[Effects of density on growth and gene transcription characteristics of Rehmannia glutinosa].

The present study analyzed the effects of planting density on the development, quality, and gene transcription characte-ristics of Rehmannia glutinosa using 85-5 and J9 as materials with three planting densities of 5 000, 25 000, and 50 000 plants/Mu(1 Mu≈667 m~2). The agronomic characteristics of leaves and tuberous roots, the content of catalpol and acteoside, and the changes of gene expression were determined. The results showed that the leaf size, the diameter of tuberous root, leaf biomass, tuberous root number, and tuberous root biomass per plant at low density were significantly higher than those of medium and high densities. The content of catalpol and acteoside in leaves was higher at high density. The content of catalpol in tuberous roots was higher at low density, and the change trend was similar to that in leaves, while the content of acteoside in tuberous roots was higher at high density. Transcriptome analysis found that about 1/2 of the expansin genes could change regularly in response to density treatment, which was rela-ted to the development of tuberous roots. The change trend of the gene expression of multiple catalytic enzymes involved in the biosynthesis of catalpol and acteoside was consistent with that of their content, which was presumedly involved in the accumulation and regulation of density-responsive medicinal components. Based on the analysis of the development, medicinal components, and gene expression characteristics of R. glutinosa at different densities, this study is expected to provide an important basis for regulating the quality and yield of medicinal materials of R. glutinosa by managing the planting density.

Bone Microenvironment and Osteosarcoma Metastasis.

The bone microenvironment is an ideal fertile soil for both primary and secondary tumors to seed. The occurrence and development of osteosarcoma, as a primary bone tumor, is closely related to the bone microenvironment. Especially, the metastasis of osteosarcoma is the remaining challenge of therapy and poor prognosis. Increasing evidence focuses on the relationship between the bone microenvironment and osteosarcoma metastasis. Many elements exist in the bone microenvironment, such as acids, hypoxia, and chemokines, which have been verified to affect the progression and malignance of osteosarcoma through various signaling pathways. We thoroughly summarized all these regulators in the bone microenvironment and the transmission cascades, accordingly, attempting to furnish hints for inhibiting osteosarcoma metastasis via the amelioration of the bone microenvironment. In addition, analysis of the cross-talk between the bone microenvironment and osteosarcoma will help us to deeply understand the development of osteosarcoma. The cellular and molecular protagonists presented in the bone microenvironment promoting osteosarcoma metastasis will accelerate the exploration of novel therapeutic strategies towards osteosarcoma.

Recombinant Irisin Prevents the Reduction of Osteoblast Differentiation Induced by Stimulated Microgravity through Increasing ß-Catenin Expression.

Background: Irisin, a novel exercise-induced myokine, was shown to mediate beneficial effects of exercise in osteoporosis. Microgravity is a major threat to bone homeostasis of astronauts during long-term spaceflight, which results in decreased bone formation. Methods: The hind-limb unloading mice model and a random position machine are respectively used to simulate microgravity in vivo and in vitro. Results: We demonstrate that not only are bone formation and osteoblast differentiation decreased, but the expression of fibronectin type III domain-containing 5 (Fdnc5; irisin precursor) is also downregulated under simulated microgravity. Moreover, a lower dose of recombinant irisin (r-irisin) (1 nM) promotes osteogenic marker gene (alkaline phosphatase (Alp), collagen type 1 alpha-1(ColIα1)) expressions, ALP activity, and calcium deposition in primary osteoblasts, with no significant effect on osteoblast proliferation. Furthermore, r-irisin could recover the decrease in osteoblast differentiation induced by simulated microgravity. We also find that r-irisin increases ß-catenin expression and partly neutralizes the decrease in ß-catenin expression induced by simulated microgravity. In addition, ß-catenin overexpression could also in part attenuate osteoblast differentiation reduction induced by simulated microgravity. Conclusions: The present study is the first to show that r-irisin positively regulates osteoblast differentiation under simulated microgravity through increasing ß-catenin expression, which may reveal a novel mechanism, and it provides a prevention strategy for bone loss and muscle atrophy induced by microgravity.

