,8]^[3][8]. This can result in a reduction in bone formation and bone microarchitecture, leading to additional vertebral fractures and interfering with bone healing and remodeling after fracture [6,9]^[3][9].

Fracture healing reproduces skeletal development and growth with intricate interactions between cells, growth factors, and extracellular matrices. There are usually four stages to fracture repair: (1) inflammation, (2) soft callus formation, (3) hard callus formation, and (4) remodeling (Figure 1) ^[10]. Each stage is associated with specific cellular and molecular events [11–13]^[11][12][13]. However, in practice, the events are insufficiently described, and there can be extensive redundancies across the different stages. Further, studies over many years have examined the molecular and cellular forces driving the central process [14–16]^[14][15][16]. When considering cellular processes, vascular cells, inflammatory cells, osteochondral precursors, and osteoclasts maintain an important role in the bone repair process. When considering molecular processes, fracture repair is helped by inflammation-promoting cytokines and three main factors: osteogenesis-promoting factor, growth factor, and angiogenesis factor ^[17]. These factors help set up relevant morphogenetic disciplines by promoting growth or differentiation and recruiting cells. After that, the damaged soft tissue undergoes a repair and the fracture is covered by soft callus and, subsequently, hard callus.

Figure 1. Models of the fracture healing process.

In the final stage of the fracture healing process, remodeling the bone-hardening callus into original cerebellar and cortical structures occurs, and is also known as secondary osteoplasty ^[13]. This bone remodeling process destroys mineralized bone followed by bone matrix formation that then becomes mineralized. This resorption and formation of bone are highly connected, allowing for skeletal integrity. However, with some pathological conditions such as osteoporosis, bone remodeling steps can uncouple, which can lead to increased fragility and decreased bone strength ^[18]. To sustain homeostasis of the bone mass, the remodeling of bone integrates extremely regulated steps that rely on the roles and interactions of the osteoblastic and osteoclastic lineages (Figure 2).

Figure 2. The bone healing process after a fracture. Bone remodeling after fracture is initiated by osteoclast activation, and it solubilizes the bone mineral and degrades the matrix. Osteoclasts originate from hematopoietic stem cells which fuse to form multinucleated cells (activated form of osteoclasts). Monocytes/macrophages remove debris, followed by a bone formation phase performed by osteoblasts, producing osteoid matrix which will mineralize.

Bone cell activation and differentiation are regulated at the molecular level by an intricate signaling network that contains systemic hormones and local secreted molecules. It is well known that many probabilistic and environmental stresses can be modulated through phylogenetic determination and gene expression [19–21]^[19][20][21]. Pathological processes for osteopathy with many factors, such as osteopenia or osteoporosis, can have crucial epigenetic elements. Epigenetic elements can present promising areas of research linking disease risk to genetics and gene expression. New treatment for OVF can be developed in virtue of a greater understanding of epigenetic and molecular regulation of bone cell function and differentiation.

Recently, a new paradigm of epigenetics has advanced. New discipline attempts to identify the delivery of stable phenotypes without altering DNA sequences due to interactions between the genome and internal and external environments. Within epigenetic mechanisms, those that are notable are modifications of histones, DNA methylation, and non-coding RNAs (Figure 3) ^[22]. Epigenetics can control transcription activity without altering DNA sequences ^[23] through an enzymatic alteration of 5-cytosine in DNA ^[24], microRNA ^[25], histone modifications ^[26], and remodeling of chromatins. DNA methyltransferases (DNMT) can catalyze methylation of CpG islands of DNA, which can weaken genome stability and reduce the activity of gene transcription ^[27]. MicroRNA (miRNA) can interrupt mRNA targets and reduce the translation of protein. In addition, it can initiate a post-transcriptional signaling pathway and promote the expression of protein [28,29]^[28][29]. Histone acetyltransferases (HATs) can control histone acetylation, which is necessary to maintain transcription; however, histone deacetylases (HDACs) can eliminate acetyl group from histones, which can favor the formation of heterochromatin to repress the activity of promoters ^[30].

Figure 3. The mechanisms of epigenetics. DNA methylation inhibits DNA transcription. Histone modification, which modifies the interaction of DNA with histones or other DNA-binding protein complexes, regulates gene expression in a more dynamic and changing manner than does DNA methylation. Non-coding RNAs may be large (>200 nucleotides) or small (18–200 nucleotides). mRNA is one of the small non-coding RNAs. miRNAs suppress gene expression by selectively binding to the 3′ non-coding region (3′UTR) of their mRNA targets through base-paring. miRNAs can negatively regulate gene expression by two different post-transcriptional mechanisms. RISC: RNA induced silencing complex.

