Circular RNAs in the management of human osteoporosis

Abstract

Background

Osteoporosis (OP) is a metabolic bone disease producing reduction in bone mass with consequent bone fragility. Circular ribonucleic acid (CircRNA) is a form of RNA that forms a loop structure rather than a linear one. CircRNA can be used for therapeutic purposes, including molecular targets or to test new therapies.

Sources of data

A systematic search of different databases to July 2024 was performed to define the role of circRNA in OP therapy. Seventeen suitable studies were identified.

Areas of agreement

CircRNAs may be useful in studying metabolic processes in OP and identify possible therapeutic targets and new drug therapies.

Areas of controversy

The metabolic processes involved in OP are regulated by many genes and cytokines that can be targeted by CircRNAs. However, it is not easy to predict whether the in vitro responses of the studied CircRNAs and their interaction with drugs are also applicable in vivo.

Growing points

Metabolic processes can be affected by gene dysregulation of CircRNAs on various growth factors. Areas timely for developing research: Despite the predictability of CircRNA pharmacological response in vitro, such pharmacological response cannot be expected to be replicated in vivo.

Data availability

The data that support the findings of this study are available from the corresponding author.

Introduction

Osteoporosis (OP), a condition affecting adults and postmenopausal women, weakens bones, increasing the risk of fractures [1–7]. This disorder disrupts the balance between bone anabolism and catabolism, with bone resorption outpacing formation [8–10]. While aging plays a role in this process, it is not the only factor behind OP development [1,11–19]. Management modalities for OP focus on strengthening bones through supplements such as calcium and vitamin D and drugs targeting bone breakdown [1,20–32]. However, these treatments may not work for all patients and have side effects. Recent research has turned to exploring circular ribonucleic acid (circRNA) in cells to uncover targets for more effective therapies [33,34].

CircRNA (Fig. 1) is a form of RNA that forms a loop structure rather than a linear one [33,34].

This occurs when the ends, at 3′ and 5′ commonly found in RNA molecules, are connected. This arrangement gives circRNA peculiar characteristics that have only recently come to light [33,34]. Additionally, some circRNA have displayed potential as regulators of gene expression. The complete biological role of circRNAs is still not entirely understood [33,34].

Given the lack of 5′ or 3′ ends, circRNAs are resistant to degradation by exonucleases, making them likely more stable than the majority of linear RNAs in cells [33,35]. Their stability has associated them with diseases, including cancer, where they hold functions of protein production that escape the classical pathways of physiological regulation and homeostasis [33,35].

CircRNAs play a role in regulating and triggering T-cell responses [33,35]. They impact the immunity and aging process of a cluster of differentiation 8 (CD8) + T cells [33,35]. CircRNAs are essential for macrophage differentiation and polarization [33,35]. When paired with soluble protein antigens, circRNAs act as stimulants that trigger adaptive immunity without needing a specific delivery method [33,35]. Treatments involving dendritic cell differentiation and maturation lead these cells to release a variety of cytokines and chemokines while expressing genes for interleukin 1 beta (IL 1β), IL 6, and tumor necrosis factor alfa (TNFα) [33,35]. Immunization with cirRNA-containing antigen sequences leads to enhanced CD8+ T-cell mediated responses against target antigens [33,35].

Given their durability and extended storage capabilities, circRNAs offer benefits when used as markers and vehicles that express genes of interest [33,35]. In OP, an unbalance occurs between bone breakdown and formation influenced by activation that impacts the functions of osteoblasts and osteoclasts [33,35–40]. Scientists investigating circRNAs have studied their effects on these bone cells, identifying particular targets with predictable outcomes [33,35]. These discoveries aim to establish targeted treatments to manage OP or to pinpoint factors for measures. This systematic review examines the present published research on employing circRNAs to manage OP, underscoring their promising role in developing more efficient and precise therapeutic approaches.

Methods

The review adheres to the preferred reporting items for systematic reviews and meta-analyses (PRISMA) (Fig. 2) [1,41–43].

This study includes all previously published investigations that meet the predetermined criteria to assess the potential involvement of circRNA in OP therapy.

The present investigation included studies carried out in languages other than English. Narrative and systematic reviews, meta-analyses, technical notes, and case studies were not included.