A novel long noncoding RNA AK016739 inhibits osteoblast differentiation and bone formation.

The incidence of postmenopausal osteoporosis research 50% in middle-aged and older women, however, effects of existing therapy are not ideal. Emerging evidence have proved that long noncoding RNAs (lncRNAs) was correlated with multiple physiological and pathology processes including development, carcinogenesis, and osteogenesis. However, reports on lncRNAs regulating bone formation were relatively limited. In this study, we screened osteogenic lncRNAs through mRNA/lncRNA microarray combined with gene coexpression analysis. The biological function of the screened lncRNA was assessed both in vitro and in vivo. The effects of the lncRNA on osteogenic transcription factors were also evaluated. We identified AK016739, which was correlated with osteogenic differentiation and enriched in skeletal tissues of mice. The expression levels of AK016739 in bone-derived mesenchymal stem cells were increased with age and negatively correlated with osteogenic differentiation marker genes. Experiments showed that AK016739 inhibited osteoblast differentiation, and in vivo inhibition of AK016739 by its small interfering RNA would rescue bone formation in ovariectomized osteoporosis mice model. In addition, AK016739 suppressed both expression levels and activities of osteogenic transcription factors. This newly identified lncRNA AK016739 has revealed a new mechanism of osteogenic differentiation and provided new targets for treatment of skeletal disorders.

Silencing of lncRNA AK045490 Promotes Osteoblast Differentiation and Bone Formation via ß-Catenin/TCF1/Runx2 Signaling Axis.

Osteoporosis, a disease characterized by both loss of bone mass and structural deterioration of bone, is the most common reason for a broken bone among the elderly. It is known that the attenuated differentiation ability of osteogenic cells has been regarded as one of the greatest contributors to age-related bone formation reduction. However, the effects of current therapies are still unsatisfactory. In this study we identify a novel long noncoding RNA AK045490 which is correlated with osteogenic differentiation and enriched in skeletal tissues of mice. In vitro analysis of bone-derived mesenchymal stem cells (BMSCs) showed that AK045490 inhibited osteoblast differentiation. In vivo inhibition of AK045490 by its small interfering RNA rescued bone formation in ovariectomized osteoporosis mice model. Mechanistically, AK045490 inhibited the nuclear translocation of ß-catenin and downregulated the expression of TCF1, LEF1, and Runx2. The results suggest that Lnc-AK045490 suppresses ß-catenin/TCF1/Runx2 signaling and inhibits osteoblast differentiation and bone formation, providing a novel mechanism of osteogenic differentiation and a potential drug target for osteoporosis.

[Molecular cloning and expression analysis of iridoid synthase genes from Rehmannia glutinosa].

Iridoid synthase( IS),the key enzyme in the natural biosynthesis of vegetal iridoids,catalyzes the irreversible cyclization of 10-oxogeranial to epi-iridodial. In this study,we screened the Rehmannia glutinosa transcriptome data by BLASTn with Catharanthus roseus CrIS cDNA,and found four c DNA fragments with length of 1 527,1 743,1 425,1 718 bp,named RgIS1,RgIS2,RgIS3 and RgIS4,respectively. Bioinformatics analysis revealed that the four iridoid synthase genes encoding proteins with 389-392 amino acid residues,protein molecular weights were between 44. 30-44. 74 k Da,and theoretical isoelectric points were between 5. 30 and 5. 87. Subcellular localization predictions showed that the four iridoid synthase were distributed in the cytoplasm. Structure analysis revealed that R. glutinosa iridoid synthases contain six conserved short-chain dehydrogenase/reductase( SDR) motifs,and their 3 D models were composed typical dinucleotide-binding " Rossmann" folds covered by helical C-terminal extensions. Using the amino acid sequences of four R. glutinosa iridoid synthases,phylogenetic analysis was performed,the result indicated that RgIS3,CrIS and Olea europaea OeIS were grouped together,the other R. glutinosa iridoid synthases and fifteen proteins in other plants had close relationship. Real-time fluorescent quantitative PCR revealed that RgIS1 and RgIS3 highly expressed in unfold leaves,however,RgIS2 and RgIS4 highly expressed in stems and tuberous roots,respectively. RgIS3 showed higher expression levels in non-radial striations( nRS) of the two cultivars,and RgIS1 and RgIS2 had higher expression levels in nRS of QH,while RgIS4 had less expression levels in nRS of QH1. RgIS1,RgIS2 and RgIS3 were up-regulated by Me JA treatment,although the time and degree of response differed. Our findings are helpful to reveal molecular function of R. glutinosa iridoid synthases and provide a clue for studing the molecular mechanism of iridoid biosynthesis.

