Inhibition of SIRT1 by miR-138-5p provides a mechanism for inhibiting osteoblast proliferation and promoting apoptosis under simulated microgravity.

Microgravity can inhibit osteoblast proliferation and promote apoptosis, which is related to a reduction in mechanical stress on the bones and results in disuse osteoporosis, but the detailed mechanism is still unclear. In this study, we first demonstrated that miR-138-5p was upregulated, inhibited osteoblast proliferation and induced osteoblast apoptosis under simulated microgravity. Moreover, miR-138-5p silencing partially mitigated the effects of proliferation and apoptosis of MC3T3-E1 cells. Our study further showed that sirtuin 1 (SIRT1) was downregulated and negatively correlated with the expression of miR-138-5p under simulated microgravity, which indicated that miR-138-5p inhibited osteoblast proliferation and promoted osteoblast apoptosis by targeting SIRT1. Thus, the miR-138-5p/SIRT1 pathway should be considered for preventative treatment of disuse osteoporosis.

Histone methyltransferase Setdb1 mediates osteogenic differentiation by suppressing the expression of miR-212-3p under mechanical unloading.

Emerging evidence indicates that multiple mechanisms are involved in bone loss induced by mechanical unloading. Thus far, few study has established the pathophysiological role of histone modification for osteogenic differentiation under mechanical unloading. Here we demonstrated that the histone H3 lysine 9 (H3K9) methyltransferase Setdb1, which was sensitive to mechanical unloading, was increased during osteogenic differentiation of MC3T3-E1 cells for the first time. Knockdown of Setdb1 significantly blocked osteoblast function in vivo and in vitro. Through bioinformatics analysis of candidate miRNAs regulated by H3K9me3, we further identified that Setdb1 inhibited the expression of miR-212-3p by regulating the formation of H3K9me3 in the promoter region. Mechanically, we revealed that miR-212-3p was upregulated under mechanical unloading and suppressed osteogenic differentiation by directly downregulating High mobility group box 1 protein (Hmgb1) expression. Furthermore, we verified the molecular mechanism of the SETDB1/miR-212-3p/HMGB1 pathway in hFOB cells under mechanical unloading. In summary, these data demonstrate the essential function of the Setdb1/miR-212-3p/Hmgb1 pathway in osteogenic differentiation under mechanical unloading, and present a potential protective strategies against bone loss induced by mechanical unloading.

Exosomes from Microvascular Endothelial Cells under Mechanical Unloading Inhibit Osteogenic Differentiation via miR-92b-3p/ELK4 Axis.

Mechanical unloading-related bone loss adversely harms astronauts' health. Nevertheless, the specific molecular basis underlying the phenomenon has not been completely elucidated. Although the bone microvasculature contributes significantly to bone homeostasis, the pathophysiological role of microvascular endothelial cells (MVECs) in bone loss induced by mechanical unloading is not apparent. Here, we discovered that MC3T3-E1 cells could take up exosomes produced by MVECs under clinorotation-unloading conditions (Clino Exos), which then prevented MC3T3-E1 cells from differentiating into mature osteoblasts. Moreover, miR-92b-3p was found to be highly expressed in both unloaded MVECs and derived exosomes. Further experiments demonstrated that miR-92b-3p was transferred into MC3T3-E1 cells by exosomes, resulting in the suppression of osteogenic differentiation, and that encapsulating miR-92b-3p inhibitor into the Clino Exos blocked their inhibitory effects. Furthermore, miR-92b-3p targeted ELK4 and the expression of ELK4 was lessened when cocultured with Clino Exos. The inhibitor-92b-3p-promoted osteoblast differentiation was partially reduced by siRNA-ELK4. Exosomal miR-92b-3p secreted from MVECs under mechanical unloading has been shown for the first time to partially attenuate the function of osteoblasts through downregulation of ELK4, suggesting a potential strategy to protect against the mechanical unloading-induced bone loss and disuse osteoporosis.

Bioinformatic analysis of the RNA expression patterns in microgravity-induced bone loss.

