Noncoding RNAs in skeletal development and disorders.

Protein-encoding genes only constitute less than 2% of total human genomic sequences, and 98% of genetic information was previously referred to as "junk DNA". Meanwhile, non-coding RNAs (ncRNAs) consist of approximately 60% of the transcriptional output of human cells. Thousands of ncRNAs have been identified in recent decades, and their essential roles in the regulation of gene expression in diverse cellular pathways associated with fundamental cell processes, including proliferation, differentiation, apoptosis, and metabolism, have been extensively investigated. Furthermore, the gene regulation networks they form modulate gene expression in normal development and under pathological conditions. In this review, we integrate current information about the classification, biogenesis, and function of ncRNAs and how these ncRNAs support skeletal development through their regulation of critical genes and signaling pathways in vivo. We also summarize the updated knowledge of ncRNAs involved in common skeletal diseases and disorders, including but not limited to osteoporosis, osteoarthritis, rheumatoid arthritis, scoliosis, and intervertebral disc degeneration, by highlighting their roles established from in vivo, in vitro, and ex vivo studies.

[Latest Findings on the Role of RUNX1 in Bone Development and Disorders]. / RUNX1åœ¨éª¨å‘è‚²åŠéª¨ç›¸å ³ç–¾ç— ä¸­çš„ä½œç”¨ç ”ç©¶è¿›å±•.

Runt-related transcription factor (RUNX1) is a transcription factor closely involved in hematopoiesis. RUNX1 gene mutation plays an essential pathogenic role in the initiation and development of hematological tumors, especially in acute myeloid leukemia. Recent studies have shown that RUNX1 is also involved in the regulation of bone development and the pathological progression of bone-related diseases. RUNX1 promotes the differentiation of mesenchymal stem cells into chondrocytes and osteoblasts and modulates the maturation and extracellular matrix formation of chondrocytes. The expression of RUNX1 in mesenchymal stem cells, chondrocytes, and osteoblasts is of great significance for maintaining normal bone development and the mass and quality of bones. RUNX1 also inhibits the differentiation and bone resorptive activities of osteoclasts, which may be influenced by sexual dimorphism. In addition, RUNX1 deficiency contributes to the pathogenesis of osteoarthritis, delayed fracture healing, and osteoporosis, which was revealed by the RUNX1 conditional knockout modeling in mice. However, the roles of RUNX1 in regulating the hypertrophic differentiation of chondrocytes, the sexual dimorphism of activities of osteoclasts, as well as bone loss in diabetes mellitus, senescence, infection, chronic inflammation, etc, are still not fully understood. This review provides a systematic summary of the research progress concerning RUNX1 in the field of bone biology, offering new ideas for using RUNX1 as a potential target for bone related diseases, especially osteoarthritis, delayed fracture healing, and osteoporosis.

Current Status of Next-Generation Sequencing in Bone Genetic Diseases.

The development of next-generation sequencing (NGS) has dramatically increased the speed and volume of genetic analysis. Furthermore, the range of applications of NGS is rapidly expanding to include genome, epigenome (such as DNA methylation), metagenome, and transcriptome analyses (such as RNA sequencing and single-cell RNA sequencing). NGS enables genetic research by offering various sequencing methods as well as combinations of methods. Bone tissue is the most important unit supporting the body and is a reservoir of calcium and phosphate ions, which are important for physical activity. Many genetic diseases affect bone tissues, possibly because metabolic mechanisms in bone tissue are complex. For instance, the presence of specialized immune cells called osteoclasts in the bone tissue, which absorb bone tissue and interact with osteoblasts in complex ways to support normal vital functions. Moreover, the many cell types in bones exhibit cell-specific proteins for their respective activities. Mutations in the genes encoding these proteins cause a variety of genetic disorders. The relationship between age-related bone tissue fragility (also called frailty) and genetic factors has recently attracted attention. Herein, we discuss the use of genomic, epigenomic, transcriptomic, and metagenomic analyses in bone genetic disorders.

