Effects of miRNAs, lncRNAs and circRNAs on osteoporosis as regulatory factors of bone homeostasis (Review).

Osteoporosis is a common metabolic bone disorder typically characterized by decreased bone mass and an increased risk of fracture. At present, the detailed molecular mechanism underlying the development of osteoporosis remains to be elucidated. Accumulating evidence shows that non‑coding (nc)RNAs, such as microRNAs (miRNAs), long ncRNAs (lncRNAs) and circular RNAs (circRNAs), play significant roles in osteoporosis through the post‑transcriptional regulation of gene expression as regulatory factors. Previous studies have demonstrated that ncRNAs participate in maintaining bone homeostasis by regulating physiological and developmental processes in osteoblasts, osteoclasts and bone marrow stromal cells. In the present review, the latest research investigating the involvement of miRNAs, lncRNAs and circRNAs in regulating the differentiation, proliferation, apoptosis and autophagy of cells that maintain the bone microenvironment in osteoporosis is summarized. Deeper insight into the aspects of osteoporosis pathogenesis involving the deregulation of ncRNAs could facilitate the development of therapeutic approaches for osteoporosis.

Introduction

Osteoporosis is a highly prevalent skeletal disorder associated with the ageing of the global population that imposes a considerable burden on health care and society, and is characterized by the deterioration of bone tissue and increased bone fragility due to the loss of bone mass and microstructure attributed to various factors, including menopause, ageing and related adverse reactions to medications (1). Bone and fat mass imbalance constitute a typical feature of the pathogenesis of osteoporosis (2). In Europe, an estimated 22 million women and 5.5 million men suffer from osteoporosis (3), and an estimated 10 million individuals over the age of 50 have osteoporosis in the USA (4). As the population of the world rapidly ages, an increasing number of individuals will suffer from osteoporosis.

Bone homeostasis is maintained mainly by intricate mechanisms synchronizing osteoblast (OB) activation with osteoclast (OC) activation, thus coupling bone formation with bone resorption (5). OBs, the cells responsible for bone formation, are believed to originate from bone marrow stromal cells (BMSCs). Osteoclasts are derived from mononuclear haematopoietic myeloid lineage cells that influence bone resorption. In addition to OBs and OCs, BMSCs, adipocytes and chondrocytes present in the microenvironment also participate in bone homeostasis (6). BMSCs, which are a key cause of osteoporosis, play an important role in maintaining the balance between bone formation and resorption. Previous findings have shown that BMSCs can normally differentiate into OBs, chondrocytes and adipocytes, but in the elderly, the differentiation of BMSCs into OBs decreases. Such changes lead to a decrease in bone formation, which, in turn, leads to osteoporosis, and the underlying mechanism remains to be elucidated (7).

Non-coding (nc)RNAs, including microRNAs (miRNAs/miRs), long ncRNAs (lncRNAs) and circular RNAs (circRNAs), play an important role as regulators in cellular processes, such as cell proliferation, differentiation, apoptosis and autophagy. miRNAs constitute a class of ncRNAs that are ~22 nucleotides in length, can recognize the 3′-untranslated regions (UTRs) of target mRNAs by means of complementary base pairing and degrade or repress the gene expression of target mRNAs at the post-transcriptional level (8). lncRNAs are a type of ncRNA with a length >200 nucleotides that can bind substrates through their own nucleotide sequence or folded secondary structure and regulate gene expression through multiple mechanisms at the transcriptional and post-transcriptional levels (9). circRNAs, which are formed mainly by reverse splicing, are endogenous covalent closed circRNA molecules that can act as miRNA sponges to regulate miRNA-related cellular processes (9).

Accumulating evidence has confirmed the crucial roles of ncRNAs in bone metabolism by regulating the differentiation, proliferation, apoptosis and autophagy of bone homeostasis-related cells. Jin et al (10) identified 260 circRNAs, 70 lncRNAs and 13 miRNAs that were differentially expressed between patients with postmenopausal osteoporosis and healthy controls using Illumina-based deep sequencing. Eskildsen et al (11) showed that miRNA-138 was related to the osteogenic differentiation of hMSCs and that the overexpression of miR-138 inhibited the OB differentiation of hMSCs. The lncRNA Xist was found to sponge miR-19a-3p to repress BMSC osteogenic differentiation in ageing-induced osteoporosis (12). Owing to the development of deep sequencing technology, an increasing number of biologically significant ncRNAs affecting bone metabolism and osteoporosis have been studied. Although the current therapies for osteoporosis, such as bisphosphonates, denosumab, teriparatide and selective oestrogen receptor modulators, are effective in restoring bone strength, they inadvertently reduce bone strain and are generally inadequate in preventing hip and non-vertebral fractures (13). Emerging evidence has indicated that ncRNAs play a crucial role in bone remodelling and degeneration in osteoporosis (9). In the present review, the latest studies are summarized to clarify the important role of miRNAs, lncRNAs and circRNAs involved in the regulation of OBs, OCs and BMSCs that affect bone metabolism in osteoporosis, which could provide a theoretical basis for exploring the pathogenesis and potential of clinical treatment for osteoporosis.

Mechanisms by which ncRNAs regulate BMSCs in osteoporosis

BMSCs have the capacity to differentiate into various bone-related cells, such as OBs, adipocytes and chondrocytes. Emerging studies have revealed that the abnormal differentiation capacities of BMSCs play a crucial role in this critical pathogenesis of osteoporosis (14). However, in the elderly, the degree of osteoblastic differentiation of BMSCs is lower than that of adipogenic differentiation, leading to a decrease in bone formation. Therefore, the number and function of BMSCs play a key role in osteoporosis, and the fine regulation of BMSCs plays an important role in maintaining bone homeostasis (7). Previous findings showed that peroxisome proliferator-activated receptor γ (PPARγ), core binding factor α1, osterix and runt-related transcription factor 2 (Runx2) are crucial regulators of differentiation towards adipogenesis or osteogenesis (15), and thus far, the most explored signalling pathway associated with OB differentiation is the Runx2 axis. Recent research indicates that epigenetic regulation is crucial and contributes to osteoporosis by regulating the differentiation, proliferation and apoptosis of BMSCs, and it is necessary to further study the role of ncRNA-mediated autophagy in osteoporosis (9).