Mesenchymal Stem Cell Migration during Bone Formation and Bone Diseases Therapy.

During bone modeling, remodeling, and bone fracture repair, mesenchymal stem cells (MSCs) differentiate into chondrocyte or osteoblast to comply bone formation and regeneration. As multipotent stem cells, MSCs were used to treat bone diseases during the past several decades. However, most of these implications just focused on promoting MSC differentiation. Furthermore, cell migration is also a key issue for bone formation and bone diseases treatment. Abnormal MSC migration could cause different kinds of bone diseases, including osteoporosis. Additionally, for bone disease treatment, the migration of endogenous or exogenous MSCs to bone injury sites is required. Recently, researchers have paid more and more attention to two critical points. One is how to apply MSC migration to bone disease therapy. The other is how to enhance MSC migration to improve the therapeutic efficacy of bone diseases. Some considerable outcomes showed that enhancing MSC migration might be a novel trick for reversing bone loss and other bone diseases, such as osteoporosis, fracture, and osteoarthritis (OA). Although plenty of challenges need to be conquered, application of endogenous and exogenous MSC migration and developing different strategies to improve therapeutic efficacy through enhancing MSC migration to target tissue might be the trend in the future for bone disease treatment.

Molecular Regulation of Catalpol and Acteoside Accumulation in Radial Striation and non-Radial Striation of Rehmannia glutinosa Tuberous Root.

Rehmannia glutinosa L., a perennial plant of Scrophulariaceae, is one of the most commonly used herbs in traditional Chinese medicine (TCM) that have been widely cultivated in China. However, to date, the biosynthetic pathway of its two quality-control components, catalpol and acteoside, are only partially elucidated and the mechanism for their tissue-specific accumulation remains unknown. To facilitate the basic understanding of the key genes and transcriptional regulators involved in the biosynthesis of catalpol and acteoside, transcriptome sequencing of radial striation (RS) and non-radial striation (nRS) from four R. glutinosa cultivars was performed. A total of 715,158,202 (~107.27 Gb) high quality reads obtained using paired-end Illumina sequencing were de novo assembled into 150,405 transcripts. Functional annotation with multiple public databases identified 155 and 223 unigenes involved in catalpol and acteoside biosynthesis, together with 325 UGTs, and important transcription factor (TF) families. Comparative analysis of the transcriptomes identified 362 unigenes, found to be differentially expressed in all RS vs. nRS comparisons, with 143 upregulated unigenes, including those encoding enzymes of the catalpol and acteoside biosynthetic pathway, such as geranyl diphosphate synthase (RgGPPS), geraniol 8-hydroxylase (RgG10H), and phenylalanine ammonia-lyase (RgPAL). Other differentially expressed unigenes predicted to be related to catalpol and acteoside biosynthesis fall into UDP-dependent glycosyltransferases (UGTs), as well as transcription factors. In addition, 16 differentially expressed genes were selectively confirmed by real-time PCR. In conclusion, a large unigene dataset of R. glutinosa generated in the current study will serve as a resource for the identification of potential candidate genes for investigation of the tuberous root development and biosynthesis of active components.

The Impact of Oxidative Stress on the Bone System in Response to the Space Special Environment.