Researchers have linked microgravity in space to the significant imbalance between bone formation and bone resorption that induces persistent bone loss in load-bearing bones. However, the underlying molecular mechanisms are still unclear, which hinders the development of therapeutic measures. The aim of this study was to identify hub genes and explore novel molecular mechanisms underlying microgravity-induced bone loss using transcriptome datasets obtained from the GEO and SRA databases. In summary, comparative RNA expression pattern studies that differ in species (Homo or Mus), models (in vitro or in vivo), microgravity conditions (real microgravity or ground-based simulators) and microgravity duration showed that it is difficult to reach a consistent conclusion about the pathogenesis of microgravity-induced bone loss across these studies. Even so, we identified 11 hub genes and some miRNA-mRNA interactions mainly based on the GSE100930 dataset. Also, the expression of CCL2, ICAM1, IGF1, miR-101-3p and miR-451a markedly changed under clinorotation-microgravity condition. Remarkedly, ICAM1 and miR-451a were key mediators of the osteogenesis of hMSCs under clinorotation-microgravity condition. These findings provide novel insights into the molecular mechanisms of bone loss during microgravity and could indicate potential targets for further countermeasures against this condition.

Innovative immune mechanisms and antioxidative therapies of intervertebral disc degeneration.

Intervertebral disc degeneration (IDD) is the basic pathological process of many degenerative diseases of the spine, characterized by series of symptoms, among which low back pain (LBP) is the most common symptom that patients suffer a lot, which not only makes patients and individual families bear a huge pain and psychological burden, but also consumes a lot of medical resources. IDD is usually thought to be relevant with various factors such as genetic predisposition, trauma and aging, and IDD progression is tightly relevant with structural and functional alterations. IDD processes are caused by series of pathological processes, including oxidative stress, matrix decomposition, inflammatory reaction, apoptosis, abnormal proliferation, cell senescence, autophagy as well as sepsis process, among which the oxidative stress and inflammatory response are considered as key link in IDD. The production and clearance of ROS are tightly connected with oxidative stress, which would further simulate various signaling pathways. The phenotype of disc cells could change from matrix anabolism-to matrix catabolism- and proinflammatory-phenotype during IDD. Recent decades, with the relevant reports about oxidative stress and inflammatory response in IDD increasing gradually, the mechanisms researches have attracted much more attention. Consequently, this study focused on the indispensable roles of the oxidative stress and inflammatory response (especially macrophages and cytokines) to illustrate the origin, development, and deterioration of IDD, aiming to provide novel insights in the molecular mechanisms as well as significant clinical values for IDD.

Methyltransferase Setdb1 Promotes Osteoblast Proliferation by Epigenetically Silencing Macrod2 with the Assistance of Atf7ip.

Bone loss caused by mechanical unloading is a threat to prolonged space flight and human health. Epigenetic modifications play a crucial role in varied biological processes, but the mechanism of histone modification on unloading-induced bone loss has rarely been studied. Here, we discovered for the first time that the methyltransferase Setdb1 was downregulated under the mechanical unloading both in vitro and in vivo so as to attenuate osteoblast proliferation. Furthermore, we found these interesting processes depended on the repression of Macrod2 expression triggered by Setdb1 catalyzing the formation of H3K9me3 in the promoter region. Mechanically, we revealed that Macrod2 was upregulated under mechanical unloading and suppressed osteoblast proliferation through the GSK-3ß/ß-catenin signaling pathway. Moreover, Atf7ip cooperatively contributed to osteoblast proliferation by changing the localization of Setdb1 under mechanical loading. In summary, this research elucidated the role of the Atf7ip/Setdb1/Macrod2 axis in osteoblast proliferation under mechanical unloading for the first time, which can be a potential protective strategy against unloading-induced bone loss.

Circulating Exosomes from Mice with LPS-Induced Bone Loss Inhibit Osteoblast Differentiation.

Osteoimmunology focuses on the intermodulation between bone and the immune system. Lipopolysaccharide (LPS)-induced bone loss models are commonly used to investigate the interface between inflammation and osteoporosis. Circulating exosomes can regulate physiological and pathological processes through exosomal microRNAs and proteins. In this study, we observed reduced osteoblast number and bone formation in LPS-induced bone loss mice (LPS mice). Levels of circulating exosomes were increased by ~ twofold in LPS mice, and the expression of exosomal miRNAs was significantly changed. miRNAs (miRNA-125b-5p, miRNA-132-3p, and miRNA-214-3p) that were reported to inhibit osteoblast activity were significantly increased in the serum exosomes and bone tissues of LPS mice. Additionally, LPS-induced increases in exosomes significantly inhibited the osteogenic differentiation of MC3T3-E1 cells.