Role of irisin in bone diseases.

Bone diseases are common among middle-aged and elderly people, and harm to activities of daily living (ADL) and quality of life (QOL) for patients. It is crucial to search for key regulatory factors associated with the development of bone diseases and explore potential therapeutic targets for bone diseases. Irisin is a novel myokine that has been discovered in recent years. Accumulating evidence indicates that irisin has beneficial effects in the treatment of various diseases such as metabolic, cardiovascular and neurological disorders, especially bone-related diseases. Recent studies had shown that irisin plays the role in various bone diseases such as osteoarthritis, osteoporosis and other bone diseases, suggesting that irisin may be a potential molecule for the prevention and treatment of bone diseases. Therefore, in this review, by consulting the related domestic and international literature of irisin and bone diseases, we summarized the specific regulatory mechanisms of irisin in various bone diseases, and provided a systematic theoretical basis for its application in the diagnosis and treatment of the bone diseases.

[Regulation of non-coding RNA in type H vessels angiogenesis of bone].

Objective: To summarize the regulatory effect of non-coding RNA (ncRNA) on type H vessels angiogenesis of bone. Methods: Recent domestic and foreign related literature about the regulation of ncRNA in type H vessels angiogenesis was widely reviewed and summarized. Results: Type H vessels is a special subtype of bone vessels with the ability to couple bone formation. At present, the research on ncRNA regulating type H vessels angiogenesis in bone diseases mainly focuses on microRNA, long ncRNA, and small interfering RNA, which can affect the expressions of hypoxia inducible factor 1α, platelet derived growth factor BB, slit guidance ligand 3, and other factors through their own unique ways of action, thus regulating type H vessels angiogenesis and participating in the occurrence and development of bone diseases. Conclusion: At present, the mechanism of ncRNA regulating bone type H vessels angiogenesis has been preliminarily explored. With the deepening of research, ncRNA is expected to be a new target for the diagnosis and treatment of vascular related bone diseases.

Differential Expression of Non-Coding RNAs in Stem Cell Development and Therapeutics of Bone Disorders.

Stem cells' self-renewal and multi-lineage differentiation are regulated by a complex network consisting of signaling factors, chromatin regulators, transcription factors, and non-coding RNAs (ncRNAs). Diverse role of ncRNAs in stem cell development and maintenance of bone homeostasis have been discovered recently. The ncRNAs, such as long non-coding RNAs, micro RNAs, circular RNAs, small interfering RNA, Piwi-interacting RNAs, etc., are not translated into proteins but act as essential epigenetic regulators in stem cells' self-renewal and differentiation. Different signaling pathways are monitored efficiently by the differential expression of ncRNAs, which function as regulatory elements in determining the fate of stem cells. In addition, several species of ncRNAs could serve as potential molecular biomarkers in early diagnosis of bone diseases, including osteoporosis, osteoarthritis, and bone cancers, ultimately leading to the development of new therapeutic strategies. This review aims to explore the specific roles of ncRNAs and their effective molecular mechanisms in the growth and development of stem cells, and in the regulation of osteoblast and osteoclast activities. Furthermore, we focus on and explore the association of altered ncRNA expression with stem cells and bone turnover.

Targeting strategies for bone diseases: signaling pathways and clinical studies.