The space special environment mainly includes microgravity, radiation, vacuum and extreme temperature, which seriously threatens an astronaut's health. Bone loss is one of the most significant alterations in mammalians after long-duration habitation in space. In this review, we summarize the crucial roles of major factors-namely radiation and microgravity-in space in oxidative stress generation in living organisms, and the inhibitory effect of oxidative stress on bone formation. We discussed the possible mechanisms of oxidative stress-induced skeletal involution, and listed some countermeasures that have therapeutic potentials for bone loss via oxidative stress antagonism. Future research for better understanding the oxidative stress caused by space environment and the development of countermeasures against oxidative damage accordingly may facilitate human beings to live more safely in space and explore deeper into the universe.

Study on Fatigue Performance of Pulsed Tungsten Inert Gas Welding Joint of Duplex Stainless Steel Thin Tube.

To solve the shortage of austenite phase precipitation caused by nitrogen loss in the welding process of UNS S2205 duplex stainless steel (DSS), shielding gas nitriding was investigated by adding different N2 contents in Ar shielding gas during the welding process. A good thin-walled pipe butt joint was formed using the pulsed tungsten inert gas (P-TIG) welding method with Ar-N2 shielding gas. High cycle fatigue tests of the weld joints were conducted to study the effect of shielding gas nitriding on the fatigue properties. Fatigue tests at three stress levels of 225 MPa, 270 MPa, and 360 MPa were carried out on the weld joints with different N2 contents, and the fatigue samples were all fractured in the high temperature heat-affected zone (H-HAZ). Within the current process parameters, the fatigue life of the 4 vol.% N2 welded joints was optimal. Fatigue striations appeared in the fatigue crack propagation zone, and the transient fracture zone was similar to the tensile fracture. Under the low-stress level, the area of the crack propagation zone under 4 vol.% N2 was the highest, the tear ridges all expanded around the crack source area, and the fatigue crack propagation zone presented a radial distribution. The proliferation and expansion of dislocations were mainly carried out in the austenite grains, and the dislocation density of the fatigue specimens under 4 vol.% N2 was smaller than that of the Ar specimens. Shielding gas nitriding effectively improved the balance of the two-phase ratio and the hardness of austenite phase, optimized the internal slip system, inhibited the proliferation of dislocations in the austenite phase, and improved the fatigue life of weld joints.

Transfer RNAs-derived small RNAs and their application potential in multiple diseases.

The role of tRNAs is best known as adapter components of translational machinery. According to the central dogma of molecular biology, DNA is transcribed to RNA and in turn is translated into proteins, in which tRNA outstands by its role of the cellular courier. Recent studies have led to the revision of the canonical function of transfer RNAs (tRNAs), which indicates that tRNAs also serve as a source for short non-coding RNAs called tRNA-derived small RNAs (tsRNAs). tsRNAs play key roles in cellular processes by modulating complicated regulatory networks beyond translation and are widely involved in multiple diseases. Herein, the biogenesis and classification of tsRNAs were firstly clarified. tsRNAs are generated from pre-tRNAs or mature tRNAs and are classified into tRNA-derived fragments (tRFs) and tRNA halves (tiRNA). The tRFs include five types according to the incision loci: tRF-1, tRF-2, tRF-3, tRF-5 and i-tRF which contain 3' tiRNA and 5' tiRNA. The functions of tsRNAs and their regulation mechanisms involved in disease processes are systematically summarized as well. The mechanisms can elaborate on the specific regulation of tsRNAs. In conclusion, the current research suggests that tsRNAs are promising targets for modulating pathological processes, such as breast cancer, ischemic stroke, respiratory syncytial virus, osteoporosis and so on, and maintain vital clinical implications in diagnosis and therapeutics of various diseases.

MACF1 promotes osteoblastic cell migration by regulating MAP1B through the GSK3beta/TCF7 pathway.