Effects of miRNAs on BMSC regulation

miRNAs play crucial roles in regulating BMSC differentiation, proliferation and apoptosis. Several studies have proven the important roles of miRNAs in regulating the osteoclastic, osteoblastic, adipogenic and chondrogenic differentiation of BMSCs to maintain bone metabolic homeostasis (11,16). miRNAs involved in metabolic homeostasis and bone formation are novel targets for the treatment of bone-related diseases. As previously reported, miR-31a-5p derived from aged BMSCs reduced osteoblastogenesis via the SATB2 pathway and promoted osteoclastogenesis via the RhoA pathway in ageing bone tissue, leading to osteoporotic bone loss. The application of antagomiR-31a-5p reduced age-associated bone loss, suggesting that it is a potential biological therapy for age-related osteoporosis (17). Moura et al (18) reported that miR-99a-5p was significantly downregulated during the early stages of human primary MSC osteogenic differentiation and during MC3T3 osteogenic differentiation. It was found that the inhibition of miR-99a-5p promoted osteogenic differentiation and that the expression of miR-99a-5p was increased during OC differentiation, indicating that miR-99a-5p is a positive regulator of osteoclastogenic differentiation and a target candidate for osteoporosis (18). Zhou et al (19) found that the expression of miR-1286 in the serum of patients with osteoporosis was significantly higher than that in the serum of healthy controls. This level of expression decreased as the number of days of osteogenic differentiation of human amnion-derived mesenchymal stem cells increased, indicating that miR-1286 participates in the regulation of osteogenic differentiation and can inhibit the osteogenic differentiation of hMSCs by binding frizzled class receptor 4, leading to the development of osteoporosis. miR-199a-3p was documented as a biomarker of osteoporosis in a model of ovariectomized mice (20). A previous study highlighted the fact that miR-199a-3p plays a crucial role during adipocyte differentiation in ovariectomized mice (21). In addition, Chen et al (22) revealed that miR-199a-3p participates in the inhibition of cardiomyocyte differentiation in stem cells via the MEF2C pathway. Recently, Wu et al (23) found that miR-199a-3p expression was increased in ovariectomized mice and that the upregulation of miR-199a-3p inhibited the osteogenic differentiation of BMSCs in vitro. Furthermore, it was revealed that miR-199a-3p regulated the osteogenic differentiation of BMSCs mainly by targeting Kdm3a (23). miR-23 was found to be upregulated in osteoporosis patients compared with healthy populations, and downregulated in the osteogenic differentiation of hBMSCs (24). Further experiments indicated that miR-23 could bind MEF2C to inhibit osteogenic differentiation (24). Yin et al (25) reported that the level of miR-129-5p expression in all osteoporosis models was changed and that the overexpression of miR-129-5p repressed OB differentiation in the MC3T3-E1 cell line and bone formation in an animal model. It was also found that miR-129-5p could regulate transcription factors in the Wnt/β-catenin pathway by binding Tcf4. Moreover, a miR-129-5p inhibitor could rescue the effect on osteoporosis, providing a novel target candidate for the condition.

In addition to mediating the inhibition of OB differentiation, miRNAs also play key regulatory roles in promoting OB differentiation. miR-218 has been reported to function as a specific suppressor in various types of cancer, such as nasopharyngeal and colorectal cancer (26,27). Recently, the role of miR-218 in postmenopausal osteoporosis was reported. miR-218-5p was found to be downregulated in BMSCs during OB differentiation, and miR-218-5p could regulate its target COL1A1 to promote OB differentiation, indicating that this miRNA may be a target for the treatment of postmenopausal osteoporosis (28). Similarly, Zhang et al (29) showed that miR-664a-5p expression was upregulated during the osteogenic differentiation of human BMSCs and that this miRNA could bind to the mRNA of HMGA2, a direct target of miR-664a-5p, to promote the osteogenic differentiation of BMSCs, which may provide new effective methods for osteoporosis therapy. Qi et al (30) reported that miR-199a-5p, which is upregulated during the induction of OB differentiation in hBMSCs, increased ALP activity and the calcification of hBMSCs, and promoted the OB differentiation of hBMSCs by targeting TET2 to downregulate its expression.

Zhang et al (31) identified that the expression of miR-199b-5p was increased during the transforming growth factor-β (TGF-β)-induced chondrogenic differentiation of MSCs, the latter of which was regulated by miR-199b-5p by targeting JAG1. In addition, miR-8485 derived from chondrocytes were able to activate the Wnt/β-catenin pathways to promote the chondrogenic differentiation of BMSCs (32). Shen et al (33) observed that the expression of miRNA-23c was significantly decreased during the process of chondrocyte differentiation of MSCs. The study found that miRNA-23c can regulate FGF2 expression to inhibit chondrogenic differentiation. SMADs are crucial mediators of canonical TGF-β. Similarly, SMADs are involved in the process of chondrogenesis. Recent findings have shown that miR-134 expression was downregulated during chondrogenesis and that miR-134, by interacting with SMAD6, acted as a negative regulator during the chondrogenic differentiation of BMSCs (34).

It has been reported that miR-149-3p inhibits the expression of fat mass- and obesity-associated genes by targeting the 3′-UTR of FTO mRNA. Additionally, miR-149-3p anti-miRNA oligonucleotides can promote the adipogenic differentiation of BMSCs and repress the osteoblastic differentiation of BMSCs, indicating that miR-149-3p, a prospective candidate target for the treatment of osteoporosis, can regulate the adipogenic differentiation of BMSCs through the miR-149-3p/FTO regulatory pathway (35). Several studies have shown that PPARγ, a key regulatory factor in adipocyte formation, is highly expressed in the early stage of adipogenesis (36,37). Lin et al (38) demonstrated that the expression level of miR-130a was decreased during ageing and that the decreased expression of miR-130 caused the upregulation of PPARγ, a direct target of miR-130, leading to the adipogenic differentiation of BMSCs. Jamali et al first reported the role of miR-100-3p, a regulatory factor of cellular apoptosis and proliferation in gastric cancer (39), in the regulation of the adipogenic differentiation of hMSCs and found that miR-100-3p plays an important role in regulating the adipogenic differentiation in hMSCs through the PI3K/AKT pathway by targeting PIK3R1 (40). Previous findings have shown that miRNAs also play a role in promoting the adipogenic differentiation of BMSCs. Zhu et al (41) reported that miR-20a-5p was upregulated during the adipogenic differentiation of BMSCs and could bind Klf3, an inhibitory factor of adipogenic differentiation, to promote adipocyte differentiation.

