Identification of TNFRSF21 as an inhibitory factor of osteosarcoma based on a necroptosis-related prognostic gene signature and molecular experiments.

BACKGROUND: Osteosarcoma is one of the most common malignant bone tumors with bad prognosis. Necroptosis is a form of programmed cell death. Recent studies showed that targeting necroptosis was a new promising approach for tumor therapy. This study aimed to establish a necroptosis-related gene signature to evaluated prognosis and explore the relationship between necroptosis and osteosarcoma. METHODS: Data from The Cancer Genome Atlas was used for developing the signature and the derived necroptosis score (NS). Data from Gene Expression Omnibus served as validation. Principal component analysis (PCA), Cox regression, receiver operating characteristic (ROC) curves and Kaplan-Meier survival analysis were used to assess the performance of signature. The association between the NS and osteosarcoma was analyzed via gene set enrichment analysis, gene set variation analysis and Pearson test. Single-cell data was used for further exploration. Among the genes that constituted the signature, the role of TNFRSF21 in osteosarcoma was unclear. Molecular experiments were used to explore TNFRSF21 function. RESULTS: Our data revealed that lower NS indicated more active necroptosis in osteosarcoma. Patients with lower NS had a better prognosis. PCA and ROC curves demonstrated NS was effective to predict prognosis. NS was negatively associated with immune infiltration levels and tumor microenvironment scores and positively associated with tumor purity and stemness index. Single-cell data showed necroptosis heterogeneity in osteosarcoma. The cell communication pattern of malignant cells with high NS was positively correlated with tumor progression. The expression of TNFRSF21 was down-regulated in osteosarcoma cell lines. Overexpression of TNFRSF21 inhibited proliferation and motility of osteosarcoma cells. Mechanically, TNFRSF21 upregulated the phosphorylation levels of RIPK1, RIPK3 and MLKL to promote necroptosis in osteosarcoma. CONCLUSIONS: The necroptosis prognostic signature and NS established in this study could be used as an independent prognostic factor, TNFRSF21 may be a necroptosis target in osteosarcoma therapy.

Comparison of Direct Pars Repair Techniques for Spondylolysis in Young Patients: Pedicle Screw Hook System versus Pedicle Screw Rod.

Background: Lumbar spondylolysis (LS) poses a potential threat, and there is a need to evaluate and compare the effectiveness of direct pars repair techniques. Objective: To assess and compare the clinical and radiographic outcomes of direct pars repair techniques using the pedicle screw hook system (PSHS) and the pedicle screw rod system (PSRS) in young symptomatic patients with lumbar spondylolysis. Methods: A retrospective study was conducted to compare clinical and radiological data in young symptomatic LS patients after surgery. Records of 45 post-surgery LS patients with a minimum 24-month follow-up (January 2014 to June 2019) were reviewed. A total of 26 patients underwent PSHS, and 19 had PSRS. Treatment outcomes were analyzed using the visual analog pain scale (VAS), Oswestry disability index (ODI), MacNab criteria, lumbar fusion status, and Pfirrmann grading standards. Patient baseline characteristics were also compared between the two groups. Results: No disc degeneration was observed in either PSHS or PSRS groups at 24 months postoperatively, according to the Pfirrmann grading scale. The PSRS group outperformed the PSHS group in operative time, intraoperative blood loss, postoperative drainage, length of hospital stays, ODI, VAS values at 3 months postoperatively, and fusion status at 6 months postoperatively. No notable differences were observed in other parameters during the 24-month follow-up period, and no significant surgical complications were recorded. Conclusions: Direct pars repair techniques using PSHS and PSRS yielded satisfactory clinical and radiographic results in young patients with symptomatic LS. PSRS, compared to PSHS, demonstrated greater effectiveness in young individuals with LS and promoted early recovery.

Combined use of tranexamic acid and rivaroxaban in posterior lumbar interbody fusion safely reduces blood loss and transfusion rates without increasing the risk of thrombosis-a prospective, stratified, randomized, controlled trial.