RATIONALE: The migration of osteoblastic cells to bone formation surface is an essential step for bone development and growth. However, whether the migration capacity of osteoblastic cells is compromised during osteoporosis occurrence and how it contributes to bone formation reduction remain unexplored so far. In this work, we found, as a positive regulator of cell migration, microtubule actin crosslinking factor 1 (MACF1) enhanced osteoblastic cells migration. We also examined whether MACF1 could facilitate osteoblastic cells' migration to bone formation surface to promote bone formation through another cytoskeleton protein, microtubule associated protein 1 (MAP1B). METHODS: Preosteoblast cell line MC3T3-E1 with different MACF1 level was used for in vitro and in vivo cell migration assay; Primary cortical bone derived mesenchymal stem cells (C-MSCs) from bone tissue of MACF1 conditional knock out (cKO) mice was used for in vitro cell migration assay. Cell migration ability in vitro was evaluated by wound healing assay and transwell assay and in vivo by bone marrow cavity injection. Small interfering RNA (siRNA) was used for knocking down Map1b in MC3T3-E1 cell. Lithium chloride (LiCl) and Wortmannin (Wort) were used for inhibiting/activating GSK3ß pathway activity. Luciferase report assay was performed for detection of transcriptional activity of TCF7 for Map1b; Chromatin immunoprecipitation (ChIP) was engaged for the binding of TCF7 to Map1b promoter region. RESULTS: We found MACF1 enhanced MC3T3-E1 cell and C-MSCs migration in vitro through promoting microtubule (MT) stability and dynamics, and increased the injected MC3T3-E1 cell number on bone formation surface, which indicated a promoted bone formation. We further authenticated that MAP1B had a similar function to MACF1 and was regulated by MACF1 in osteogenic cell, and silencing map1b repressed MC3T3-E1 cell migration in vitro. Mechanistically, by adopting MC3T3-E1 cell with different MACF1 level or treated with LiCl/Wort, we discovered that MACF1 decreased the levels of 1265 threonine phosphorylated MAP1B (p[T1265] MAP1B) through inhibiting GSK3ß activity. Additionally, total MAP1B mRNA expression level was upregulated by MACF1 through strengthening the binding of TCF7 to the map1b promoter sequence. CONCLUSION: Our study uncovered a novel role of MACF1 in bone formation and MAP1B regulation, which suggested that MACF1 could be a potential therapeutic target for osteoporosis.

MiR-138-5p Targets MACF1 to Aggravate Aging-related Bone Loss.

Senile osteoporosis is one of the major health problems in an aging society. Decreased bone formation due to osteoblast dysfunction may be one of the causes of aging-related bone loss. With increasing evidence suggesting that multiple microRNAs (miRNAs) play important roles in osteoblast function, the relationship between miRNAs and senile osteoporosis has become a popular research topic. Previously, we confirmed that mechanoresponsive miR-138-5p negatively regulated bone anabolic action. In this study, the miR-138-5p level was found to be negatively correlated with BMD and osteogenic markers in bone specimens of senile osteoporotic patients by bioinformatic analysis and experimental verification. Furthermore, high miR-138-5p levels aggravated the decrease of aged osteoblast differentiation in vitro and led to worse bone loss in aged osteoblastic miR-138-5p transgenic mice in vivo. We also previously identified that the target of miR-138-5p, microtubule actin cross-linking factor 1 (MACF1), could attenuate senile osteoporosis. Here, miR-138-5p was demonstrated to regulate aged osteoblast differentiation by targeting MACF1. Finally, the therapeutic inhibition of miR-138-5p counteracted the decrease in bone formation and aging-related bone loss in aged mice. Overall, our results highlight the crucial roles and the molecular mechanism of miR-138-5p in aging-related bone loss and may provide a powerful therapeutic target for ameliorating senile osteoporosis.

Polyvinylamine with moderate binding affinity as a highly effective vehicle for RNA delivery.