HDAC6 Negatively Regulates miR-155-5p Expression to Elicit Proliferation by Targeting RHEB in Microvascular Endothelial Cells under Mechanical Unloading.

Mechanical unloading contributes to significant cardiovascular deconditioning. Endothelial dysfunction in the sites of microcirculation may be one of the causes of the cardiovascular degeneration induced by unloading, but the detailed mechanism is still unclear. Here, we first demonstrated that mechanical unloading inhibited brain microvascular endothelial cell proliferation and downregulated histone deacetylase 6 (HDAC6) expression. Furthermore, HDAC6 promoted microvascular endothelial cell proliferation and attenuated the inhibition of proliferation caused by clinorotation unloading. To comprehensively identify microRNAs (miRNAs) that are regulated by HDAC6, we analyzed differential miRNA expression in microvascular endothelial cells after transfection with HDAC6 siRNA and selected miR-155-5p, which was the miRNA with the most significantly increased expression. The ectopic expression of miR-155-5p inhibited microvascular endothelial cell proliferation and directly downregulated Ras homolog enriched in brain (RHEB) expression. Moreover, RHEB expression was downregulated under mechanical unloading and was essential for the miR-155-5p-mediated promotion of microvascular endothelial cell proliferation. Taken together, these results are the first to elucidate the role of HDAC6 in unloading-induced cell growth inhibition through the miR-155-5p/RHEB axis, suggesting that the HDAC6/miR-155-5p/RHEB pathway is a specific target for the preventative treatment of cardiovascular deconditioning.

The small protein MafG plays a critical role in MC3T3-E1 cell apoptosis induced by simulated microgravity and radiation.

Microgravity and radiation exposure-induced bone damage is one of the most significant alterations in astronauts after long-term spaceflight. However, the underlying mechanism is still largely unknown. Recent ground-based simulation studies have suggested that this impairment is likely mediated by increased production of reactive oxygen species (ROS) during spaceflight. The small Maf protein MafG is a basic-region leucine zipper-type transcription factor, and it globally contributes to regulation of antioxidant and metabolic networks. Our research investigated the role of MafG in the process of apoptosis induced by simulated microgravity and radiation in MC3T3-E1 cells. We found that simulated microgravity or radiation alone decreased MafG expression and elevated apoptosis in MC3T3-E1 cells, and combined simulated microgravity and radiation treatment aggravated apoptosis. Meanwhile, under normal conditions, increased ROS levels facilitated apoptosis and downregulated the expression of MafG in MC3T3-E1 cells. Overexpression of MafG decreased apoptosis induced by simulated microgravity and radiation. These findings provide new insight into the mechanism of bone damage induced by microgravity and radiation during space flight.

The combined effects of simulated microgravity and X-ray radiation on MC3T3-E1 cells and rat femurs.

Microgravity is well-known to induce Osteopenia. However, the combined effects of microgravity and radiation that commonly exist in space have not been broadly elucidated. This research investigates the combined effects on MC3T3-E1 cells and rat femurs. In MC3T3-E1 cells, simulated microgravity and X-ray radiation, alone or combination, show decreased cell activity, increased apoptosis rates by flow cytometric analysis, and decreased Runx2 and increased Caspase-3 mRNA and protein expressions. In rat femurs, simulated microgravity and X-ray radiation, alone or combination, show increased bone loss by micro-CT test and Masson staining, decreased serum BALP levels and Runx2 mRNA expressions, and increased serum CTX-1 levels and Caspase-3 mRNA expressions. The strongest effect is observed in the combined group in MC3T3-E1 cells and rat femurs. These findings suggest that the combination of microgravity and radiation exacerbates the effects of either treatment alone on MC3T3-E1 cells and rat femurs.

MiR-103-3p targets the m6 A methyltransferase METTL14 to inhibit osteoblastic bone formation.