Two investigators performed a systematic search independently of each other until July 2024. They reviewed the full-text archives of Embase, Google Scholar, Scopus, and PubMed. During the search, different combinations of the following key terms were employed: OP, OP therapy, circular RNA, circRNA, RNA silencing, and RNA interference, without any restriction on the year of publication. The two researchers performed an independent review of the titles and abstracts to eliminate any duplicates. They then assessed the articles that met the requirements against the predetermined criteria for inclusion. If the titles and abstracts did not provide enough information to determine whether to include or exclude a study, the entire article was thoroughly examined. The bibliography of the listed articles was extensively examined to identify additional related articles. Discussions with the senior investigator allowed to address any disagreements.

Seventeen papers met the criteria for inclusion and were therefore included in the analysis. Details of the search are described in the flowchart presented in Fig. 2.

Results

A total of 374 studies were identified. 291 duplicates were removed, and 178 articles were obtained. 82 articles were removed after reading the title and abstract. A further 79 articles were removed because of incomplete data and inappropriate information.

The remaining 17 articles were analyzed and included in Table 1. The articles were analyzed according to the function of the circRNA being studied.

CircRNAs and osteoclastogenesis

Patients with OP usually exhibit impaired bone fracture healing because of diminished osteogenesis and inhibited angiogenesis [59]. Recent studies have highlighted the role of circRNAs in osteoclastogenesis [59]. One such circRNA, circ_0029463, is elevated in OP patients and during osteoclastogenesis [59]. Circ_0029463 was necessary for osteoclast differentiation, whereas its knockdown would hinder it [59]. Researchers additionally identified circ_0029463, microRNA (miR)-134-5p, and adeno-associated virus (Rab)27a in the context of a regulatory network driving osteoclast differentiation [59]. This investigation indicates the possibility to use non-coding RNAs such as circRNAs as potential therapeutic targets in the medical management of OP [59].

Circa_0006859 has been investigated for its role in postmenopausal OP (PMOP) [57], as it inhibits the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs), exacerbating the condition. Targeting miR-642b-5p and miR-483-3p then increases Ephrin A2 (EFNA2) and dedicator of cytokinesis 3 (DOCK3) levels [57]. These molecular interactions are crucial for the Wnt (Wingless/Integrated) -signaling pathway, which is necessary for bone health. Targeting hydroxylase activity (has)_circ_0006859 might, therefore, serve as a possible therapeutic approach to PMOP, as it plays a significant role in BMSCs dysfunction associated with OP [57].

Recent investigations have also revealed the role of circ- Procollagen-Lysine,2-Oxoglutarate 5-Dioxygenase 2(Plod2) in postmenopausal OP. Circ-Plod2 was downregulated in the BMSCs of ovariectomized rats, which are a model for PMOP [56]. This downregulation was linked to an increase in the myeloperoxidase (Mpo) gene [56]. Through its interactions with the RNA-binding protein insulin growth factors binding protein 2 (IGF2BP2), Circ-Plod2 destabilizes Mpo mRNA, stimulating osteogenic differentiation of BMSCs [56]. This interaction highlights the crucial role of circ-Plod2 in the activity of BMSCs and the development of postmenopausal OP. It also suggests that circ-Plod2 could be used as a biomarker and therapeutic target for POMP [56].

Examining the role of circular Forkhead box protein P1 (circFOXP1) in OP, the protein was significantly down-regulated in the bone tissues of OP patients [44]. By absorbing miR-33a-5p, circFOXP1 stimulates osteogenesis and controls the expression of FOXP1 [44]. This axis supports osteogenic development and in vivo bone regeneration of human adipose-derived mesenchymal stem cells (hASCs) [44]. These results shed light on the regulatory mechanisms underpinning osteogenesis and also suggest circFOXP1 as an intriguing target for the management of OP [44].

These studies collectively indicate the complex and critical functions that circRNAs play in osteoclastogenesis and the broader pathophysiology of OP. Research has identified novel pathways for therapeutic intervention by unraveling molecular mechanisms involving circ_0029463, circ_0006859, circ-Plod2, and circFOXP1 [44], which might significantly enhance the management of OP and related bone conditions.

CircRNAs as biomarkers

The role of circRNAs extends beyond their involvement in osteoclastogenesis; they also serve as promising biomarkers for OP [49]. For instance, circRNA hsa_circ_0006215 is an important variable in bone development and repair, specifically in the setting of elderly OP [49]. Decreased levels of hsa_circ_0006215 in OP patients suggest that it plays a role in the condition [49]. This circRNA promotes osteogenic development of BMSCs regulating key proteins such as runt-related transcription factor 2 (RUNX2) and vascular endothelial growth factor (VEGF) through competitive binding to miR-942-5p [49]. Such interactions point to hsa_circ_0006215 as a potential as a target of treatment for promoting bone health [49].