Polymeric carriers for RNA therapy offer potential advantages in terms of low immunogenicity, promoting modifiability and accelerating intracellular transport. However, balancing high transfection efficacy with low toxicity remains challenging with polymer-based vehicles; indeed, polyethyleneimine (PEI) remains the "gold standard" polymer for this purpose despite its significant toxicity limitations. Herein, we demonstrate the potential of polyvinylamine (PVAm), a commodity high-charge cationic polymer used in the papermaking industry and has similar structure with PEI, as an alternative carrier for RNA delivery. High levels of transfection of normal, tumor, and stem cells with a variety of RNA cargoes including small interfering RNA (siRNA), microRNA (miRNA), and recombinant RNA can be achieved in vitro under the proper complex conditions. While, both the anti-tumor effect achieved in a xenograft osteosarcoma model and lipid-lowering activity observed in a hyperlipidemia mice indicate the potential for highly effective in vivo activity. Of note, both the transfection efficiency and the cytotoxicity of PVAm compare more favorably with those of PEI, with PVAm offering the additional advantages of simpler purification and significantly lower cost. In addition, the mechanism for the difference in transfection efficiency between PVAm and PEI is explored by molecular docking as well as analyzing the process of association and dissociation between polymers (PVAm and PEI) and nucleic acids. Our research provides a novel, non-toxic, and cost-effective carrier candidate for next generation RNA therapy, and elucidates the potential mechanism of PVAm for its efficient delivery of RNA.

Targeting long noncoding RNA PMIF facilitates osteoprogenitor cells migrating to bone formation surface to promote bone formation during aging.

Rationale: The migration of mesenchymal osteoprogenitor cells (OPCs) to bone formation surface is the initial step of osteoblastogenesis before they undergo osteoblast differentiation and maturation for governing bone formation. However, whether the migration capacity of OPCs is compromised during aging and how it contributes to the aging-related bone formation reduction remain unexplored. In the present study, we identified a migration inhibitory factor (i.e., long noncoding RNA PMIF) and examined whether targeting lnc-PMIF could facilitate osteoprogenitor cells migrating to bone formation surface to promote bone formation during aging. Methods: Primary OPCs from young (6-momth-old) and aged (18-momth-old) C57BL/6 mice and stable lnc-PMIF knockdown/overexpression cell lines were used for in vitro and in vivo cell migration assay (i.e., wound healing assay, transwell assay and cell intratibial injection assay). RNA pulldown-MS/WB and RIP-qPCR were performed to identify the RNA binding proteins (RBPs) of lnc-PMIF. Truncations of lnc-PMIF and the identified RBP were engaged to determine the interaction motif between them by RNA pulldown-WB and EMSA. By cell-based therapy approach and by pharmacological approach, small interfering RNA (siRNA)-mediated lnc-PMIF knockdown were used in aged mice. The cell migration ability was evaluated by transwell assay and cell intratibial injection assay. The bone formation was evaluated by microCT analysis and bone morphometry analysis. Results: We reported that the decreased bone formation was accompanied by the reduced migration capacity of the bone marrow mesenchymal stem cells (BMSCs, the unique source of OPCs in bone marrow) in aged mice. We further identified that the long non-coding RNA PMIF (postulated migration inhibitory factor) (i.e., lnc-PMIF) was highly expressed in BMSCs from aged mice and responsible for the reduced migration capacity of aged OPCs to bone formation surface. Mechanistically, we found that lnc-PMIF could bind to human antigen R (HuR) for interrupting the HuR-ß-actin mRNA interaction, therefore inhibit the expression of ß-actin for suppressing the migration of aged OPCs. We also authenticated a functionally conserved human lncRNA ortholog of the murine lnc-PMIF. By cell-based therapy approach, we demonstrated that replenishing the aged BMSCs with small interfering RNA (siRNA)-mediated lnc-PMIF knockdown could promote bone formation in aged mice. By pharmacological approach, we showed that targeted delivery of lnc-PMIF siRNA approaching the OPCs around the bone formation surface could also promote bone formation in aged mice. Conclusion: Toward translational medicine, this study hints that targeting lnc-PMIF to facilitate aged OPCs migrating to bone formation surface could be a brand-new anabolic strategy for aging-related osteoporosis.

MACF1 promotes preosteoblast migration by mediating focal adhesion turnover through EB1.