Histone methyltransferases catalyze the methylation of histone lysine residues to repress transcription of the gene ^[31]. Histone demethylases can eliminate the methyl group from lysine, which reverses the transcription of the gene ^[32]. Beyond enzymatic modification, some metabolites, including succinate, butyrate, and propionate, can trigger histone butyrylation, propionylation, succinylation, and crotonylation ^[33]. A collective review is necessary of the medical role of mRNA, methylated DNA, and histone modification for bone metabolism and osteoporosis occurrence. This article highlights how DNA methylation, histone modification, and non-coding RNA affect osteoporosis, bone remodeling, and treatment of OVF.

2. Noncoding RNA

Noncoding RNA research, particularly in the musculoskeletal area, is still an unexploited field with a new class being updated regularly. Within the non-coding RNA area, microRNA (miRNA) is the most studied category. miRNA is a small non-coding RNA molecule (containing about 22 nucleotides) found in plants, animals, and some viruses, that modulates gene expression. RNA interference is a powerful mechanism for gene silencing, underlying many aspects of eukaryotic biology. At the molecular level, RNA interference is mediated by a ribonucleoprotein complex called RNA-induced silencing complex (RISC) and can be programmed to target virtually all nucleic acid sequences for silencing. RISC’s ability to find target RNA has been adopted many times in evolution, producing a wide range of gene silencing pathways [79]^[34]. Several new functions of miRNAs have been proposed, and several novel classes of larger noncoding RNA with a crucial transcriptional role have been recently demonstrated, including small interfering RNA (siRNA), Piwi-interacting RNAs (piRNAs), long non-coding RNAs (lncRNA), and others (Table 1) [80]^[35].

Table 1. The various non-coding RNAs (ncRNA) and their characteristics.

2.1. MicroRNAs (miRNAs)

miRNAs are small, non-coding RNA molecules. The size of mature miRNAs ranges between 21–23 nucleotides. Their role is to modulate gene expression by post-transcriptional silencing, either by moderating the cleavage of a targeted transcript or by translational inhibition. In addition, miRNA is involved in the epigenetic regulation of bone remodeling (Table 2 and Figure 3).

 Table 2. The pathway/affected molecule of miRNAs and their effects on bone biology.

Hmga2 (high-mobility group AT-hook 2), EGFR (epidermal growth factor receptor), HB-EGF (heparin-binding EGF-like growth factor), NOTCH1 (notch homolog 1, translocation-associated (Drosophila)), RUNX (runt-related transcription factor), PI3K (phosphatidylinositol 3-kinase), AKT (protein kinase B), BMP (bone morphogenetic protein), TGF-β (transforming growth factor beta), DKK1 (Dickkopf Wnt signaling pathway inhibitor 1), SFRP1 (secreted frizzled-related protein 1), NKIRAS2 (NFKB Inhibitor Interacting Ras Like 2), Dlx (Distal-Less Homeobox), GSK-3β (Glycogen synthase kinase 3β), BMPR (bone morphogenetic protein receptor), ATF4 (activating transcription factor 4), TWIST (Twist-related protein), RANKL (receptor activator of nuclear factor kappa-Β ligand), PDCD4 (Programmed cell death protein 4), TNF (tumor necrosis factor), IL (interleukin), M-CSF (macrophage colony-stimulating factor), TRAP (tartrate-resistant acid phosphatase), Bcl-2 (B-cell lymphoma), LEPR (leptin receptor), AMPK (5' adenosine monophosphate-activated protein kinase), OPG (Osteoprotegerin), Bax (Bcl-2-associated X protein), TAB 1 (TGF-beta activated kinase 1 binding protein 1), Foxo3 (forkhead box O3), Maml1 (mastermind-like protein 1), HO-1 (heme oxygenase-1), Pten (phosphatase and tensin homolog), DC-STAMP (dendritic cell-specific transmembrane protein), CTGF/CCN (connective tissue growth factor), RhoA (ras homolog gene family, member A), PTHrP (parathyroid hormone-related peptide), MMP-13 (matrix metallopeptidase 13), Calcr (calcitonin receptor), EphA2 (ephrin type-A receptor 2).

Figure 4. The epigenetic regulation of miRNA in bone remodeling.