Circ_0001795 has received some attention in OP research [53]. This circRNA is dramatically downregulated in OP patients, and its overexpression promotes osteogenic differentiation of human bone marrow stromal cells (hBMSCs). Circ_0001795 serves as a molecular sponge for miR-339-5p, which targets yes-associated protein 1 (YAP1) [53]. Downregulation of YAP1, which is mediated by miR-339-5p, has been associated with OP progression [53]. Thus, the circ_0001795/miR-339-5p/YAP1 axis plays a critical role in regulating osteogenic differentiation, setting circ_0001795 as an avenue for therapy of OP [53].

A separate study explored the potential of circular RNA hsa circ 0001445 as a biomarker to diagnose OP in postmenopausal women [46]. The association between bone density ratings and lower levels of hsa circ 0001445 in OP patients, as opposed to healthy controls, was highlighted [46]. Based on that study, hsa circ 0001445 has the potential to be employed as a non-invasive tool and may allow to monitor anti-osteoporotic therapy [46]. This underscores the importance of identifying novel biomarkers for early identification and evaluation of treatments for OP [46].

These studies collectively underline the possible application of circRNAs as biomarkers for OP. The finding of specific circRNAs that are dysregulated in OP patients and an understanding of their roles in bone metabolism has allowed researchers to develop innovative diagnostic and therapeutic strategies to manage OP.

CircRNAs in osteogenic differentiation

The involvement of circRNAs in osteogenic differentiation is a rapidly expanding field of research with considerable promise for understanding and treating OP [54]. Circ_0019693 is one of the circRNAs that promotes osteogenic differentiation and angiogenesis in bone marrow stem cells [54]. Targeting miR-942-5p and Purkinje cell protein 4(PCP4) may enable novel alternatives to therapy for OP [54]. Circ_0019693 helps to regulate bone growth and repair by regulating these molecular pathways [54].

Circ_0027885 has been implicated in the regulation of osteogenic differentiation; its levels are low in OP patients [60]. Circ_0027885 enhances osteogenic differentiation by upregulating RUNX2, a key transcription factor for osteogenesis, in hBMSCs [60]. Bioinformatic analysis identified circ_0027885 as a sponge for miR-203-3p. Overexpression of circ_0027885 decreases miR-203-3p levels, which promotes osteogenesis by downregulating RUNX2, and this reduces osteogenic differentiation [60]. The circ_0027885/miR-203-3p/RUNX2 loop thus gives important information into the pathogenesis of OP [60].

Another notable breakthrough in this field is hsa_circ_0114581, a crucial circRNA in osteogenic differentiation via transcriptome analysis. This circRNA sequesters miR-155-5p, resulting in higher heterogeneous ribonuclear protein A3 (HNRNPA3) transcription in BMSCs [55]. This study indicates a distinctive regulatory pathway in the pathogenesis of OP, giving potential paths for therapeutic intervention and future enquiry into the molecular underpinnings of bone disorders [55].

Another study covers the function of circRNA in the osteogenic development of BMSCs for OP treatment [51]. With high-throughput sequencing identifying differently expressed circRNAs during BMSCs osteogenic differentiation, circ_0005564 was revealed as a positive putative regulator [51]. Data on osteogenic markers and mineralized nodule development support the hypothesis that circ 0005564 is a therapeutic target for OP, and it is confirmed to have a functional role [51].

Hsa_circ_0006766 is likewise recognized for its regulatory function in the osteogenic differentiation of human bone marrow mesenchymal stem cells (hBM-MSCs) [50]. This circRNA influences osteogenic differentiation by targeting miR-4739 and regulating Notch2, a gene crucial to bone formation [50]. The regulatory nexus involving Hsa_circ_0006766 and miR-4739 conveys that this circRNA might potentially be considered a therapeutic target for OP [50]. The study demonstrates the role of circRNAs in bone metabolism and the differentiation of stem cells, offering new insights into bone disease as well as prevention strategies for OP [50].

CircRNA_0048211 plays a major part in PMOP through the establishment of a regulatory loop with miRNA-93-5p and BMP2, which controls osteogenesis in human bone marrow stem cells. Overexpression of circ_0048211 boosts osteogenic gene expression, alkaline phosphatase (ALP) activity, and mineralization, thereby presenting fresh targets for PMOP medical care. The modulation of this regulatory loop could lead to significant advancements in managing the progression of PMOP [45].