Since the proposal of Paul Ehrlich's magic bullet concept over 100 years ago, tremendous advances have occurred in targeted therapy. From the initial selective antibody, antitoxin to targeted drug delivery that emerged in the past decades, more precise therapeutic efficacy is realized in specific pathological sites of clinical diseases. As a highly pyknotic mineralized tissue with lessened blood flow, bone is characterized by a complex remodeling and homeostatic regulation mechanism, which makes drug therapy for skeletal diseases more challenging than other tissues. Bone-targeted therapy has been considered a promising therapeutic approach for handling such drawbacks. With the deepening understanding of bone biology, improvements in some established bone-targeted drugs and novel therapeutic targets for drugs and deliveries have emerged on the horizon. In this review, we provide a panoramic summary of recent advances in therapeutic strategies based on bone targeting. We highlight targeting strategies based on bone structure and remodeling biology. For bone-targeted therapeutic agents, in addition to improvements of the classic denosumab, romosozumab, and PTH1R ligands, potential regulation of the remodeling process targeting other key membrane expressions, cellular crosstalk, and gene expression, of all bone cells has been exploited. For bone-targeted drug delivery, different delivery strategies targeting bone matrix, bone marrow, and specific bone cells are summarized with a comparison between different targeting ligands. Ultimately, this review will summarize recent advances in the clinical translation of bone-targeted therapies and provide a perspective on the challenges for the application of bone-targeted therapy in the clinic and future trends in this area.

Effect of HPSE and HPSE2 SNPs on the Risk of Developing Primary Paraskeletal Multiple Myeloma.

Multiple myeloma (MM) is a plasma cell malignancy that is accompanied by hypercalcemia, renal failure, anemia, and lytic bone lesions. Heparanase (HPSE) plays an important role in supporting and promoting myeloma progression, maintenance of plasma cell stemness, and resistance to therapy. Previous studies identified functional single nucleotide polymorphisms (SNPs) located in the HPSE gene. In the present study, 5 functional HPSE SNPs and 11 novel HPSE2 SNPs were examined. A very significant association between two enhancer (rs4693608 and rs4693084), and two insulator (rs4364254 and rs4426765) HPSE SNPs and primary paraskeletal disease (PS) was observed. SNP rs657442, located in intron 9 of the HPSE2 gene, revealed a significant protective association with primary paraskeletal disease and lytic bone lesions. The present study demonstrates a promoting (HPSE gene) and protective (HPSE2 gene) role of gene regulatory elements in the development of paraskeletal disease and bone morbidity. The effect of signal discrepancy between myeloma cells and normal cells of the tumor microenvironment is proposed as a mechanism for the involvement of heparanase in primary PS. We suggest that an increase in heparanase-2 expression can lead to effective suppression of heparanase activity in multiple myeloma accompanied by extramedullary and osteolytic bone disease.

Integration Analysis of circRNA-miRNA-mRNA and Identification of Critical Networks in Valgus-Varus Deformity (Gallus gallus).

Valgus-valgus deformity (VVD) is a common leg deformity in broilers with inward or outward deviation of the tibiotarsus and tarsometatarsus. The competing endogenous RNA (ceRNA) network plays an essential role in the study of leg disease. However, its role in the etiology and pathogenesis of VVD remains unclear. Here, based on case (VVD) and control (normal) group design, we performed analyses of differentially expressed circRNAs (DEcircRNAs), differentially expressed miRNAs (DEmiRNAs) and differentially expressed mRNAs (DEmRNAs). Transcriptome data derived 86 DEcircRNAs, 13 DEmiRNAs and 410 DEmRNAs. Functional analysis showed that DEmRNAs were significantly enriched in cell cycle, apoptosis, ECM-receptor interaction, FoxO signaling pathway and protein processing synthesis. DEcirc/miRNA-associated DEmRNAs were associated with skeletal and muscle growth and development pathways, including mTOR, Wnt, and VEGF signaling pathways. Subsequently, a circRNA-miRNA-mRNA regulatory network was constructed based on the ceRNA hypothesis, including 8 circRNAs, 6 miRNAs, and 31 mRNAs, which were significantly enriched in the skeletal developmental pathway. Finally, two key mRNAs (CDC20 and CTNNB1) and their regulatory axes were screened by the PPI network and cytohubba. The expression levels of CDC20 and CTNNB1 in cartilage and seven other tissues were also quantified by qPCR. In conclusion, we analyzed the functions of DEmRNA, DEcircRNA and DEmiRNA and constructed the hub ceRNA regulatory axis, and obtained two hub genes, CDC20 and CTNNB1. The study more deeply explored the etiology and pathogenesis of VVD and lays the foundation for further study of the role of the ceRNA network on skeletal development.