Emerging evidence has shown that miRNAs play a crucial role in BMSC proliferation. Cui et al (42) studied the regulatory role of miR-146a in BMSC proliferation for the first time. It was found that the knockdown of miR-146a could promote BMSC proliferation and that miR-146a could suppress the expression of SNHG7 and EPB41L4A-AS1, leading to the inhibition of BMSC proliferation. Kong et al (43) reported that miR-126 plays a key role in promoting the proliferation and migration of BMSCs through the PI3K/AKT and MEK1/ERK1 signalling pathways. The level of miP-144 in clinical serum samples obtained from patients with postmenopausal osteoporosis was found to be higher than that in normal populations, and further studies showed that miR-144 could promote proliferation and inhibit apoptosis in BMSCs to regulate osteoporosis via the Wnt/β-catenin pathway (44).

Accumulating studies indicate the crucial regulatory roles of exosomal miRNAs in various bone diseases. Yang et al (45) showed that miR-1263 derived from exosomes of HUCMSCs played a role in inhibiting apoptosis in BMSCs, and further experiments verified that miR-1263 directly targeted the 3′-UTR of Mob1 to suppress the expression of Mob1 to inhibit BMSC apoptosis in disuse osteoporosis by activating Yes-associated protein (YAP). Previous studies reported that miR-181c could regulate the viability of cancer cells under reactive oxygen species (ROS) stress by sustaining the function of mitochondria (46,47). Fan et al (48) revealed that miR-181c could reverse the effects of oxidative stress on BMSCs and attenuate oxidative stress-mediated BMSC apoptosis mainly through the AMPK-Mfn1 pathway. Increasing studies indicate that BMSCs are relatively radiosensitive. Ionizing radiation could induce BMSC apoptosis via ROS generation and accumulation in mitochondria. Liu et al (49) found that miR-22 was significantly upregulated in BMSCs after ionizing radiation exposure and that the overexpression of miR-22 in BMSCs accelerated the ionizing radiation-induced accumulation of mitochondrial ROS, thus resulting in cellular apoptosis. Furthermore, the study verified that the apoptosis of BMSCs induced by mitochondrial ROS generation is mainly dependent on the miR-22/Redd1 pathway (Table I).

Table I.

Roles of miRNAs in the regulation of bone marrow stromal cells.

First author, year	miRNA	Target gene	Role	(Refs.)
Xu et al, 2018	miR-31a-5p	SATB2, RhoA	Promote osteoclastic differentiation	(17)
Moura et al, 2020	miR-99a-5p	NA	Promote osteoclastic differentiation and inhibit osteoblastic differentiation	(18)
Zhou et al, 2020	miR-1286	FZD4	Inhibit osteoblastic differentiation	(19)
Wu et al, 2021	miR-199a-3p	Kdm3a	Inhibit osteoblastic differentiation	(23)
Jiang et al, 2020	miR-23	MEF2C/MAPK signalling pathway	Inhibit osteoblastic differentiation	(24)
Yin et al, 2020	miR-129-5p	Tcf4, Wnt/β-catenin pathway	Inhibit osteoblastic differentiation	(25)
Kou et al, 2020	miR-218-5p	COL1A1	Promote osteoblastic differentiation	(28)
Zhang et al, 2020	miR-664a-5p	HMGA2	Promote osteoblastic differentiation	(29)
Qi et al, 2020	miR-199a-5p	TET2	Promote osteoblastic differentiation	(30)
Zhang et al, 2020	miR-199b-5p	JAG1	Promote chondrogenic differentiation	(31)
Li et al, 2020	miR-8485	Wnt/β-catenin pathways	Promote chondrogenic differentiation	(32)
Shen et al, 2019	miRNA-23c	FGF2	Inhibit chondrogenic differentiation	(33)
Xu and Wu 2019	miR-134	SMAD6	Inhibit chondrogenic differentiation	(34)
Li et al, 2019	miR-149-3p	FTO	Inhibit adipogenic differentiation	(35)
Lin et al, 2019	miR-130	PPARγ	Inhibit adipogenic differentiation	(38)
Wang et al, 2020	miR-100-3p	PI3K/AKT pathway	Inhibit adipogenic differentiation	(40)
Zhu et al, 2018	miR-20a-5p	Klf3	Promote adipogenic differentiation	(41)
Cui et al, 2020	miR-146a	EPB41L4A-AS1/SNHG7	Inhibit proliferation	(42)
Kong et al, 2020	miR-126	PI3K/AKT and MEK1/ERK1 pathway	Promote proliferation and migration	(43)
Tang et al, 2019	miR-144	Sfrp1/Wnt/β-catenin	Promote proliferation and inhibit apoptosis	(44)
Yang et al, 2020	miR-1263	Mob1/Hippo signalling pathway	Inhibit apoptosis	(45)
Fan et al, 2020	miR-181c	AMPK-Mfn1	Inhibit apoptosis	(48)
Liu et al, 2019	miR-22	Redd1	Promote apoptosis	(49)

miRNA/miR, microRNA; NA, not applicable.

Effects of lncRNAs on regulation of BMSCs

Numerous studies have concluded that lncRNAs play various vital roles in cell biology (50–52). Recent findings have indicated that lncRNAs are involved in BMSC differentiation and proliferation in osteoporosis. The switch between the adipogenic and osteogenic differentiation of BMSCs plays an important role in ageing-induced osteoporosis. miR-19a-3p is able to regulate OB differentiation in BMSCs by regulating Hoxa5 expression, and lncRNA Xist, as a sponge of miR-19a-3p, plays a crucial role in OB differentiation by binding miR-19a-3p in BMSCs (12). Recently, Zhang et al (53) found that the expression of the lncRNA LOXL1-AS1 was high in the peripheral blood from patients with osteoporosis and that this gradually decreased during the process of osteogenic differentiation of hBMSCs. Furthermore, it was revealed that LOXL1-AS1 inhibited osteogenic differentiation but promoted adipocytic differentiation in hBMSCs, mainly by sponging miR-196a-5p to regulate Hmga2 expression (53). A report showed that the expression of the lncRNA HCG18 was increased in BMSCs from osteoporosis patients and that HCG18 expression was significantly decreased during the process of the differentiation of OB BMSCs, indicating that HCG18 plays an important role in BMSC differentiation (54). Subsequently, the study reported that HCG18, as a regulator of osteogenic differentiation, repressed the osteogenic differentiation of BMSCs induced by osteoporosis through the miR-30a-5p/NOTCH1 pathway (54).