Hsa_circ_0076690 has been studied for its potential as a diagnostic biomarker as well as for its function in regulating osteogenic differentiation in human bone marrow stem cells via miR-152 sponging [47]. These findings may pave the way for new approaches to manage OP, underscoring the vital role of circRNAs in bone health and disease [47].

The function of circular RNA circ-3626 in bone formation has been addressed by outlining its regulatory contribution to the miR-338-3p/RUNX2 pathway in BMSCs [58]. The study highlights the potential of circ-3626 to improve osteogenic ability and bone regeneration, particularly in OP linked to aging [58]. It implies that circ-3626 might be a cutting-edge therapeutic target to treat and prevent age-related bone loss [58].

Another study focuses on the regulation of microRNA-145 and core-binding factor subunit beta (CBFB) by circular RNA_0062582, in addition to its function in osteogenic development of hBMSCs [52]. MiR-145, a target of circ_0062582, can specifically target CBFB, and circ_0062582 promotes osteogenic differentiation by upregulating related proteins [52]. Emphasizing the critical role of the circ_0062582/miR-145/CBFB pathway in bone regeneration and OP etiology, circ_0062582 is a potential biomarker and therapeutic target for OP therapy [52], though additional in vivo research is needed [52].

Lastly, hsa_circ_0076906 has been identified as a key player in preventing OP by functioning as a sponge for miR-1305, which governs osteoglycin (OGN) production and induces osteogenic differentiation in human-derived mesenchymal stem cells [48]. This finding offers fascinating insights into the management of OP, highlighting the potential of hsa_circ_0076906 in bone tissue regeneration and the growing field of gene therapy [48].

These studies collectively illustrate the multifaceted roles that circRNAs play in osteogenic differentiation and their potential as therapeutic targets for OP. Studies are uncovering new avenues for therapeutic intervention that could significantly improve the management of OP and related bone diseases by elucidating the molecular mechanisms involving several circRNAs.

Discussion

OP is a serious condition given its potential to cause structural damage to bones, increasing the likelihood of fractures and interfering with mobility, resulting in hospitalization and increased mortality [61,62]. Osteoporotic fractures occur following alterations in the microstructure of bone tissue produced by the interaction of various systemic factors [63]. Both males and females experience a decrease in bone density throughout their lives [64,65]. However, women experience a more rapid loss of bone density and are more prone to fractures caused by post-menopausal fragility [66]. Other factors include cellular processes linked to imbalances between osteoclasts and osteoblasts. Calcium and vitamin D play a key role in this context [67].

Although fractures remain the most noticeable symptom of OP, patients with OP shall have suffered from this condition for an extended period prior to experiencing a fracture [68,69].

Profound molecular and genetic factors have been associated with OP to identify a possible therapeutic strategy that focuses on preventing and minimizing the risk of bone injuries.

Many types of cells have been studied, including human cells, mesenchymal stem cells, Bone Marrow Mesenchymal Progenitors, osteoblasts, osteoclasts, and myoblasts, to draw attention to the biological pathways and the specific molecular targets that may be regulated by circRNAs [70].

Presently, therapy for OP involves the administration of antiresorptive drugs such as calcitonin, estrogen, bisphosphonates, and anabolic pharmaceuticals such as teriparatide [71,72].

Drugs for OP often produce suboptimal compliance of patients, a high incidence of serious side effects, and comparatively modest efficacy [73]. Bisphosphonate can cause a range of adverse effects, including typical gastrointestinal problems and serious complications such as osteonecrosis of the mandible [74]. Prolonged use of oral bisphosphonate medications has been linked to an increased risk of fractures and esophageal carcinoma [75]. Hence, bisphosphonate therapy is advised for no more than five years [76]. Denosumab, a monoclonal antibody specifically targeting the receptor activator of nuclear factor kappa ligand (RANKL), first entered the market in 2010 [77]. Denosumab is the first innovative drug for OP, wherein experts attempt to identify and target new therapeutic goals [78]. This work aims to identify new targets for therapy through the use of circRNAs.

The epidermal growth factor receptor (EGFR) interacts with both epidermal growth factor (EGF) and transforming growth factor α (TGFα). This interaction triggers the activation of the receptor, leading to its homodimerization, with a group of proteins that includes human epidermal growth factor receptor 2 (ERBB 2), human epidermal growth factor receptor 3 (ERBB 3), and human epidermal growth factor receptor 4 (ERBB 4) [79]. This activation process stimulates the tyrosine kinase domain activity, resulting in the phosphorylation and recruitment of proteins such Son of Sevenless (SOS), which then activates Rat virus (RAS) [79].