Micro-RNA: A Future Approach to Personalized Diagnosis of Bone Diseases.

Osteoporosis is a highly prevalent bone disease worldwide and the most studied bone-associated pathological condition. Although its diagnosis makes use of advanced and clinically relevant imaging and biochemical tools, the information suffers from several limitations and has little or no prognostic value. In this context, circulating micro-RNAs represent a potentially attractive alternative or a useful addition to the diagnostic arsenal and offer a greater prognostic potential than the conventional approaches. These short non-coding RNA molecules act as inhibitors of gene expression by targeting messenger RNAs with different degrees of complementarity, establishing a complex multilevel network, the basis for the fine modulation of gene expression that finally regulates every single activity of a cell. Micro-RNAs may passively and/or actively be released in the circulation by source cells, and being measurable in biological fluids, their concentrations may be associated to specific pathophysiological conditions. Mounting, despite debatable, evidence supports the use of micro-RNAs as markers of bone cell metabolic activity and bone diseases. Indeed, several micro-RNAs have been associated with bone mineral density, fractures and osteoporosis. However, concerns such as absence of comparability between studies and, the lack of standardization and harmonization of the methods, limit their application. In this review, we describe the pathophysiological bases of the association between micro-RNAs and the deregulation of bone cells activity and the processes that led to the identification of potential micro-RNA-based markers associated with metabolic bone diseases.

The effects of epigenetic modifications on bone remodeling in age-related osteoporosis.

PURPOSE: As the population ages, there is an increased risk of fracture and morbidity diseases associated with aging, such as age-related osteoporosis and other bone diseases linked to aging skeletons. RESULTS: Several bone-related cells, including multipotent bone mesenchymal stem cells, osteoblasts that form bone tissue, and osteoclasts that break it down, are in symbiotic relationships throughout life. Growing evidence indicates that epigenetic modifications of cells caused by aging contribute to compromised bone remodeling and lead to osteoporosis. A number of epigenetic mechanisms are at play, including DNA/RNA modifications, histone modifications, microRNAs (miRNAs), and long noncoding RNAs (lncRNAs), as well as chromatin remodeling. CONCLUSION: In this review, we summarized the epigenetic modifications of different bone-related cells during the development and progression of osteoporosis associated with aging. Additionally, we described a compensatory recovery mechanism under epigenetic regulation that may lead to new strategies for regulating bone remodeling in age-related osteoporosis.

Autosomal recessive LRP1-related syndrome featuring cardiopulmonary dysfunction, bone dysmorphology, and corneal clouding.

We provide the first study of two siblings with a novel autosomal recessive LRP1-related syndrome identified by rapid genome sequencing and overlapping multiple genetic models. The patients presented with respiratory distress, congenital heart defects, hypotonia, dysmorphology, and unique findings, including corneal clouding and ascites. Both siblings had compound heterozygous damaging variants, c.11420G > C (p.Cys3807Ser) and c.12407T > G (p.Val4136Gly) in LRP1, in which segregation analysis helped dismiss additional variants of interest. LRP1 analysis using multiple human/mouse data sets reveals a correlation to patient phenotypes of Peters plus syndrome with additional severe cardiomyopathy and blood vessel development complications linked to neural crest cells.

Epigenetic therapy targeting bone marrow mesenchymal stem cells for age-related bone diseases.

As global aging accelerates, the prevention and treatment of age-related bone diseases are becoming a critical issue. In the process of senescence, bone marrow mesenchymal stem cells (BMSCs) gradually lose the capability of self-renewal and functional differentiation, resulting in impairment of bone tissue regeneration and disorder of bone tissue homeostasis. Alteration in epigenetic modification is an essential factor of BMSC dysfunction during aging. Its transferability and reversibility provide the possibility to combat BMSC aging by reversing age-related modifications. Emerging evidence demonstrates that epigenetic therapy based on aberrant epigenetic modifications could alleviate the senescence and dysfunction of stem cells. This review summarizes potential therapeutic targets for BMSC aging, introduces some potential approaches to alleviating BMSC aging, and analyzes its prospect in the clinical application of age-related bone diseases.

