An Overview of Long Noncoding RNAs Involved in Bone Regeneration from Mesenchymal Stem Cells

Bone regeneration is very important for the recovery of some diseases including osteoporosis and bone fracture trauma. It is a multiple-step- and multiple-gene-involved complex process, including the matrix secretion and calcium mineralization by osteoblasts differentiated from mesenchymal stem cells (MSCs) and the absorption of calcium and phosphorus by osteoclasts differentiated from hematopoietic stem cells. Long noncoding RNAs (lncRNAs) are a family of transcripts longer than 200 nt without or with very low protein-coding potential. Recent studies have demonstrated that lncRNAs are widely involved in the regulation of lineage commitment and differentiation of stem cells through multiple mechanisms. In this review, we will summarize the roles and molecular mechanism of lncRNAs including H19, MALAT1, MODR, HOTAIR, DANCR, MEG3, HoxA-AS3, and MIAT in osteogenesis ossification; lncRNA ZBED3-AS1 and CTA-941F9.9, DANCR, and HIT in chondrogenic differentiation; and lncRNA DANCR in osteoclast differentiation. These findings will facilitate the development and application of novel molecular drugs which regulate the balance of bone formation and absorption.


Introduction
The bone regeneration after bone fracture trauma or other diseases is a process participated by a well-organized system of the synergistic effect of MSCs, immune cells, and osteoclasts. Osteoclasts absorb the organic and inorganic compounds released from the impaired bone, during which the degraded compound matrix goes into the bloodstream in the form of Ca2 + , (PO4)3 − , and so on for recycling [1,2]. Meanwhile, the cytokines after the damage process initiate the osteogenic differentiation of MSCs. MSCs gradually differentiate into osteoprogenitors, preosteoblasts, and osteoblasts. The well-differentiated osteoblasts synthesize and secrete the matrix and thus induce the initiation of bone formation. MSC-mediated bone regeneration and osteoclastmediated bone resorption are the two core processes of bone regeneration and repair. The process of osteogenic differentiation of MSCs is mainly regulated by tissue-specific transcriptional regulators and epigenetic factors [3,4]. On the one hand, in the corporate induction of BMPs, Wnt/β-FGF, and other growth factors, related molecules in the signal pathways such as BMPs/Smads [5], Wnt/β-catenin [6] and MAPK/p38 [7], and transcription factors RUNX2 [8] and OSX [9] are activated, increasing the expression of osteoblast-specific genes (OPN, OCN, ALP, and COL1A1); eventually, MSCs differentiate into osteoblasts. On the other hand, epigenetic modulation including DNA methylation, histone modification, and noncoding RNA regulation also exerts a role in the regulation of osteogenic differentiation of MSCs. The regulation of DNA methylation and histone modification has been well understood. For example, Dansranjavin et al. found that the osteocalcin of undifferentiated stem cells was hypermethylated. However, in mature osteocytes, the degree of methylation was reduced and the expression levels of osteocalcin were increased [10]. Hsiao et al. observed that transfection of human bone marrow MSCs with the methylated thyroid hormone receptor interactor 10 (Trip 10) promoter resulted in cytosine methylation at the promoter region and downregulation of Trip10 expression, in which accelerating MSCs differentiate into neurons and osteoblasts [11]. Histone acetylation and methylation are another important epigenetic mechanisms in the process of osteogenesis [12][13][14][15]. Studies have shown that the BMP signaling pathway promotes osteogenic differentiation by regulating the acetylation of H3K9. Shen et al. observed that H3K4 methylation decreased while H3K9 acetylation increased during the osteogenic differentiation of ROS17/ 2.8 osteosarcoma cells and normal osteoblasts by using CHIP-seq techniques [16]. The roles and related mechanisms of miRNAs in bone development and balance have been reviewed [17][18][19]. However, the regulation of lncRNAs on bone regeneration has not well summarized.
Long noncoding RNAs (lncRNAs) belong to a family of transcripts longer than 200 nt without or with very low protein-coding potential. In the human genome, 15,787 lncRNA transcripts from 14,470 lncRNA genes have been identified, while the GENCODE annotation is constantly being updated [20,21]. It is believed that lncRNAs are a transcriptional noise for a long time, which are a byproduct of RNA polymerase II transcription without biological function. However, recent studies have found that lncRNAs play a crucial role in regulating nuclear chromatin structure and gene expression in the developmental process and are also an active participant in disease occurrence and development [22][23][24]. Except extensive and constitutive expression of partial lncRNAs, most lncRNAs are specifically expressed during the cell tissue developmental stage. In general, the general expression levels of lncRNA are lower than those of mRNA. Some lncRNAs are located in the cell nucleus and some in the cytoplasm. Compared with miRNA, the interspecies homology similarity of lncRNAs is relatively lower, but there is a certain degree of conservation in its promoter region and exon area, which indicates the function of lncRNAs is relatively conservative. The transcripts produced from the 4~9% sequence of the mammalian genome sequence are lncRNAs (corresponding protein-coding portion is 1%). Despite that the recent advances on lncRNA have progressed rapidly, the functions of most of lncRNAs are still unclear.