RUNX2 is a central transcription factor that regulates osteogenesis, particularly osteoblast differentiation, as previously described. The genomic target of RUNX2 is osteoblast-specific cis-acting element 2 (OSE2), an approximately 18-bp, highly conserved DNA sequence located in the promoter region of many osteogenic genes, such as secreted phosphoprotein 1 (SPP1), collagen 1 alpha 1 (COL1A1), alkaline phosphatase (ALP), and others [81]^[36]. Beyond the epigenetic controls of RUNX2 transcription, many miRNAs have been shown to be activators or attenuators of its expression. Lineage progression in osteoblast and chondrocyte is strictly controlled by a cell fate-determining transcription factor, RUNX2. During osteogenesis differentiation and cartilage formation, miRNAs (miR-23a, miR-30c, miR-34c, miR-133a, miR-135a, miR-137, miR-204, miR-205, miR-217, miR-218, and miR-338) are generally reversely expressed to RUNX2. All RUNX2 targeting miRNA (except miR-218) significantly inhibits osteoblast differentiation and can reverse its effect by corresponding anti-miRNA [82]^[37]. Attenuation of miRNA in protein translation has emerged as a crucial regulator of mesenchymal cell differentiation into osteoblast lines. The deletion of the dicer enzyme in the osteogenesis factor by Col1a1-Cre hindered the survival of the fetus after E14.5. This suggests that dicer-generated miRNA is essential for promoting prenatal osteoblast differentiation and suppressing bone development in adults [83]^[38].

Members of the miR-30 family are known as crucial modulators in osteogenic differentiation. miR-26a modulates the expression of the Smad1 protein during the osteoblastic differentiation of human adipose tissue-derived stem cells. Inhibition of miR-26a could increase osteoblast differentiation [84]^[39]. miR-135b can control osteoblast differentiation of unrestricted somatic cells (USSCs) by regulating the expression of bone-related genes. A reduction in the expression of osteogenesis markers IBSP and Osterix is known to be involved in bone mineralization in the osteogenesis of USSCs which overexpress miR-135b [85]^[40]. Exogenous miR-125b transfection inhibits osteoblast differentiation. In contrast, when endogenous miR-125b is blocked by the transfection of antisense RNA molecules, ALP activity after BMP-4 treatment is increased [86]^[41]. Both miR-141 and -200a remarkably modulated BMP-2-induced pre-osteoblast differentiation by the translational repression of Dlx5 [87]^[42]. In addition, miR-204/211 acts as a crucial negative regulator of RUNX2, which inhibits bone formation and promotes adipogenic differentiation of mesenchymal progenitor cells and BM-MSCs [88]^[43].

A few miRNAs can be promoters of bone formation. miR-218 is induced during osteoblast differentiation and has strong osteogenesis characteristics. miR-218 promotes the involvement and differentiation of bone marrow stromal cells by activating positive Wnt signaling loops [89]^[44]. miR-218 expression is promoted by the expression of the Wnt pathway gene Wnt3a, which forms an amplification circuit [90]^[45]. MiR-449a inhibits HDAC1 expression and regulates histone acetylation. Accordingly, silencing of endogenous HDAC1 expression by exogenous miR-449a maintains a histone acetylated state, stimulates RUNX2 gene expression, and rapidly promotes osteoblast differentiation [91]^[46].

miRNA-directed regulation also targets osteoclasts. Expression of miR-503, targeting RANK, is strongly reduced in circulating CD14+ macrophages of human osteoporosis patients in comparison to normal patients, and it silences miR-503 with an antagomir that promotes RANK expression and osteoclast differentiation, promotes bone resorption, and decreases bone mass in an ovariectomized mouse model [92]^[47]. MiR-223 is strongly expressed in rheumatoid arthritis (RA) synovial membrane, and excessive expression of miR-223 inhibits osteoclast formation in vitro [93]^[48]. In addition, MiR-223 expression was significantly higher in the synovial membrane of RA patients and ankle joints of collagen-induced arthritis (CIA) mice than in OA patients and normal mice. The knockdown of miR-233 by lentiviral-mediated silencing reduced the arthritis score, histological score, miR-223 expression, osteoclast formation, and bone erosion in mice with CIA [94]^[49].

Several miRNAs have recently drawn scrutiny in the bone field as potential clinical biomarkers for OVF. Ahn identified the TT genotype of miR-149aT>C and suggested it may contribute to decreased susceptibility to OVF in Korean postmenopausal women. The miR-146aCG/miR-196a2TC combined genotype and the miR-146aG/-149T/-196a2C/-449G allele combination may promote increased susceptibility to OVF [4]. In addition, Zarecki et al. showed seven significantly (p<0.05) up-regulated miRNAs (miR-375, miR-532-3p, miR-19b-3p, miR-152-3p, miR-23a-3p, miR-335-5p, miR-21-5p) in patients with OVF [95]^[50].