Microtubule actin crosslinking factor 1 (MACF1) is a widely expressed cytoskeletal linker and plays an essential role in various cells' functions by mediating cytoskeleton organization and dynamics. However, the role of MACF1 on preosteoblast migration is not clear. Here, by using MACF1 knockdown and overexpressed MC3T3-E1 cells, we found MACF1 positively regulated preosteoblast migration induced by cell polarization. Furthermore, immunofluorescent staining showed that MACF1 increased end-binding protein (EB1) distribution on microtubule (MT), and decreased EB1 distribution on focal adhesion (FA) complex. Moreover, upregulation of MACF1 activated Src level and enhanced the colocalization of EB1 with activated Src. In addition, MACF1 diminished colocalization of EB1 with adenomatous polyposis coli (APC), which induced EB1 release from FA and promoted FA turnover. These results indicated an important role and mechanism of MACF1 in regulating preosteoblast migration through promoting FA turnover by mediating EB1 colocalization with Src and APC, which inferred that MACF1 might be a potential target for preventing and treating bone disorders.

miR-129-5p Inhibits Bone Formation Through TCF4.

Osteoporosis is a frequently occurring bone disease in middle-aged and aged men and women. However, current therapies on this disease are still not ideal. MicroRNAs (miRNAs) are a class of endogenous non-protein-coding RNA with a length of 18-25 nucleotides. miRNAs have been identified as important regulators for development, metabolism, carcinogenesis, and bone formation. miR-129-5p has been reported as a regulator of cancer and neuroscience, whereas studies about its function on bone formation is still limited. In this study, we investigated the function and mechanism of miR-129-5p on osteoblast differentiation and bone formation. We have assessed the expression of miRNAs in bone mesenchymal stem cells from aging and menopause osteoporosis C57BL6 mice. The expression of miR-129-5p was altered in all osteoporosis models. Besides, the expression of miR-129-5p was negatively correlated with osteoblastic differentiation markers in the femur tissues of C57BL/6 mice of different ages. We further demonstrated that overexpression of miR-129-5p inhibited osteoblast differentiation in MC3T3-E1 cell line, as well as bone formation of C57BL/6 mice. On the other hand, down-regulation of miR-129-5p enhanced osteoblast differentiation and bone formation. We also found that miR-129-5p inhibited Wnt/ß-catenin pathway in osteoblast. The target gene of miR-129-5p has been forecasted and proved as Tcf4. We further found that plasmid containing Tcf4-3' UTR sequence enhanced osteoblast differentiation, as well as Wnt/ß-catenin pathway in MC3T3-E1 cells. To further investigate the rescue effect of miR-129-5p inhibitor, we manufactured bioengineered novel recombinant miR-129-5p inhibitor through Escherichia coli system and then tested its function. The results showed that the novel recombinant miR-129-5p inhibitor promoted osteoblast differentiation and greatly ameliorated menopause osteoporosis in C57BL6 mice. In conclusion, we have discovered miR-129-5p as an inhibitor of bone formation. miR-129-5p inhibited downstream transcription factors of Wnt/ß-catenin pathway through targeting Tcf4. Moreover, novel recombinant miR-129-5p inhibitor showed rescue effect on osteoporosis. This study has revealed a new mechanism of osteogenic differentiation and provided novel therapeutic strategies for treatment of skeletal disorders.

LncRNA, Important Player in Bone Development and Disease.

BACKGROUND: Bone is an important tissue and its normal function requires tight coordination of transcriptional networks and signaling pathways, and many of these networks/ pathways are dysregulated in pathological conditions affecting cartilage and bones. Long non-coding RNA (lncRNA) refers to a class of RNAs with a length of more than 200 nucleotides, lack of protein-coding potential, and exhibiting a wide range of biological functions. Although studies on lcnRNAs are still in their infancy, they have emerged as critical players in bone biology and bone diseases. The functions and exact mechanism of bone-related lncRNAs have not been fully classified yet. OBJECTIVE: The objective of this article is to summarize the current literature on lncRNAs on the basis of their role in bone biology and diseases, focusing on their emerging molecular mechanism, pathological implications and therapeutic potential. DISCUSSION: A number of lncRNAs have been identified and shown to play important roles in multiple bone cells and bone disease. The function and mechanism of bone-related lncRNA remain to be elucidated. CONCLUSION: At present, majority of knowledge is limited to cellular levels and less is known on how lncRNAs could potentially control the development and homeostasis of bone. In the present review, we highlight some lncRNAs in the field of bone biology and bone disease. We also delineate some lncRNAs that might have deep impacts on understanding bone diseases and providing new therapeutic strategies to treat these diseases.