Classification and Characteristics of lncRNAs
According to the genomic location, lncRNAs can be classified into five types: sense, antisense, bidirectional, intronic, and intergenic [3,4,25]. Many lncRNAs have conserved secondary structures, alternative splicing, and subcellular localization. The conservativeness and specificity indicate that they are functional [26]. lncRNAs possess the following characteristics: (1) The length of transcripts is 200-100,000 nt, with a similar structure to that of the mRNA. After splicing, there is a structure with a poly(A) tail and a promoter. During differentiated processes, there are a dynamic expression mechanism and alternative splicing that form different lncRNAs [27]. (2) Generally, lncRNAs have noncoding potentials, but some lncRNAs can encode some short peptides [28]. (3) They have low conservation [29]. (4) They are tissue-specific and spatiotemporal-specific. The amount of lncRNAs expressed in different tissues was different, and the expression of lncRNAs was different in the same tissues but different status [30]. (5) The abundance of different lncRNAs is various in different cells [31].

Modes of Action of lncRNAs
With the gradual knowledge of lncRNA functions, the mechanism of lncRNA interaction with targets has become a hot topic. Early identification of in situ regulation is the only mechanism in which lncRNAs silence the transcription of adjacent genes by recruiting chromatin-modifying complexes. The mechanism of lncRNAs is very complex and has not yet been fully understood. According to the current research, the mechanism of lncRNAs could be summarized as the four levels (epigenetic, transcriptional, and posttranscriptional regulation and other specific regulation modes).

lncRNAs Mediate Epigenetic
Modifications. lncRNAs can recruit a chromatin remodeling complex to specific sites and then regulate the expression of targeting genes. For example, HOTAIR derived from the HOXC loci recruits the chromatin remodeling complex PRC2 and locates it to the HOXD site, thereby inducing the parent genetic silencing of the HOXD loci [32][33][34]. Similarly, lncRNAs Xist [35] and Kcnq1ot [36,37] can be recruited by the remodeling complexes such as methyltransferase Ezh2 or G9a to realize epigenetic silence of related genes.
3.2. lncRNAs Regulate Transcriptional Expression. lncRNAs can silence gene expression at the transcriptional level through a variety of mechanisms. lncRNAs can interfere with the transcription of adjacent genes. For example, in yeast, the transcription of the SER3 gene is affected by its upstream lncRNA SRG1 [38]. lncRNAs can interfere with gene expression by blocking the promoter region. For instance, lncRNA DHFR can form an RNA-DNA3 helix structure in the promoter region of the DHFR gene [39], inhibiting the binding of the transcription factor TFIID and thereby inhibiting DHFR gene expression. Moreover, lncRNA can interact with RNA-binding proteins and target to the promoter region, regulating gene expression. For instance, lncRNA located in the upstream of the CCND1 promoter can regulate the activity of the RNA-binding protein TLS and affect the expression of CCND1 [40]. Besides, lncRNAs regulate the activity of transcription factors. lncRNA Evf2 can form transcriptional complexes with the transcription factor Dlx2 to activate Dlx6 expression [41,42]. At last, lncRNAs can control gene expression by regulating the basic transcription factor. For instance, Alu RNA can realize extensive gene suppression by inhibiting RNA polymerase II [43].

lncRNAs Mediate Posttranscriptional Regulation.
lncRNA can form double-stranded RNA complexes with mRNA at the posttranscriptional level to mask the major cis-acting elements of mRNA, thereby regulating gene expression. For example, lncRNA Zeb2 (Sip1) is able to form a double strand at the 5 ′ end shear site of an intron of the mRNA transcribed by the HOX site, thereby preventing the intron from being sheared. The region contains ribosomebinding sites which are necessary for the expression of Zeb2 protein, and Zeb2 antisense RNA can increase the expression of Zeb2 protein in this way. This example shows that lncRNAs can guide alternative splicing of mRNA isoforms. lncRNAs compete with mRNA to bind miRNA-binding sites, leading to the upregulation of miRNA target molecules. LncMD as a spongy molecule isolates miR-125b binding to the target molecule IGF mRNA, promoting MSC differentiation into muscle cells [44].