Impaired osteoblast function is involved in osteoporosis, and microRNA (miRNA) dysregulation may cause abnormal osteoblast osteogenic activity. However, the influence of miRNA on osteoblast activity and the underlying mechanisms remain elusive. In this study, miR-103-3p was found to be negatively correlated with bone formation in bone specimens from elderly women with fractures and ovariectomized (OVX) mice. Additionally, miR-103-3p directly targeted Mettl14 to inhibit osteoblast activity, and METTL14-dependent N6 -methyladenosine (m6 A) methylation inhibited miR-103-3p processing by the microprocessor protein DGCR8 and promoted osteoblast activity. Moreover, miR-103-3p inhibited bone formation in vivo, and therapeutic inhibition of miR-103-3p counteracted the decreased bone formation in OVX mice. Further, METTL14 was negatively correlated with miR-103-3p but positively correlated with bone formation in bone specimens from elderly women with fractures and OVX mice. Collectively, our results highlight the critical roles of the miR-103-3p/METTL14/m6 A signaling axis in osteoblast activity, identifying this axis as a potential target for ameliorating osteoporosis.

Bone-targeted lncRNA OGRU alleviates unloading-induced bone loss via miR-320-3p/Hoxa10 axis.

Unloading-induced bone loss is a threat to human health and can eventually result in osteoporotic fractures. Although the underlying molecular mechanism of unloading-induced bone loss has been broadly elucidated, the pathophysiological role of long noncoding RNAs (lncRNAs) in this process is unknown. Here, we identified a novel lncRNA, OGRU, a 1816-nucleotide transcript with significantly decreased levels in bone specimens from hindlimb-unloaded mice and in MC3T3-E1 cells under clinorotation-unloading conditions. OGRU overexpression promoted osteoblast activity and matrix mineralization under normal loading conditions, and attenuated the suppression of MC3T3-E1 cell differentiation induced by clinorotation unloading. Furthermore, this study found that supplementation of pcDNA3.1(+)-OGRU via (DSS)6-liposome delivery to the bone-formation surfaces of hindlimb-unloaded (HLU) mice partially alleviated unloading-induced bone loss. Mechanistic investigations demonstrated that OGRU functions as a competing endogenous RNA (ceRNA) to facilitate the protein expression of Hoxa10 by competitively binding miR-320-3p and subsequently promote osteoblast differentiation and bone formation. Taken together, the results of our study provide the first clarification of the role of lncRNA OGRU in unloading-induced bone loss through the miR-320-3p/Hoxa10 axis, suggesting an efficient anabolic strategy for osteoporosis treatment.

Targeted overexpression of the long noncoding RNA ODSM can regulate osteoblast function in vitro and in vivo.

Ameliorating bone loss caused by mechanical unloading is a substantial clinical challenge, and the role of noncoding RNAs in this process has attracted increasing attention. In this study, we found that the long noncoding RNA osteoblast differentiation-related lncRNA under simulated microgravity (lncRNA ODSM) could inhibit osteoblast apoptosis and promote osteoblast mineralization in vitro. The increased expression level of the lncRNA ODSM partially reduced apoptosis and promoted differentiation in MC3T3-E1 cells under microgravity unloading conditions, and the effect was partially dependent on miR-139-3p. LncRNA ODSM supplementation in hindlimb-unloaded mice caused a decrease in the number of apoptotic cells in bone tissue and an increase in osteoblast activity. Furthermore, targeted overexpression of the lncRNA ODSM in osteoblasts partially reversed bone loss induced by mechanical unloading at the microstructural and biomechanical levels. These findings are the first to suggest the potential value of the lncRNA ODSM in osteoporosis therapy and the treatment of pathological osteopenia.

Targeted silencing of miRNA-132-3p expression rescues disuse osteopenia by promoting mesenchymal stem cell osteogenic differentiation and osteogenesis in mice.