Circulating miR-181c-5p and miR-497-5p Are Potential Biomarkers for Prognosis and Diagnosis of Osteoporosis.

CONTEXT: Osteoporosis is a degenerative bone disease in aging men and women. MiRNAs associated with progressive bone loss in osteoporosis had not been clearly demonstrated. OBJECTIVE: The evaluation of the differentially expressed miRNAs in the bone tissue and serum of osteoporotic women with aging. METHODS: MiRNAs GeneChip and real-time PCR were used to screen differently expressed miRNAs in bone tissues of 21 osteoporotic women ages 60-69 years and 80-89 years. Identified miRNAs were detected in the serum of the validation cohort, which consisted of 14 healthy premenopausal women and 86 postmenopausal women with osteopenia or osteoporosis. MiR-181c-5p and miR-497-5p expression were validated in aging and OVX mice models, and osteoblasts. Their role in osteogenesis was validated in vitro. RESULTS: Twenty-four miRNAs showed the highest differential expression in bone tissues of osteoporotic women in initial screening. Among them, four miRNAs were identified both in the bone tissue and serum in the validation cohort. The levels of miR-181c-5p and miR-497-5p were decreased in the serum of postmenopausal women with osteopenia or osteoporosis, but increased in subjects treated with bisphosphonate plus calcitriol. MiR-181c-5p and miR-497-5p were significantly downregulated in the bone tissue of aging and OVX mice models, and upregulated during the osteogenic differentiation of hFOB1.19 and MC3T3-E1 cells. Overexpression of miR-181c-5p and miR-497-5p promoted the differentiation and mineralization of osteoblasts. CONCLUSIONS: MiR-181c-5p and miR-497-5p are involved in bone metabolism and associated with progressive bone loss of due to osteoporosis, suggesting that circulating miR-181c-5p and miR-497-5p might act as potential biomarkers for monitoring the effects of antiosteoporotic therapies or the diagnostic approach.

Silencing of miR-138-5p sensitizes bone anabolic action to mechanical stimuli.

Emerging evidence is revealing that microRNAs (miRNAs) play essential roles in mechanosensing for regulating osteogenesis. However, no mechanoresponsive miRNAs have been identified in human bone specimens. Methods: Bedridden and aged patients, hindlimb unloaded and aged mice, and Random Positioning Machine and primary aged osteoblasts were adopted to simulate mechanical unloading conditions at the human, animal and cellular levels, respectively. Treadmill exercise and Flexcell cyclic mechanical stretching were used to simulate mechanical loading in vivo and in vitro, respectively. Results: Here, we found increased miR-138-5p levels with a lower degree of bone formation in bone specimens from bedridden and aged patients. Loss- and gain-of-function studies showed that miR-138-5p directly targeted microtubule actin crosslinking factor 1 (MACF1) to inhibit osteoblast differentiation under different mechanical conditions. Regarding translational medicine, bone-targeted inhibition of miR-138-5p attenuated the decrease in the mechanical bone anabolic response in hindlimb unloaded mice. Moreover, bone-targeted inhibition of miR-138-5p sensitized the bone anabolic response to mechanical loading in both miR-138-5p transgenic mice and aged mice to promote bone formation. Conclusion: These data suggest that miR-138-5p as a mechanoresponsive miRNA accounts for the mechanosensitivity of the bone anabolic response and that inhibition of miR-138-5p in osteoblasts may be a novel bone anabolic sensitization strategy for ameliorating disuse or senile osteoporosis.