2.2. Long Non-Coding (lnc) RNAs

The lncRNAs, large non-coding RNAs > 200 nucleotides in length, play crucial roles in many activities of life [96]^[51], such as epigenetic regulation, dose compensation effects, and regulation of cell differentiation (Table 3). Abnormalities of lncRNAs can cause disease, and many reports have demonstrated that lncRNAs are closely associated with the underlying mechanism in the pathogenesis of osteoporosis.

Table 3. The pathway/affected molecule or lncRNAs and their effects in bone biology.

HIF1A-AS (Hypoxia‑inducible factor‑1A-antisense RNA), SIRT (Sirtuin), EZH2 (enhancer of zeste homolog 2), H3K27me3 (polycomb repressive complex 2 methylates lysine 27 of histone H3-mediated trimethylation), RUNX2 (runt-related transcription factor 2), MAPK (mitogen-activated protein kinas), TCF1 (transcription factor 1), LEF1 (lymphoid enhancer-binding factor 1), TF (transcription factor), BMP (bone morphogenic protein), IGF (insulin-like growth factor), PTEN (phosphatase and tensin homolog), NIP45 ( Nuclear Factor Of Activated T Cells (NFAT) 1interacting protein), RANKL (receptor activator of nuclear factor kappa-Β ligand), CTSK (cathepsin), DDL3 (delta-like 3), HDAC4 (histone deacetylase 4), NF-κB (nuclear factor-κB), MAPK (mitogen-activated protein kinase), IL (interleukin).

Many lncRNAs have demonstrated an ability to promote osteoblast differentiation, and it has been hypothesized that they could help treat osteoporosis. The histone decarboxylase SIRT1 was shown to be a crucial positive regulator of bone mass and osteoblastogenesis. Nevertheless, the expression of SIRT1 was inversely proportional to lncRNA HIF1A-AS1 expression, which can suggest a role of lncRNA HIF1A-AS1 in osteogenic differentiation [97]^[52]. In addition, lncRNA plays a crucial role in gene regulation and is involved in a variety of cellular processes. HoxA-AS3 is increased when inducing adipogenesis of MSC and it interacts with Enhanced Of Zeste 2 (EZH2) and is required for H3K27 trimethylation of the main osteogenesis transcription factor RUNX2. As a result, lncRNA HoxA-AS3 is a crucial molecule in osteoblast differentiation [98]^[53]. In contrast to lncRNA HoxA-AS3, lncRNA-differentiation antagonizing non-protein coding RNA (DANCR) was reported to recruit EZH2 in the promotion of H3K27 trimethylation through the interaction with a 305-nt transcript and enhancer of zestehomolog2, which ultimately inhibited transcription of the target gene RUNX2 and osteogenic differentiation [99]^[54]. DANCR also modulated the proliferation and osteogenic differentiation of hBM-MSCs via the inactivation of the p38 MAPK signaling pathway [100]^[55]. The decline in anti-differentiation noncoding RNA (ANCR) accelerated the growth of periodontal ligament stem cells (PDLSC). Further, the down-regulated ANCR promotes osteogenesis differentiation of PDLSC by up-regulating the osteogenesis differentiation marker gene [101]^[56]. In addition, inhibition of lncRNAs, such as HOX transcript antisense RNA (HOTAIR), promoted ALP activity and increased the number of osteogenesis marker genes and calcified nodules in BM-MSC. However, the over-expression of HOTAIR showed the opposite effect. HOTAIR inhibited expression levels of Wntβ-catenin pathway-related proteins [102]^[57]. Furthermore, low expression of lncRNAp21 activates the Wntβ-catenin signaling pathway by increasing E2 secretion, ultimately stimulating osteogenesis, and increasing osteogenesis differentiation of MSCs in osteoporosis rat models [103]^[58]. The DKK4 gene encodes a protein that belongs to the Dickkopf family. Downregulating lncRNA H19 reduces the expression level of Dkk4, which inhibits the Wnt/β-catenin signaling pathway and negatively regulates osteogenic differentiation [104]^[59]. lncRNA AK045490 correlates with osteogenesis differentiation and strengthens skeletal tissue of mice. In the in vitro analysis of BM-MSC, AK045490 inhibited osteoblast differentiation. In vivo inhibition of AK045490 by siRNA saved bone formation in the oophorectomy osteoporosis mouse model. AK045490 inhibits nuclear dislocation of β-catenin and inhibits expression of TCF1, LEF1, and RUNX2 [105]^[60]. In a similar fashion, lncRNA AK016739 inhibits osteogenic differentiation and bone formation since it inhibits osteoblastic transcription factors [106]^[61]. By inhibiting lncRNA-UCA1, the BMP-2 (Smad158) signaling pathway in osteoblasts is activated to promote osteoblast proliferation and differentiation [107]^[62]. Moreover, the expression of serum lncRNA MEG3 in fracture patients is remarkably increased. Because LncRNA MEG3 can promote osteoblast proliferation and differentiation by activating the Wntβ-catenin signaling pathway, it is expected to become a new target for promoting fracture healing [108]^[63]. In contrast, downregulating lncRNA MEG3 inhibits osteogenic differentiation by promoting the expression of IGF1 [109]^[64]. The considerations outlined here suggest that many lncRNAs have inhibitory effects on osteogenic differentiation. As a result, silencing the expression of these specific lncRNAs using specifically targeted drugs may be possible, which may limit osteoporosis development.