BACKGROUND: Skeletal unloading can induce severe disuse osteopenia that often occurs in spaceflight astronauts or in patients subjected to prolonged bed-rest or immobility. Previously, we revealed a mechano-sensitive factor, miRNA-132-3p, that is closely related to the osteoblast function. The aim of this study was to investigate whether miRNA-132-3p could be an effective target for treating disuse osteopenia. METHODS: The 2D-clinostat device and the hindlimb-unloaded (HU) model were used to copy the mechanical unloading condition at the cellular and animal levels, respectively. Mimics or inhibitors of miRNA-132-3p were used to interfere with the expression of miRNA-132-3p in bone marrow-derived mesenchymal stem cells (BMSCs) in vitro for analyzing the effects on osteogenic differentiation. The special in vivo antagonists of miRNA-132-3p was delivered to the bone formation regions of HU mice for treating disuse osteopenia by a bone-targeted (AspSerSer)6-cationic liposome system. The bone mass, microstructure, and strength of the hindlimb bone tissue were analyzed for evaluating the therapeutic effect in vivo. RESULTS: miRNA-132-3p expression was declined under normal conditions and increased under gravitational mechanical unloading conditions during osteogenic differentiation of BMSCs in vitro. The upregulation of miRNA-132-3p expression resulted in the inhibition of osteogenic differentiation, whereas the downregulation of miRNA-132-3p expression enhanced osteogenic differentiation. The inhibition of miRNA-132-3p expression was able to attenuate the negative effects of mechanical unloading on BMSC osteogenic differentiation. Most importantly, the targeted silencing of miRNA-132-3p expression in the bone tissues could effectively preserve bone mass, microstructure, and strength by promoting osteogenic differentiation and osteogenesis in HU mice. CONCLUSION: The overexpression of miRNA-132-3p induced by mechanical unloading is disadvantageous for BMSC osteogenic differentiation and osteogenesis. Targeted silencing of miRNA-132-3p expression presents a potential therapeutic target for the prevention and treatment of disuse osteoporosis.

MiR-30 family members inhibit osteoblast differentiation by suppressing Runx2 under unloading conditions in MC3T3-E1 cells.

Disuse osteoporosis is common in prolonged therapeutic bed rest, space flight and immobilization due to limb fracture, which is related to reduction of mechanical stress on bone. Mechanical unloading can inhibit the differentiation of osteoblasts, but the detailed mechanism is still unclear. Runt-related transcription factor-2 (Runx2), is an important transcription factor, which plays a crucial role in disuse osteoporosis induced by unloading conditions. In this study, we found that Runx2-targeting mechano-sensitive miR-30 family members, miR-30b, miR-30c, miR-30d and miR-30e increased significantly, and were negatively correlated with the expression of Runx2 under unloading condition. Further studies found that the four miRNAs inhibited the expression of Runx2 and osteoblast differentiation under normal loading, and the knockdown of these miRNAs attenuated partly the inhibition of osteoblast differentiation induced by unloading condition in MC3T3-E1 cells. This study is the first to report miR-30 family members can regulate partly the dysfunction of osteoblasts under unloading condition, which is expected to be targets for the treatment of disuse osteoporosis.

miR-181c-5p mediates simulated microgravity-induced impaired osteoblast proliferation by promoting cell cycle arrested in the G2 phase.

Impaired osteoblast proliferation plays fundamental roles in microgravity-induced bone loss, and cell cycle imbalance may result in abnormal osteoblast proliferation. However, whether microgravity exerts an influence on the cell cycle in osteoblasts or what mechanisms may underlie such an effect remains to be fully elucidated. Herein, we confirmed that simulated microgravity inhibits osteoblast proliferation. Then, we investigated the effect of mechanical unloading on the osteoblast cell cycle and found that simulated microgravity arrested the osteoblast cell cycle in the G2 phase. In addition, our data showed that cell cycle arrest in osteoblasts from simulated microgravity was mainly because of decreased cyclin B1 expression. Furthermore, miR-181c-5p directly inhibited cyclin B1 protein translation by binding to a target site in the 3'UTR. Lastly, we demonstrated that inhibition of miR-181c-5p partially counteracted cell cycle arrest and decreased the osteoblast proliferation induced by simulated microgravity. In conclusion, our study demonstrates that simulated microgravity inhibits cell proliferation and induces cell cycle arrest in the G2 phase in primary mouse osteoblasts partially through the miR-181c-5p/cyclin B1 pathway. This work may provide a novel mechanism of microgravity-induced detrimental effects on osteoblasts and offer a new avenue to further investigate bone loss induced by mechanical unloading.

Simulated microgravity reduces intracellular-free calcium concentration by inhibiting calcium channels in primary mouse osteoblasts.