Li et al (55) previously found that the lncRNA GAS5 plays a crucial role in negatively regulating lipoblast/adipocyte differentiation in humans and identified that GAS5 was downregulated in bones and BMSCs from osteoporosis patients. Specifically, the study concluded that GAS5 promotes OB differentiation in BMSCs by regulating the UPF1/SMAD7 pathway and protects against osteoporosis (55). Similarly, Zheng et al (56) reported that lncSNHG5 promoted osteogenic differentiation in hBMSCs by competitively targeting miR-582-5p to regulate RUNX3 expression, indicating that SNHG5 may be a novel target candidate for osteoporosis therapy. MIR22HG expression was found to be significantly downregulated in BMSCs from osteoporotic mice and was increased during the process of human BMSC osteogenic differentiation. Mechanistically, MIR22HG promotes the osteogenic differentiation of BMSCs by downregulating phosphatase and tensin homologue and activating the AKT pathway, indicating that MIR22HG plays a crucial role in bone metabolism and may be a novel target for osteoporosis (57).

lncRNAs have emerged as crucial regulators of cell differentiation. However, the potential regulatory function of lncRNAs in BMSC chondrogenic differentiation remains poorly studied. The lncRNA ADAMTS9-AS2 was found to be upregulated during the chondrogenesis of hMSCs using microarray analysis, and ADAMTS9-AS2 acted as a ceRNA for miR-942-5p, thereby playing a crucial role in regulating chondrogenic differentiation, and promoted chondrogenic differentiation of hMSCs (58). Recent findings showed that ADAMTS9-AS2 can inhibit oesophageal cancer development by inducing CDH3 promoter methylation (59). Shu et al (60) reported that the lncRNA UCA1 was upregulated during the chondrogenic differentiation of BMSCs. Subsequently, it was found that the chondrogenic differentiation of BMSCs was promoted by UCA1 mainly through the miR-145-5p/SMAD5 and miR-124-3p/SMAD4 pathways. In another study, UCA1 was found to be significantly upregulated in patients with osteoporosis and was indicated to promote the proliferation and differentiation of OBs by regulating the BMP-2 pathway in OBs (61).

Pan et al (62) reported that the expression of lncRNA ROA, which promotes hnRNP A1 to target the PTX3 promoter, thereby activating the ERK1/2 signalling pathway and regulating BMSC adipogenic differentiation, was significantly decreased during the process of BMSC adipogenesis. The lncRNA Plnc1, which is derived from the PPAR-γ2 gene, was upregulated during the adipogenic differentiation of ST2 cells and BMSCs, and further investigation showed that Plnc1 decreased methylation of the CpG region in the PPAR-γ2 promoter, thereby promoting PPAR-γ2 transcription, indicating that Plnc1 plays an important role in promoting adipogenic differentiation (63). In addition, Zhang et al (53) showed that LOXL1-AS1 could also promote adipocytic differentiation.

The lncRNA NORAD iss decreased in steroid-induced osteonecrosis in femoral head tissues, and findings of a mechanistic study indicated that NORAD could target miR-26a-5p to promote the DEX-induced inhibition of proliferation in hBMSCs (64). However, numerous studies have concluded that NORAD also plays a crucial role in cellular processes involved in carcinogenesis, including cell proliferation, invasion and metastasis (65), indicating that the function of NORAD is complex and that further studies are needed. In another study, the inhibition of the lncRNA LINC01535 decreased the proliferation of hBMSCs, and the regulatory effect of LINC01535 on BMSCs was found to be mediated mainly by targeting miR-3619-5p (66). Gan et al (67) reported that the expression of the lncRNA H19 was significantly decreased in postmenopausal patients with osteoporosis and that the overexpression of H19 obviously inhibited BMSC proliferation by targeting miR-19b-3p, demonstrating the crucial role of H19/miR-19b-3p in osteoporosis for the first time and providing a novel target for osteoporosis. Recent evidence has indicated that autophagy has a critical effect on the pathogenesis of acute pancreatitis and that the overexpression of H19 in MSCs significantly inhibits autophagy via the FAK/PDK1/AKT/mTOR axis in rats with acute pancreatitis (68).

Li et al (69) identified that downregulated expression of the lncRNA LNC_000052 inhibited BMSC apoptosis through the PI3K/Akt pathway, and further investigations showed that LNC_000052 regulates BMSC apoptosis mainly by binding PIK3R1, which is also a target of miR-96-5p. In addition to the regulation of apoptosis, it was found that LNC_000052 plays a crucial role in BMSC proliferation and migration via the miR-96-5p-PIK3R1 axis (69). Knockdown of the lncRNA SNHG5 promoted apoptosis in hBMSCs, and further results demonstrated that SNHG5 inhibited apoptosis through the miR-582-5p/RUNX3 pathway (56). Similarly, SNHG5 has been reported to play an important role in human chronic myelogenous leukaemia by inhibiting cell apoptosis (70). Another study also reported that LINC01535 could inhibit hBMSC apoptosis by regulating miR-3619-5p (66) (Table II).

Table II.

Roles of lncRNAs in regulation of bone marrow stromal cells.