Other Specific Regulation
Modes. In addition, the renaturation (annealing) of lncRNAs has a targeting effect, allowing protein receptor complexes to recognize the mRNA transcripts of the sense chain. This mode resembles the RNA-induced silencing complex (RISC) targeting mRNA through siRNA. Double-stranded RNA derived from complementary transcripts and even lncRNA, combined with extended internal hairpin structure, can be processed into endogenous siRNA to silence gene expression.

lncRNAs in Bone Development and Homeostasis
The formation of new bone is induced from MSCs via lineage commitment, which successively form osteoprogenitor cells, preosteoblasts, mature osteoblasts, and osteocytes. These major regulatory mechanisms, including tissue-specific transcription factors and regulatory molecules, mediated bone matrix synthesis, bone remodeling, and mineralizationrelated and repair-related gene expression. These osteogenic activities are simultaneously regulated by genetic and epigenetic levels. Epigenetic regulation includes DNA methylation, histone modification, and miRNA and lncRNA regulation. miRNA regulation for bone function and repair process has been summarized before. Numerous experiments have shown that lncRNAs play a role in these processes. Classification and functional analyses also show that lncRNAs are involved in the lineage differentiation of MSCs into muscle cells, adipocytes, chondrocytes, and osteoblasts. Many scientists have focused on how lncRNAs play a role in stem cell differentiation for the past few years. Here, we will concentrate on the expression of lncRNAs in osteogenic differentiation of MSCs (Table 1). In addition, we will also analyze the role of lncRNAs in bone and cartilage differentiation, as well as the role and balance of bone, cartilage, and osteoclasts.

Global Transcriptomic Analyses Identify lncRNA Profiles
Zuo et al. [45] firstly published the earliest report about osteogenesis-related lncRNAs identified at 2013. They found that the expression profile of lncRNAs from C3H10T1/2 MSCs was changed under BMP2 induction. At the same time, they identified 116 differentially expressed lncRNAs and these lncRNAs positively regulate the expression of its adjacent genes, which indicated that lncRNAs regulated osteogenesis under the synergistic effect of adjacent genes. Song et al. utilized high-throughput RNA sequencing (RNA-seq) data to detect the expression profile of lncRNAs from immortalized MSCs which was induced by osteogenic induction medium for 28 days and thus screened 2597 mRNAs and 574 lncRNAs, of which 351 were known lncRNAs and 217 were novel lncRNAs. 32 novel lncRNAs are the precursor molecules of miR-689, miR-640, miR-601, and miR-544. They also constructed 14,275 coexpression relationships in the osteogenesis process, as well as 217 gene regulatory networks between the novel lncRNA and the mRNA [46] . Qu et al. utilized high-throughput expression profiles (30,586 lncRNAs and 26,109 coding transcripts) to screen the differentially expressed genes of human periodontal ligament stem cells (hPDLSCs), which were, respectively, cultured in growth medium and osteogenic induction medium for 14 days, and screened out 3557 differentially expressed mRNAs, among which 1578 mRNAs were upregulated, 1979 mRNAs downregulated, 994 lncRNAs upregulated, and 1177 lncRNAs downregulated. These lncRNAs (AC078851.1, RP11-45A16.4, XLOC_002932, RP4-613B23.1, and RP11-305L7.6) and mRNAs (ALP, COL1A1, and COL1A20) are upregulated and BMP5 and IL6 are downregulated as verified by Q-PCR indicating that there are 131 pairs of lncRNA-mRNA regulatory relationships and 262 pairs of positive regulatory relationships, and MAPK, VEGF, and TGF-β signaling pathways were mainly involved in the regulation during osteogenic differentiation process [47].
Zhang et al. reported the human BMSCs derived from 18-to 20-year-old healthy male bone marrow cultured in osteogenic induction medium for 7 days and screened them with high-throughput human transcription microarray (Affymetrix, covering more than 285,000 coding and 40,000 noncoding transcripts). They screened out 1269 differentially expressed mRNAs (among which 648 were upregulated and 621 were downregulated) and 1408 lncRNAs, and MAPK, JAK-STAT, Toll-like receptor, and TGF-β signal pathways were found to participate in osteogenic differentiation of hBMSCs. GPX3, TLR2, BDKRB1, FBXO5, BRCA1, MAP3K8, SCARB1, and 6 lncRNAs (XR_111050, NR_024031, FR374455, FR401275, FR406817, and FR148647) played a key role in osteogenic process, and lncRNA XR_111050 promoted osteogenic differentiation of mesenchymal stromal cells [48]. It can be confirmed that lncRNAs play an important role in osteogenesis differentiation, and the current studies have identified a number of differentially expressed lncRNAs. However, the mechanisms of most lncRNAs regulating the osteogenesis process remain to be understood and explored. is one of the highly upregulated genes during the induction of primitive stem cells with osteogenic induction medium. It is located on 11p15.5 and is 2.3 kb in length and is conserved in evolution and plays an important role in regulating biological functions. H19 is the precursor of miR-675 which can generate two mature miRNAs (miR-675-5p and miR-675-3p) by Drosha and Dicer with splicing-dependent modes. H19 and miR-675 were upregulated during osteogenic differentiation of human MSCs. miR-675 not only downregulates TGF-β1 but also inhibits Smad3 phosphorylation and downregulates HDAC4/5 leading to reduced HDACs to be recruited to the promoter of osteogenesisspecific runt-related transcription factor 2 (Runx2) [49].   MSCs [58]. The mechanism may be associated with miR-140-5p. It also has been reported that MEG3 upregulated miR-133a-3p and inhibited osteogenic differentiation in bone marrow MSCs from patients with postmenopausal osteoporosis [59] (Figure 1).
HoxA-AS3 inhibits RUNX2 transcription by binding to EZH2 and promoting H3k27 methylation. HoxA-AS3 acts as an epigenetic modified switch to inhibit osteogenic differentiation of MSCs [61] (Figure 2). Tumor necrosis factor treatment increases MIAT expression. Knockdown of MIAT can reverse the inhibition of osteogenic differentiation induction by an inflammatory factor. It acts as a sponge molecule of miR-150-5p to regulate its binding to the target gene and also acts as an endogenous competitive RNA to form AKT-miR-150-5p feedback loop to regulate oxidative stress and inflammatory factors and to stimulate the functional regulation of human lens epithelial cells [62][63][64] (Figure 2).
6.2.6. POIR. lncRNA POIR, which is located on chromosome 6 and is 786 nt in length, was found to be expressed differentially in periodontal MSCs from patients with periodontitis and healthy human. Its expression is upregulated during osteogenic differentiation of periodontal membrane stem cells (PMSCs). Further studies have shown that lncRNA POIR as an endogenous competitive RNA competes for the binding sites of miR-182, leading to an increase in its target