lncRNAs have been reported to modulate osteoclastogenesis by modulating the expression of specific target mRNAs. Plasma lncRNA TUG1 was regulated at a higher level in patients with osteoporosis than in healthy participants. A patient with osteoporosis and a healthy patient is distinguished by increasing the regulation of plasma lncRNA TUG1. LncRNA TUG1 levels increased with advances in the clinical stage. Although excessive expression of lncRNA TUG1 accelerated proliferation and inhibited apoptosis of mouse osteoclasts, lncRNA TUG1 siRNA silencing played a reverse role [110]^[65]. The expression of lncRNA AK077216 is remarkably suppressed during osteoclast formation. Up- and down-regulation of lncRNA AK077216 promotes and inhibits osteoclast differentiation, bone absorption, and expression of related genes. lncRNA AK077216 regulates the expression of NFATc1 and promotes osteoclast formation and function [111]^[66]. Downregulation of lncRNA SNHG15 inhibits osteoclasts by modulating the RANK/RANKL pathway [112]^[67]. lncRNAs also have inhibitory effects on osteoclasts. These lncRNA expression levels were inversely correlated with osteoporosis severity. lncRNA RP11-498C9.17 is strongly correlated with many epigenetic regulatory factors, including HDAC4, HMGA1, MORF4L1, and DND1. Downregulation of HDA was reported to increase osteoclast differentiation, which suggests that lncRNA RP11-498C9.17 may modulate osteoclast production via HDAC4 signaling [113]^[68]. In addition, lncRNA Bmncr inhibits RANKL-induced osteoclast differentiation [114]^[69]. Upregulation of lncRNA NONMMUT037835.2 inhibits osteoclast differentiation, and downregulation of lncRNA-NONMMUT037835.2 promotes osteoclast formation [115]^[70]. In conclusion, these lncRNAs with effects on osteoclast differentiation may demonstrate a breakthrough in treating osteoporosis. The expression level of lncRNAs promoting osteoclast differentiation is lowered and suppressed.

3. The Possibility of Epigenetics in Treatment of Osteoporosis and OVF

The epigenetic effects on bone formation and remodeling reactions may facilitate the development of epigenetic therapeutics with the potential to treat osteoporosis and OVF. A large variety of proof-of-concept studies have demonstrated the remedial effects of miRNA and epigenetic modifiers in slowing osteoporosis development. A recombinant adeno-associated virus serotype 9 (rAAV9) can deliver artificial miRNA (amiR) to osteoclast cells of patients/animals with osteoporosis to silence expression of RANK and cathepsin K (rAAV9.amiR-rank, rAAV9.amiR-ctsk), which are major osteoporosis regulators. Because rAAV9 is very effective for the transduction of osteoclasts, systemic administration of rAAV9 with amiR rank or amiR-ctsk results in a significant increase in bone mass in mice. rAAV9.amiR-ctsk suppresses bone loss and improves bone mechanical properties in postmenopausal and senile osteoporosis mouse models [116]^[71]. A circulating miR-338 cluster in the serum could maintain bone formation capacity and increased bone mass and trabecular structure in an osteoporosis mouse model [117]^[72]. miR-672-5p induced osteoblast differentiation and mineralization in ovariectomized mice [118]^[73]. Histone methyltransferase DOT1L inhibition decreases osteoclastic activity, which can delay osteoporosis progression [119]^[74]. The knockdown of EZH2 by lentivirus-expressing shRNA rescued the abnormal fate of osteoporotic MSC. The H3K27me3 inhibitor DZNep effectively derepressed Wnt signaling and improved osteogenic differentiation of osteoporotic MSCs in vitro [120]^[75]. DNA methylation inhibitor 5-aza-20-deoxycytidine improves bone mass in disuse-induced osteopenic bone development [121]^[76].