First author, year	lncRNA	Target gene	Role	(Refs.)
Chen et al, 2020	Xist	miR-19a-3p	Inhibit osteoblastic differentiation	(12)
Zhang et al, 2020	LOXL1-AS1	miR-196a-5p	Inhibit osteoblastic differentiation promote adipogenic differentiation	(53)
Che et al, 2020	HCG18	miR-30a-5p/NOTCH1	Inhibit osteoblastic differentiation	(54)
Li et al, 2020	GAS5	UPF1/SMAD7	Promote osteoblastic differentiation	(55)
Zheng et al, 2020	SNHG5	miR-582-5p/RUNX3	Promote osteoblastic differentiation, inhibit apoptosis	(56)
Jin et al, 2020	MIR22HG	PTEN/AKT	Promote osteoblastic differentiation	(57)
Huang et al, 2019	ADAMTS9-AS2	miR-942-5p	Promote chondrogenic differentiation	(58)
Shu et al, 2019	UCA1	miR-145-5p/miR-124-3p	Promote chondrogenic differentiation	(60)
Pan et al, 2020	ROA	hnRNP A1-PTX3-ERK	Inhibit adipogenic differentiation	(62)
Zhu et al, 2019	Plnc1	PPAR-γ2	Promote adipogenic differentiation	(63)
Fu et al, 2021	NORAD	miR-26a-5p	Promote proliferation	(64)
Zhao et al, 2020	LINC01535	miR-3619-5p	Promote proliferation, inhibit apoptosis	(66)
Gan et al, 2020	H19	miR-19b-3p	Inhibit proliferation	(67)
Li et al, 2020	LNC_000052	PIK3R1	Promote apoptosis, inhibit proliferation	(69)

lncRNA, long non-coding RNA; miR, microRNA.

Effects of circRNAs on BMSC regulation

circRNAs are transcribed from exons that have a cell- or tissue-specific expression profile, and circRNA expression in tissues is highly stable due to their resistance to RNase degradation. Emerging studies have revealed that circRNAs play a crucial role in the regulation of cellular functions by sponging miRNAs or interacting with RNA-binding proteins (71,72). However, few studies have demonstrated the role of circRNAs in regulating BMSCs in osteoporosis. Wang et al (73) found that circ_0006393 expression was decreased in patients with glucocorticoid-induced osteoporosis, and further experiments revealed that the increased expression of circ_0006393 promoted the expression of osteogenesis-associated genes by targeting miR-145-5p. An abnormally low expression level of circ_0076906 was reported for the first time in osteoporosis; however, the roles of this circRNA in regulating osteoporosis are poorly understood (74). Wen et al (75) found that circ_0076906 promoted the osteogenic differentiation of hMSCs and relieved osteoporosis by binding miR-1305 to regulate osteoglycin expression. Shen et al (76) showed that circFOXP1 was significantly decreased in bone tissues from patients with osteoporosis, and in vitro and in vivo analyses indicated that circFOXP1 can sponge miR-33a-5p, promote the osteogenic differentiation of human adipose-derived mesenchymal stem cells and prevent osteoporosis by regulating FOXP1, revealing that circFOXP1 can be used as a novel candidate therapeutic target for osteoporosis. In another study, circFOXP1 was highly expressed in MSCs compared with differentiated mesodermal derivatives, and it was indicated to play a critical role in sustaining the identity of MSCs by regulating the Wnt and EGFR pathways (77). In addition, circFOXP1 was involved in the regulation of cancer cell proliferation (78). Liu et al (79) screened the differential expression of circRNAs in postmenopausal patients with osteoporosis using RNA-seq and found that cir0007059 expression was increased in patients and during the OC differentiation of hBMSCs. Furthermore, circRNAs play a crucial role in OC differentiation by regulating the miR-378/BMP-2 pathway (79). In addition, circ_0007059 plays a critical role in lung cancer cell proliferation (80). Huang et al (81) found that YAP1 can promote BMSC and MC3T3-E1 osteogenic differentiation, and that circ_0024097, which is derived from YAP1, can target miR-376b-3p to regulate YAP1 expression, leading to increased osteogenic differentiation.

Zhang et al (82) documented for the first time that circ-DAB1 is upregulated during the osteogenic differentiation of BMSCs. Chia et al (83) later verified via RT-qPCR that circ-DAB1 expression is significantly increased during the osteogenic differentiation of BMSCs. A mechanistic study revealed that circ-DAB1 increases the proliferation and osteogenic differentiation of BMSCs through the NOTCH/RBPJ pathway (83). Chen et al (84) screened differentially expressed circRNAs in BMSCs from patients with steroid-induced osteonecrosis in the femoral head and found that the circRNA CDR1as was upregulated. It was revealed that circRNA CDR1as plays an important role in regulating the adipogenic and osteogenic differentiation of BMSCs (84). However, whether the circRNA CDR1as also participates in the regulation of BMSCs from patients with osteoporosis needs to be determined.

Recently (85), circRNA_25487 was found to be upregulated in the peripheral blood of patients with trauma-induced osteonecrosis of the femoral head (TIONFH), and the functions of circRNA_25487 in bone repair in TIONFH were studied using BMSCs. Zhang et al (85) revealed that circRNA_25487 promotes apoptosis and inhibits proliferation in BMSCs by binding miR-134-3p to promote p21 expression, which promotes bone repair in TIONFH. However, the role of circRNA_25487 in osteoporosis remains to be elucidated via further research.

Autophagy has pivotal functions in sustaining cell homeostasis by removing damaged macromolecules and organelles during oxidative stimulation or starvation (86). Autophagy is crucial for recycling cell components and promoting BMSC osteogenesis by eliminating ROS, which help maintain bone homeostasis (49). Previous findings have shown the relationship between circRNAs and autophagy in a number of conditions, such as sciatic nerve injury and thyroid cancer (87). However, the correlations between circRNAs and autophagy in osteoporosis remain unclear (Table III).

Table III.

Roles of circRNAs in the regulation of bone marrow stromal cells.

First author, year	circRNA	Target gene	Role	(Refs.)
Wang et al, 2019	circ_0006393	miR-145-5p	Promote osteoblastic differentiation	(73)
Wen et al, 2020	circ_0076906	miR-1305	Promote osteoblastic differentiation	(75)
Shen et al, 2020	circFOXP1	miR-33a-5p	Promote osteoblastic differentiation	(76)
Huang et al, 2020	circ-0024097	miR-376b-3p/YAP1	Promote osteoblastic differentiation	(81)
Chia et al, 2020	circ-DAB1	NOTCH/RBPJ	Promote proliferation and osteoblastic differentiation	(83)
Chen et al, 2020	circ-CDR1	miR-7-5p/WNT5B	Promote adipogenic and inhibit osteoblastic differentiation	(84)
Zhang et al, 2021	circ_25487	miR-134-3p/p21	Promote apoptosis and inhibit proliferation	(85)

circRNA, circular RNA; miR, microRNA.