PURPOSE: This prospective, stratified, randomized, single-blind, placebo-controlled multicentre study investigated the safety and effectiveness of reducing blood loss and preventing venous thromboembolism (VTE) during posterior lumbar interbody fusion (PLIF) in patients with stenosis or spondylolisthesis using the combination of tranexamic acid (TXA) and rivaroxaban. METHODS: The Autar score was evaluated in patients after admission. Patients with an Autar score ≤ 10 were randomized to group A or B. Group A was the placebo-controlled group. Patients in group B were treated with 1 g TXA via intravenous injection and 1 g TXA for external use. Patients with an Autar score > 10 were randomized to group C or D. Patients in group C were treated with 10-mg rivaroxaban qd for 35 days after surgery. Patients in group D received the same treatment as those in group B intra-operatively and as those in group C post-operatively. RESULTS: A total of 599 patients from eight hospitals participated in this clinical trial. The total blood loss, intra-operative blood loss, and drainage volume were reduced by the administration of TXA (group A vs group B, P < 0.01; group C vs group D, P < 0.01), and the blood transfusion rate was also decreased (group A vs group B, P < 0.01; group C vs group D, P < 0.01). There were no significant differences (P > 0.05) in the VTE incidence rates among group A and group B. In patients with high-risk thrombosis, the number of patients with VTE was only three and seven after the application of rivaroxaban. Epidural haematoma was not discovered in any patients in our trial. CONCLUSION: The combined application of tranexamic acid and rivaroxaban significantly reduced the amount of blood loss and the transfusion rate during PLIF surgery and avoided an increase in the probability of thrombosis and the occurrence of epidural haematoma. TRIAL REGISTRATION NUMBER AND DATE OF REGISTRATION: ChiCTR-1800016430 2018-06-01.

MicroRNA-645 promotes metastasis of osteosarcoma via targeting tumor suppressor NM23 nucleoside diphosphate kinase 2.

Osteosarcoma is the most common non-hematological primary bony malignancy in children and young adults with tumor metastasis being a common event at diagnosis. Understanding the pathogenesis of metastatic osteosarcoma may help identify potential therapeutic targets. In this study, we found that the level of microRNA-645 (miR-645) in osteosarcoma tumor tissues was significantly increased compared with their paired non-tumorous tissues, and was associated with histologic grade, TNM staging, lymph metastasis and distant metastasis. Knockdown of miR-645 caused a remarkable inhibition of migration of osteosarcoma U2OS cells. Furthermore, miR-645 inhibited NME2 (nucleoside diphosphate kinase 2) expression through directly binding to its 3' untranslated region. In human osteosarcoma tissues, we also found that NME2 was significantly decreased in tumor tissues, and its level was negatively correlated with miR-645. In addition, silencing NME2 attenuated the decreased cell migration by knockdown of miR-645, suggesting that it was involved in the miR-645 induced cell migration of osteosarcoma cells. Taken together, we found that miR-645 was up-regulated in osteosarcoma tissues and could promote osteosarcoma cell migration through directly inhibiting the tumor suppressor NME2. Our data provide novel insight into the role of miR-645 in osteosarcoma and indicate that miR-645 might be a potential therapeutic target of osteosarcoma.

Highly Enantioselective Rhodium-Catalyzed Addition of Arylboroxines to Simple Aryl Ketones: Efficient Synthesis of Escitalopram.

Highly enantioselective additions of arylboroxines to simple aryl ketones have been achieved for the first time with a Rh/(R,R,R,R)-WingPhos catalyst, thus providing a range of chiral diaryl alkyl carbinols with excellent ee values and yields. (R,R,R,R)-WingPhos has been proven to be crucial for the high reactivity and enantioselectivity. The method has enabled a new, concise, and enantioselective synthesis of the antidepressant drug escitalopram.

Current evidences on the XPG Asp1104His polymorphism and melanoma susceptibility: a meta-analysis based on case-control studies.

Previous studies evaluating the association between the XPG Asp1104His polymorphism and melanoma susceptibility remained controversial. To draw a more precise estimation of the relationship, a total of eight published case-control studies containing 5,212 cases and 7,045 controls were included for meta-analysis. Overall, a significant association was found between the XPG Asp1104His polymorphism and melanoma susceptibility for the dominant model (OR = 2.42, 95 % CI = 2.26-2.60). In subgroup analysis by source of control, there was an obvious association was found among Population-based subgroup for the dominant model CC+GC vs GG (OR 2.51, 95 % CI 2.28-2.77), among the Hospital-based subgroup, an obvious association was also found for the dominant model CC+GC vs GG (OR 2.34, 95 % CI 2.12-2.58). This meta-analysis suggested that the XPG Asp1104His polymorphism was a risk factor for melanoma susceptibility.