RAS activates the mitogen-activated protein kinase (MAPK), which plays a role in the differentiation of osteoclasts and osteoblasts in bone tissue [80].

Transforming growth factor beta-2 (TGF-β2), a gene that encodes for TGFβ, a serine/threonine protein kinase, controls protein phosphorylation processes within the cell nucleus, leading to increased proliferation of osteocytes and osteoblasts [81].

Furthermore, research has been conducted on IGF [82]. This hormone, also referred to as somatomedine, has interesting properties. It is synthesized by the liver and differentiated chondroblasts [82]. Structurally similar to insulin, IGF has anabolic activities [82]. The bone is stimulated to produce aggrecan, type VI, and IX collagen, and binding proteins that support cell growth, influencing the quality and structure of the bone [82]. CircRNAs are increasingly recognized as regulators in both health and pathological conditions. A study observed changes in expression in the hBMSCs undergoing osteogenesis at specific time points—days 7 and 14 compared to day 0 [83,84]. CircRNAs are involved in osteoblastic differentiation of BMSCs, significantly affecting the process of bone formation [83,84]. For example, Zhang et al. found changes in circRNA levels during osteogenic induction of BMSCs, while Liu et al. emphasized the role of circ transcription elongation factor 4(AFF4), an increased circRNA that promotes osteogenesis in BMSCs by activating the SMAD1/5 signaling pathway through the miR 135a 5p/fibronectin type III domain-containing protein 5(FNDC5)/Irisin mechanism [85].

Circ 3626 increased during osteogenesis, which boosts the bone-forming potential of hBMSCs [86], underscoring the function of circ 3626 in bone development in vivo in mice [86].

The authors used adeno-associated viruses (AAVs) in gene therapy for BMSCs using circ 3626 [86]. Li et al. showed that AAV-mediated delivery of Krüppel-like factor 7(KLF7) targets BMSCs to enhance nerve regeneration and functional recovery [86]. The use of AAV to deliver circ 3626 increased bone density and improved bone structure in mice, demonstrating the potential of circ 3626 to prevent bone loss and improve bone strength through new bone formation [58].

Further exploration into this mechanism revealed that circ 3626 boosts the expression of RUNX2, a transcription factor in bone formation, by interacting with miR 3383p, thus enhancing the expression of genes involved in bone formation. CircRNAs function as molecular snares that interfere with the transcription of their original genes. Circ 3626 effectively binds with miR 338 3p, which is elevated in OP and hinders bone formation. MiR 338 3p inhibits the process of bone formation by regulating RUNX2 and its downstream targets, such as Fgfr2 [87].

The significant increase in RUNX2 expression and subsequent activation of genes related to bone formation by circ 3626 in mice not only confirms its role in regenerating bones but also aligns with broader genetic mechanisms involved in controlling bone health [58].

Circ 3626 favorably impacts bone strength and may be a promising agent for bone-related conditions [87]. However, the impact of circ 3626 on activity and adipogenesis has not been explored yet [86]. While no significant negative effects were seen in mice after treatment, the broad nature of the AAV vector presents challenges for BMSC transfection in live animals [86]. Future studies should focus on the production of targeted delivery systems for circRNAs to improve accuracy and reduce side effects.

Circ 3626 promotes bone formation by influencing gene expression through miR 3383p interactions [86], introducing novel possibilities to manage metabolic bone disorders.

Conclusion

Many conditions seem to be age-related or have multifactorial causes arising from molecular and cellular imbalances. In the current therapies for OP, we intervene when the disease is already present, trying to restore normality in an already long-established condition. Using circRNAs, it is possible to study the genetic and molecular bases of OP, allowing the development of specific therapies that prevent the condition and its consequences. Several researchers have used circRNAs for various objectives: some to identify target molecules, others as therapeutic targets, and others still to evaluate the effectiveness of specific drugs. Studies on human cells in vitro have given promising results, offering hope of future drugs capable of fighting OP at its root without the side effects of current therapies.

Author contributions

Giuseppe Gargano (Data curation, Formal analysis, Writing—original draft), Simona Maria Pagano (Conceptualization, Methodology, Validation, Writing—original draft), and Nicola Maffulli (Conceptualization, Data curation, Formal analysis, Writing—original draft)

Conflict of interest

None declared.

Data availability

The authors confirm that the data supporting the findings of this study are available within the article.

References