Mechanisms by which ncRNAs regulate OBs in osteoporosis

Osteoblasts, which are derived from multipotent mesenchymal stem cells, have an important function in maintaining bone microstructure and homeostasis, and the dysregulation of OB number or activity is related to the pathophysiology of bone disorders, such as osteoporosis (88). Numerous studies have revealed that miRNAs, lncRNAs and circRNAs are crucial factors involved in OB proliferation, differentiation, autophagy and apoptosis in osteogenesis.

Effects of miRNAs on OB regulation. miRNAs have been documented to play pivotal roles in the regulation of OB biology. Numerous studies have concluded that miRNAs are involved in the regulation of the osteogenic differentiation of mesenchymal precursor cells. One previous study found that miR-197-3p expression was significantly increased in a number of cancer types, including lung cancer, indicating that miR-197-3p plays an important role in the development of tumours by promoting cell proliferation (89). You et al (90) demonstrated that miR-197-3p expression was upregulated in a rat model of osteoporosis, and further research revealed that miR-197-3p inhibits OB differentiation by regulating KLF10 in osteoporosis. Emerging evidence has elucidated that miR-122 is a diagnostic and prognostic biomarker for osteoporosis. Seeliger et al (91) reported that miR-122 expression was significantly increased in serum samples from patients with osteoporosis. A recent study reported that miR-122 was upregulated in OBs originating from ovariectomized rats, and further investigation revealed that miR-122 could inhibit OB proliferation and differentiation by activating the JNK pathway and suppressing PCP4 expression (13). Recent studies have shown that miR-205-5p is involved in regulating the proliferation of various cells, such as retinal pigment epithelial cells, thymic epithelial cells and pancreatic cancer cells (92–94). Recently, Huang et al (95) revealed that miR-205-5p expression was increased in samples from patients with osteoporosis and then was gradually downregulated during osteogenic differentiation. Moreover, it was concluded that miR-205-5p could inhibit osteogenic differentiation by regulating RUNX2 expression. Previous findings showed that miR-22-3p expression was high in extracellular vesicles originating from BMSCs, and that the abundance of miR-22-3p was also high in extracellular vesicles derived from plasma (96,97). A current study revealed that miR-22-3p, which is delivered by BMSC-derived extracellular vesicles, promotes osteogenic differentiation in BMSCs via the MYC/PI3K/AKT signalling pathway by inhibiting FTO (98).

Previously, it was revealed that miR-150-3p is a regulatory factor of inflammatory signalling pathways and that the osteogenic differentiation of hBMSCs may play an important role in modulating inflammation during bone formation (99). The latest research highlighted that miR-150-3p can promote OB proliferation in osteoporosis and identified it as a novel clue for osteoporosis therapy (100). Ma et al (101) documented that the expression of miR-497-5p, which is considered a clinically significant biomarker for osteoporosis, was downregulated in bone tissues from ageing mice and played an important role in the metabolism of bone. In addition, it was reported that miR-497 was decreased in breast cancer (102). Recently, Gu et al (103) declared that miR-497 expression is decreased in osteoporosis and that increased miR-497 promotes OB proliferation by regulating the TGF-β1/Smad signalling pathway, alleviating the progression of osteoporosis. miR-214 was previously reported to play a role in osteoporosis by regulating the expression of osterix in bones (104). Another study indicated that miR-214 could protect MC3T3-E1 OBs against apoptosis by repressing oxidative stress (105). Yang et al (106) demonstrated that the expression of miR-214 in femoral tissues from osteoporosis rats was low and that the overexpression of miR-214 could promote OB proliferation by repressing TXNIP, which could provide a novel therapeutic target for osteoporosis. miR-15b is known to participate in the occurrence of osteoporosis and plays a role in OB differentiation in bone diseases (20,107). Recent findings indicated that the expression of miR-15b was upregulated in mice with osteoporosis and that miR-15b can suppress OB proliferation, which could aggravate osteoporosis by targeting USP7 and regulating KDM6B expression, providing a new target for osteoporosis treatment (108).

The dysregulation of miR-142 has been identified in numerous diseases. miR-142 is related to malignancy in breast cancer stem cells via the WNT pathway (109). In addition, miR-142 plays a role in regulating the growth and apoptosis of airway smooth muscle cells. Importantly, miR-142 promotes the osteoclastogenesis of bone marrow-originated macrophages by regulating PTEN and the PI3K/Akt/FoxO1 axis, indicating the regulatory function of miR-142 in osteoporosis (110). Recently, Luo et al (111) revealed that miR-142 could promote apoptosis and inhibit proliferation in MC3T3-E1 cells by targeting the BMP/Smad signalling pathway. miR-491-3p expression was found to be downregulated in postmenopausal osteoporosis, and the overexpression of miR-491-3p enhanced the viability and suppressed the apoptosis of hFOB1.19 cells by regulating cathepsin S (CTSS) (112). Similarly, Zhang et al (113) revealed that miR-708 plays a role in protecting MC3T3-E1 cells by inhibiting apoptosis induced by H2O2 by targeting PTEN expression.

Autophagy is a stress-responsive catabolic process that plays a critical role in maintaining cellular and tissue homeostasis. In addition, autophagy can facilitate osteogenic differentiation to preserve bone homeostasis. However, dysregulation of autophagy in bone cells can cause a series of bone diseases, such as osteoporosis, and the activation of autophagy in OCs is related to bone loss. Recently, Lu et al (108) disclosed that the overexpression of miR-15b could depress USP7 expression, which could inhibit the autophagy, proliferation and differentiation of OBs, and lead to osteoporosis by suppressing KDM6B expression (Table IV).

Table IV.

Roles of miRNAs in the regulation of osteoblasts.

First author, year	miRNA	Target gene	Role	(Refs.)
You et al, 2021	miR-197-3p	KLF10	Inhibit differentiation	(90)
Meng et al, 2020	miR-122	PCP4/JNK	Inhibit differentiation and proliferation	(13)
Huang et al, 2020	miR-205-5p	RUNX2	Inhibit differentiation	(95)
Zhang et al, 2020	miR-22-3p	FTO/MYC/PI3K/AKT	Promote differentiation	(98)
Qiu et al, 2021	miR-150-3p	NA	Promote proliferation and differentiation	(100)
Gu et al, 2020	miR-497	TGF-β1/Smads	Promote proliferation	(103)
Yang et al, 2021	miR-214	TXNIP	Promote proliferation	(106)
Lu et al, 2021	miR-15b	USP7/KDM6B	Inhibit autophagy, proliferation, and differentiation	(108)
Luo et al, 2020	miR-142	BMP/Smad	Promote apoptosis	(111)
Hu et al, 2020	miR-491-3p	CTSS	Inhibit apoptosis	(112)
Zhang et al, 2020	miR-708	PTEN	Inhibit apoptosis	(113)

miRNA/miR, microRNA; NA, not applicable.