Calcium homeostasis in osteoblasts plays fundamental roles in the physiology and pathology of bone tissue. Various types of mechanical stimuli promote osteogenesis and increase bone formation elicit increases in intracellular-free calcium concentration in osteoblasts. However, whether microgravity, a condition of mechanical unloading, exerts an influence on intracellular-free calcium concentration in osteoblasts or what mechanisms may underlie such an effect are unclear. Herein, we show that simulated microgravity reduces intracellular-free calcium concentration in primary mouse osteoblasts. In addition, simulated microgravity substantially suppresses the activities of L-type voltage-sensitive calcium channels, which selectively allow calcium to cross the plasma membrane from the extracellular space. Moreover, the functional expression of ryanodine receptors and inositol 1,4,5-trisphosphate receptors, which mediate the release of calcium from intracellular storage, decreased under simulated microgravity conditions. These results suggest that simulated microgravity substantially reduces intracellular-free calcium concentration through inhibition of calcium channels in primary mouse osteoblasts. Our study may provide a novel mechanism for microgravity-induced detrimental effects in osteoblasts, offering a new avenue to further investigate bone loss induced by mechanical unloading.

MicroRNA-139-3p regulates osteoblast differentiation and apoptosis by targeting ELK1 and interacting with long noncoding RNA ODSM.

Recent studies have confirmed that microRNAs and lncRNAs can affect bone cell differentiation and bone formation. In this study, miR-139-3p was upregulated in the femurs of hindlimb unloading mice and MC3T3-E1 cells under simulated microgravity; this effect was related to osteoblast differentiation and apoptosis. Silencing miR-139-3p attenuated the suppression of differentiation and the promotion of MC3T3-E1 cell apoptosis induced by simulated microgravity. ELK1 is a target of miR-139-3p and is essential for miR-139-3p to regulate osteoblast differentiation and apoptosis. An osteoblast differentiation-related lncRNA that could interact with miR-139-3p (lncRNA ODSM) was identified in MC3T3-E1 cells under simulated microgravity. Further investigations demonstrated that lncRNA ODSM could promote MC3T3-E1 cell differentiation. Therefore, this research was the first to reveal the critical role of the lncRNA ODSM/miR-139-3p/ELK1 pathway in osteoblasts, and these findings suggest the potential value of miR-139-3p in osteoporosis diagnosis and therapy.

Osteoblast-targeted delivery of miR-33-5p attenuates osteopenia development induced by mechanical unloading in mice.

A growing body of evidence has revealed that microRNAs (miRNAs) play crucial roles in regulating osteoblasts and bone metabolism. However, the effects of miRNAs in osteoblast mechanotransduction remain to be defined. In this study, we investigated the regulatory effect of miR-33-5p in osteoblasts and tested its anti-osteopenia effect when delivered by an osteoblast-targeting delivery system in vivo. First, we demonstrated that miR-33-5p could promote the activity and mineralization of osteoblasts without influencing their proliferation in vitro. Then our data showed that supplementing miR-33-5p in osteoblasts by a targeted delivery system partially recovered the osteopenia induced by mechanical unloading at the biochemical, microstructural, and biomechanical levels. In summary, our findings demonstrate that miR-33-5p is a key factor in the occurrence and development of the osteopenia induced by mechanical unloading. In addition, targeted delivery of the mimics of miR-33-5p is a promising new strategy for the treatment of pathological osteopenia.

Genome­wide analysis and prediction of functional long noncoding RNAs in osteoblast differentiation under simulated microgravity.

Long noncoding RNAs (lncRNAs) have been regarded as important regulators in numerous biological processes during cell development. However, the holistic lncRNA expression pattern and potential functions during osteoblast differentiation under simulated microgravity remain unknown. In the present study, a high throughput microarray assay was performed to detect lncRNA and mRNA expression profiles during MC3TC­E1 pre­osteoblast cell osteo­differentiation under simulated microgravity. The expression of 857 lncRNAs and 2,264 mRNAs was signi-ficantly altered when MC3T3­E1 cells were exposed to simulated microgravity. A relatively consistent distribution pattern on the chromosome and a co­expression network were observed between the differentially­expressed lncRNAs and mRNAs. Genomic context analysis further identified 132 differentially­expressed lncRNAs and nearby coding gene pairs. Subsequently, 3 lncRNAs were screened out for their possible function in osteoblast differentiation, based on their co­expression association and potential cis­acting regulatory pattern with the deregulated mRNAs. The present study aimed to provide a comprehensive understanding of and a foundation for future studies into lncRNA function in mechanical signal­mediated osteoblast differentiation.