Sp7 Action in the Skeleton: Its Mode of Action, Functions, and Relevance to Skeletal Diseases.

Osteoblast differentiation is a tightly regulated process in which key transcription factors (TFs) and their target genes constitute gene regulatory networks (GRNs) under the control of osteogenic signaling pathways. Among these TFs, Sp7 works as an osteoblast determinant critical for osteoblast differentiation. Following the identification of Sp7 and a large number of its functional studies, recent genome-scale analyses have made a major contribution to the identification of a "non-canonical" mode of Sp7 action as well as "canonical" ones. The analyses have not only confirmed known Sp7 targets but have also uncovered its additional targets and upstream factors. In addition, biochemical analyses have demonstrated that Sp7 actions are regulated by chemical modifications and protein-protein interaction with other transcriptional regulators. Sp7 is also involved in chondrocyte differentiation and osteocyte biology as well as postnatal bone metabolism. The critical role of SP7 in the skeleton is supported by its relevance to human skeletal diseases. This review aims to overview the Sp7 actions in skeletal development and maintenance, particularly focusing on recent advances in our understanding of how Sp7 functions in the skeleton under physiological and pathological conditions.

Effect of Regulator of G Protein Signaling Proteins on Bone.

Regulator of G protein signaling (RGS) proteins are critical negative molecules of G protein-coupled receptor (GPCR) signaling, which mediates a variety of biological processes in bone homeostasis and diseases. The RGS proteins are divided into nine subfamilies with a conserved RGS domain which plays an important role in regulating the GTPase activity. Mutations of some RGS proteins change bone development and/or metabolism, causing osteopathy. In this review, we summarize the recent findings of RGS proteins in regulating osteoblasts, chondrocytes, and osteoclasts. We also highlight the impacts of RGS on bone development, bone remodeling, and bone-related diseases. Those studies demonstrate that RGS proteins might be potential drug targets for bone diseases.

Premature Vertebral Mineralization in hmx1-Mutant Zebrafish.

H6 family homeobox 1 (HMX1) regulates multiple aspects of craniofacial development, and mutations in HMX1 are linked to an ocular defect termed oculoauricular syndrome of Schorderet-Munier-Franceschetti (OAS) (MIM #612109). Recently, additional altered orofacial features have been reported, including short mandibular rami, asymmetry of the jaws, and altered premaxilla. We found that in two mutant zebrafish lines termed hmx1mut10 and hmx1mut150, precocious mineralization of the proximal vertebrae occurred. Zebrafish hmx1mut10 and hmx1mut150 report mutations in the SD1 and HD domains, which are essential for dimerization and activity of hmx1. In hmx1mut10, the bone morphogenetic protein (BMP) antagonists chordin and noggin1 were downregulated, while bmp2b and bmp4 were highly expressed and specifically localized to the dorsal region prior to the initiation of the osteogenic process. The osteogenic promoters runx2b and spp1 were also upregulated. Supplementation with DMH1-an inhibitor of the BMP signaling pathway-at the specific stage in which bmp2b and bmp4 are highly expressed resulted in reduced vertebral mineralization, resembling the wildtype mineralization progress of the axial skeleton. These results point to a possible role of hmx1 as part of a complex gene network that inhibits bmp2b and bmp4 in the dorsal region, thus regulating early axial skeleton development.

A neomorphic variant in SP7 alters sequence specificity and causes a high-turnover bone disorder.