Effects of lncRNAs on regulation of OBs. The inhibition of OB differentiation has been confirmed to be a crucial regulator of osteoporosis, and emerging evidence has indicated that lncRNAs may be treated as targets for osteoporosis treatment. Yin et al (114) recently revealed that the lncRNAs AK039312 and AK079370 are involved in inhibiting OB differentiation and bone formation by targeting miR-199b-5p, and that upregulated GSK-3β further suppresses the Wnt/β-catenin signalling pathway. Moreover, it was revealed that small interfering RNAs targeting AK039312 and AK079370 could relieve postmenopausal osteoporosis in mice, providing a novel direction for the treatment of osteoporosis. The lncRNA DANCR was previously found to be deregulated in human circulating monocytes and to play a key role in osteoporosis (115). Wang et al (116) reported the dysregulation of DANCR in patients with osteoporosis and an ovariectomy model, and DANCR expression in BMSCs originating from osteoporosis patients was increased. In addition, DANCR can suppress osteogenic differentiation in osteoporosis by suppressing the Wnt/β-catenin signalling pathway by regulating CTNNB1 expression (116).

The lncRNA MEG3 has previously been reported to inhibit the osteogenic differentiation of BMSCs in postmenopausal osteoporosis (117). Yang et al (106) suggested that the downregulation of MEG3 can promote the differentiation and proliferation of OBs in osteoporosis by targeting miR-214 and depressing TXNIP expression. The dysregulation of lncRNA CCAT1 is involved in a number of human diseases. Recently, Hu et al (118) revealed that the suppression of CCAT1 improved pathology and inhibited osteocyte apoptosis in bone tissues from ovariectomized rats with osteoporosis, promoted differentiation and proliferation, and depressed apoptosis in OBs derived from ovariectomized rats by promoting miR-34a-5p expression by downregulating SMURF2. Mulati et al (119) suggested that the lncRNA CRNDE, which was previously reported as a cancer-related RNA, plays a crucial role in OB proliferation and differentiation. Additionally, mechanistically, the upregulation of CRNDE can promote OB proliferation and regulate bone formation via the Wnt/β-catenin pathway (119).

Recent results have shown that iron accumulation (IA), which is a pathological risk factor among postmenopausal women, is related to postmenopausal osteoporosis and that iron accumulation leads to BMSC apoptosis by activating the caspase 3 pathway. The latest study reported that the expression of the lncRNA XIST was increased in IA mouse and cell models, indicating that XIST may play an important role in osteoporosis (120). Furthermore, knockdown of XIST can suppress OB apoptosis induced by IA via the regulation of caspase 3. Similarly, Niu et al (121) revealed that XIST, which was found to be upregulated in plasma, also promoted OB apoptosis through the miR-203-3p/ZFPM2 pathway. In addition, XIST plays a role in regulating OB differentiation by modulating the expression of miR-19a-3p, indicating that XIST performs various important regulatory functions in bone-related diseases (12). The effect of lncRNA-mediated autophagy on OBs in osteoporosis has rarely been reported and needs further exploration (Table V).

Table V.

Roles of lncRNAs in the regulation of osteoblasts.

First author, year	lncRNA	Target gene	Role	(Refs.)
Yin et al, 2021	AK039312/AK079370	miR-199b-5p	Inhibit differentiation	(114)
Wang et al, 2020	DANCR	CTNNB1	Inhibit differentiation	(116)
Yang et al, 2021	MEG3	miR-214	Inhibit proliferation and differentiation	(117)
Hu et al, 2021	CCAT1	miR-34a-5p	Inhibit proliferation and differentiation and promote apoptosis	(118)
Mulati et al, 2020	CRNDE	Wnt/β-catenin	Promote proliferation	(119)
Liu et al, 2021	XIST	miR-758-3p/miR-203-3p	Promote apoptosis	(120)
Niu et al, 2020	XIST	miR-758-3p/miR-203-3p	Promote apoptosis	(121)

lncRNA, long non-coding RNA; miR, microRNA.

Effects of circRNAs on OB regulation. circRNAs are known to play crucial roles in osteoporosis by binding miRNAs to modulate the expression of target genes at the transcriptional or post-transcriptional level. However, few studies and reports related to circRNAs regulating OB differentiation in osteoporosis, especially OB proliferation, apoptosis and autophagy, are available. Further systematic studies are needed to clarify the role of circRNAs in regulating OBs during osteoporosis. Mi et al (122) suggested that the expression of the circRNA AFF4, which is located in the cytoplasm, was upregulated during the few days after fracture in vivo, and mechanistic studies revealed that circRNA AFF4 promotes OB proliferation and suppresses apoptosis by regulating the miR-7223-5p/PIK3R1 signalling pathway. However, whether circRNA AFF4A plays a role in osteoporosis remains to be determined. A recent study reported that the expression of circ8500 was obviously increased during mineralization processes, and further experiments showed that circ8500 can facilitate OB matrix mineralization by inhibiting miR-1301-3p to promote PADI4 expression (123). Recently, Ji et al (124) revealed that circ_0026827 promotes OB differentiation via the Beclin-1-mediated autophagy pathway. Mechanistically, circ_0026827 regulates the autophagy signalling pathway by targeting miR-188-3p, suggesting novel therapeutics for osteoporosis (Table VI).

Table VI.

Roles of circRNAs in the regulation of osteoblasts.

First author, year	circRNA	Target gene	Role	(Refs.)
Mi et al, 2019	circ AFF4	miR-7223-5p	Promote proliferation and inhibit apoptosis	(122)
Zhai et al, 2020	circ-0008500	miR-1301-3p	Promote matrix mineralization	(123)
Ji et al, 2020	circ_0026827	miR-188-3p	Promote differentiation	(124)

circRNA, circular RNA; miR, microRNA.