lncRNAs Involved in Chondrogenic Differentiation
Cartilage plays a role in modeling, protecting and supplementing bone tissue during individual development. lncRNA HIT was highly expressed in E11 mouse embryos, which is located in the nucleus and formed complexes with p100 and CBP. CHIRP-seq analysis revealed that the lncRNA-HIT-p100/CBP complex was associated with multiple sites in the mouse genome and exerted its role in cartilage differentiation. Silencing HIT with specific siRNA leads to decreased p100 activity and decreased H3K27ac, so lncRNA HIT plays an integral role in cartilage differentiation [72,73] (Table 2).

lncRNAs Involved in Osteoclastogenesis
Bone formation is a dynamic and continuous experience shaping, repair, and reconstruction process.   [72,73] The research group constructed 142 pairs of correlation between lncRNA and mRNA [74] and found that lncRNAs are also involved in the regulation of hematopoietic stem cell differentiation into osteoclasts. Osteoporosis is a common disease associated with reduced bone mineralization, which is mainly due to osteoblastic bone resorption exceeding bone formation function of osteoblasts. Tong et al. reported that lncRNA DANCR was involved in mononuclear cell formation in peripheral blood and was associated with human osteoporosis. DANCR promotes IL6 and TNFα expression and increases bone resorption. These results suggest that lncRNAs are involved in bone resorption processes of osteoclasts [75] (Figure 3).

Concluding Remarks
In this manuscript, we summarized the long noncoding RNAs which play an important role in the osteogenic differentiation (H19, MALAT1, MODR, etc.), cartilage differentiation (ZBED3-AS1, DANCR, and HIT) from MSCs, and osteoclast differentiation (DANCR) from hematopoietic stem cells and mononuclear progenitor cells. The mechanism has been demonstrated. Compared with coding protein and small RNA, the knowledge of lncRNAs is only at an initial stage; functions and regulation mechanisms of which remain to be further elucidated. At present, we get to know about the functions and regulatory molecular mechanism through traditional techniques including in situ hybridization technology, overexpression technology, luciferase reporter gene system, and gene silencing technology by siRNA and Crisp/ Cas9. At the same time, the development of some new technologies, such as CLIP (cross-linking immunoprecipitation) [76][77][78], RIP (RNA-binding protein immunoprecipitation) [79], RNA pulldown [80], CLASH (cross-linking, ligation, and sequencing of hybrids) [81], and ChIRP (chromatin isolation by RNA purifications) [82], has also provided a new platform for studying the networks involving proteins and RNA. With the development of more high-throughput screening technologies, such as microarray chip hybridization, combining the new generation of high-throughput sequencing technology with bioinformatics prediction tools, people will be able to find those with important regulatory functions more quickly and efficiently. The understanding, development, and application of the novel lncRNAs in the field of regeneration and repair will also present a new blueprint for a better and healthier life.