SP7/Osterix is a transcription factor critical for osteoblast maturation and bone formation. Homozygous loss-of-function mutations in SP7 cause osteogenesis imperfecta type XII, but neomorphic (gain-of-new-function) mutations of SP7 have not been reported in humans. Here we describe a de novo dominant neomorphic missense variant (c.926 C > G:p.S309W) in SP7 in a patient with craniosynostosis, cranial hyperostosis, and long bone fragility. Histomorphometry shows increased osteoblasts but decreased bone mineralization. Mice with the corresponding variant also show a complex skeletal phenotype distinct from that of Sp7-null mice. The mutation alters the binding specificity of SP7 from AT-rich motifs to a GC-consensus sequence (typical of other SP family members) and produces an aberrant gene expression profile, including increased expression of Col1a1 and endogenous Sp7, but decreased expression of genes involved in matrix mineralization. Our study identifies a pathogenic mechanism in which a mutation in a transcription factor shifts DNA binding specificity and provides important in vivo evidence that the affinity of SP7 for AT-rich motifs, unique among SP proteins, is critical for normal osteoblast differentiation.

Deciphering spatial genomic heterogeneity at a single cell resolution in multiple myeloma.

Osteolytic lesions (OL) characterize symptomatic multiple myeloma. The mechanisms of how malignant plasma cells (PC) cause OL in one region while others show no signs of bone destruction despite subtotal infiltration remain unknown. We report on a single-cell RNA sequencing (scRNA-seq) study of PC obtained prospectively from random bone marrow aspirates (BM) and paired imaging-guided biopsies of OL. We analyze 148,630 PC from 24 different locations in 10 patients and observe vast inter- and intra-patient heterogeneity based on scRNA-seq analyses. Beyond the limited evidence for spatial heterogeneity from whole-exome sequencing, we find an additional layer of complexity by integrated analysis of anchored scRNA-seq datasets from the BM and OL. PC from OL are characterized by differentially expressed genes compared to PC from BM, including upregulation of genes associated with myeloma bone disease like DKK1, HGF and TIMP-1 as well as recurrent downregulation of JUN/FOS, DUSP1 and HBB. Assessment of PC from longitudinally collected samples reveals transcriptional changes after induction therapy. Our study contributes to the understanding of destructive myeloma bone disease.

The Contribution of Deleterious Rare Alleles in ENPP1 and Osteomalacia Causative Genes to Atypical Femoral Fracture.

CONTEXT: Atypical femoral fractures (AFFs) are very rare atraumatic or mild trauma fractures in the subtrochanteric region or femoral shaft. Some unique genetic variants in Asian populations might confer susceptibility to AFF, since the incidence of AFFs is higher in Asian populations. OBJECTIVE: Because rare variants have been found to be causative in some diseases and the roles of osteomalacia causative genes have not been reported, we investigated rare variants in genes causing abnormal mineralization. METHODS: Exome sequencing was performed to detect variants in gene coding and boundary regions, and the frequencies of deleterious rare alleles were compared between Japanese patients with AFF (n = 42) and controls of the 4.7KJPN panel of Tohoku Medical Megabank by whole genome sequencing (n = 4773). RESULTS: The frequency of the deleterious rare allele of ENPP1 was significantly increased in AFF (P = .0012, corrected P [Pc] = .0155, OR 4.73, 95% CI 2.15-10.40). In multigene panel analysis, the frequencies of deleterious rare alleles of candidate genes were increased in AFF (P = .0025, OR 2.72, 95% CI 1.49-4.93). Principal component analysis of bone metabolism markers identified a subgroup of patients with AFF with higher frequencies of deleterious rare alleles in ENPP1 (P = 4.69 × 10-5, Pc = .0006, OR 8.47, 95% CI 3.76-19.09) and the candidate genes (P = 1.08 × 10-5, OR 5.21, 95% CI 2.76-9.86). CONCLUSION: AFF is associated with genes including ENPP1 that cause abnormal mineralization, suggesting that osteomalacia is an underlying condition predisposing to AFF and that higher incident rates of AFFs in Asian populations might be explained by the genetic risk factors including ENPP1.