Mechanisms by which ncRNAs regulate OCs in osteoporosis

Osteoclasts, which are derived from the mononuclear haematopoietic lineage, are multinucleated giant cells after fusion that are regulated mainly by various cytokines, which play a crucial role in the formation of functional OCs (88). The process of osteoclastic bone resorption is related to the dysregulation of miRNA, lncRNA and circRNA expression, which, in turn, regulates the differentiation, proliferation, apoptosis and autophagy of OCs by modulating target genes.

Previously, miR-128 was reported to be involved in ageing, inflammatory signalling and inflammatory diseases. Additionally, miR-128 was involved in osteogenic/adipogenic differentiation (125). Recently, miR-128 was found to be upregulated in bone tissues from patients with postmenopausal osteoporosis, and the expression level of miR-128 was positively correlated with the expression level of nuclear factor of activated T cells 1 (125). Mechanistically, miR-128 knockdown can inhibit osteoclastogenesis by targeting sirtuin 1 and regulating the activity of NF-κB, which, in turn, markedly depresses ovariectomy-induced osteoclastogenesis and alleviates bone loss in mice (125). Similarly, miR-301-b expression was upregulated in bone tissues derived from postmenopausal patients with osteoporosis. Results of mechanistic studies showed that miR-301-b could promote osteoclastogenesis by post-transcriptionally regulating the expression of cylindromatosis, a target of miR-301-b (126). Huang et al (127) suggested that miR-25-3p plays an important role in inhibiting OC proliferation by suppressing the expression of nuclear factor I X, which is involved in the regulation of OC proliferation and differentiation.

Zhang et al (128) showed that the expression of the lncRNA Neat1 was increased during osteoclastic differentiation and that Neat1 knockdown suppressed OC formation, whereas Neat1 overexpression promoted it. Further evidence revealed that upregulated Neat1 can facilitate osteoclastogenesis in mice, providing a novel therapeutic target for osteoporosis (128). Mechanistic studies revealed that Neat1 targets miR-7 and regulates the expression of protein tyrosine kinase 2 (128). Chang et al (129) analysed lncRNA expression levels using a microarray during the process of OC differentiation and fusion, and found that the overexpression of lncRNA-NONMMUT037835.2 suppressed osteoclastic differentiation, whereas the inhibition of lncRNA-NONMMUT037835.2 facilitated OC formation and fusion. it was also revealed that lncRNA-NONMMUT037835.2 modulated osteoclastogenesis by targeting RANK and repressing the NF-κB/MAPK pathway (129). Previous findings suggested that miR-21 plays crucial roles in osteoporosis by targeting reversion-inducing cysteine-rich protein with Kazal motifs to depress the process of osteoporosis (130). A recent study indicated that miR-21 expression was decreased in plasma from patients with osteoporosis, and further evidence showed that the overexpression of miR-21 could inhibit OC apoptosis (131). In addition, the expression of GAS5 was increased in plasma from patients with osteoporosis, and GAS5 could decrease miR-21 expression to promote OC apoptosis, which, in turn, plays a protective role in osteoporosis (131) (Table VII).

Table VII.

Roles of ncRNAs in the regulation of osteoclasts.

First author, year	ncRNA	Target gene	Role	(Refs.)
Shen et al, 2020	miR-128	SIRT1	Promote differentiation	(125)
Zhu et al, 2020	miR-301-b	CYLD	Promote differentiation	(126)
Huang et al, 2020	miR-25-3p	NFIX	Inhibit proliferation	(127)
Zhang et al, 2020	lncRNA Neat1	miR-7	Promote differentiation	(128)
Chang et al, 2020	lncRNA-NONM MUT037835.2	RANK	Inhibit differentiation	(129)
Cong et al, 2020	lncRNA GAS5	miR-21	Promote apoptosis	(131)

ncRNA, non-coding RNA; lncRNA, long ncRNA; miR, microRNA.

Conclusion and perspectives

In this review, the latest evidence concerning the regulatory roles of miRNAs, lncRNAs and circRNAs involved in the modulation of BMSCs, OBs and OCs in osteoporosis was summarized. Recently, emerging studies have shown that ncRNAs are related to bone homeostasis and play essential roles in the occurrence and development of osteoporosis. In addition, some ncRNAs have therapeutic potential for osteoporosis treatment.

Although various anabolic drugs are applied in osteoporosis treatment, these agents have side effects and unwanted limitations that interrupt the quality of life of patients. Thus, it is imperative to identify the therapeutic potential of ncRNAs in osteoporosis. To the best of our knowledge, research related to the regulatory function of miRNAs in the differentiation, proliferation, apoptosis and autophagy of BMSCs, OBs and OCs has markedly increased. However, only a few studies were conducted in vivo, and functional investigations of miRNAs in bone homeostasis in vivo are needed. Both lncRNAs and circRNAs can influence the process of osteoporosis by directly binding miRNAs as sponges. Compared with miRNAs, which have been studied extensively, the lncRNAs and circRNAs involved in osteoporosis are relatively new. In particular, the role of circRNAs in maintaining bone homeostasis requires further investigation. For instance, data related to the regulation of circRNAs in OBs and OCs in osteoporosis are limited. The potential use of circRNAs as treatment options for osteoporosis is undoubtedly promising. Differentiation, proliferation, apoptosis and autophagy are important physiological processes in cells that play crucial roles in the regulation of BMSCs, OBs and OCs in the bone microenvironment. However, few studies have investigated apoptosis and autophagy in the aforementioned cells, and the importance of ncRNAs in the regulation of apoptosis and autophagy of BMSCs, OBs, and OCs needs to be further elucidated. In addition, some ncRNAs simultaneously play a specific regulatory role in various diseases and can simultaneously participate in the occurrence and development of tumours and the progression of osteoporosis. Therefore, it is necessary to explore the precise mechanism underlying the links between ncRNAs and osteoporosis and other organ diseases, which could be of great significance for the therapeutic application of ncRNA-related drugs in osteoporosis.

Although numerous recent studies have investigated the mechanisms of ncRNAs in the progression of osteoporosis, limited research concerning the important ncRNAs has been clinically translated. In addition, in vivo studies investigating differentially expressed ncRNAs in bone tissue at different stages of osteoporosis are currently insufficient. Therefore, identifying more consequential ncRNAs related to osteoporosis and carrying out meaningful clinical translational research are of great importance for understanding the pathological mechanism, prevention and treatment of osteoporosis.