The association between the APE1 Asp148Glu polymorphism and prostate cancer susceptibility: a meta-analysis based on case-control studies.

The apurinic/apyrimidinic endonuclease 1 (APE1) plays important roles in the repair of DNA damage and adducts. However, previous case-control studies on the association between the APE1 Asp148Glu polymorphism and prostate cancer susceptibility have shown contradictory results, this meta-analysis was performed to draw a more precise estimation of the relationship. A total of seven case-control studies including 1,294 cases and 1,762 controls were included for analysis. In overall, no significant associations were found in all genetic models (GG vs. TT: OR = 1.16, 95 % CI 0.89-1.52; TG vs. TT: OR = 1.04, 95 % CI 0.81-1.35; the dominant model GG + TG vs. TT: OR = 1.12, 95 % CI 0.96-1.30; the recessive model GG vs. TG + TT: OR = 0.90, 95 % CI 0.77-1.04); in the subgroup by source of control, we found a significant association for the dominant model in Hospital-based subgroup (OR = 1.34, 95 % CI 1.08-1.68), no significant associations were found in other models in the subgroups. This meta-analysis suggested that the APE1 Asp148Glu polymorphism was a risk factor for prostate cancer susceptibility in Hospital-based population.

Arsenic trioxide inhibits growth of human chondrosarcoma cells through G2/M arrest and apoptosis as well as autophagy.

It has been demonstrated that Gli1 is expressed in chondrosarcoma but not in the normal articular cartilage tissues. Downregulating Gli1 by small interfering RNA inhibited chondrosarcoma cells growth. Arsenic trioxide (ATO) has been demonstrated to suppress human cancer cell growth by targeting Gli1. The aim of this study was to investigate the effect of ATO on antineoplastic capability of chondrosarcoma cells. We found that ATO inhibited the growth of chondrosarcoma cells in dose-dependent and time-dependent manners via MTT and colony formation assays. In addition, ATO treatment induced apoptosis and promoted G2/M phase arrest in SW1353 cells as analyzed by flow cytometry assays and Western blotting. Furthermore, we observed that ATO also triggered autophagy by regulating mammalian target of rapamycin (mTOR) phosphorylation. Finally, we found that ATO-mediated cell death could be averted by autophagy inhibitor. Taken together, the current study suggested that ATO had therapeutic efficacy in human chondrosarcoma cells through the promotion of G2/M arrest and induction of both apoptosis as well as autophagy. ATO administration could be a novel therapeutic strategy for treating chondrosarcomas.

XPC Lys939Gln and Ala499Val polymorphisms in colorectal cancer susceptibility: a meta-analysis of case-control studies.

The XPC Lys939Gln and Ala499Val polymorphisms were likely to be involved with the development of colorectal cancer. However, there had been inconsistent reports of association. This meta-analysis of literatures was performed to draw a more precise estimation of the relationship. We systematically searched PubMed, Embase and Web of Science for relevant articles with a time limit of December 2012. The strength of association between the XPC Lys939Gln and Ala499Val polymorphisms and colorectal cancer susceptibility were assessed by odds ratio (OR) with the corresponding 95 % confidence interval (95 % CI). This meta-analysis including six case-control studies evaluated the associations between the two XPC polymorphisms (Lys939Gln, Ala499Val) and colorectal cancer susceptibility. For XPC Lys939Gln, no obvious associations were found for all genetic models [CC vs AA: OR (95 % CI) = 1.12 (0.94-1.32); CA vs AA: OR (95 % CI) = 1.08 (0.94-1.24); the dominant model: OR (95 % CI) = 1.09 (0.97-1.23); the recessive model: OR (95 % CI) = 1.07 (0.92-1.25)]. For XPC Ala499Val, no obvious associations were also not found for all genetic models [TT vs CC: OR (95 % CI) = 0.84 (0.65-1.10); CT vs CC: OR (95 % CI) = 1.00 (0.86-1.15); the dominant model: OR (95 % CI) = 0.98 (0.85-1.12); the recessive model: OR (95 % CI) = 0.87 (0.67-1.12)]. This meta-analysis suggested that both the XPC Lys939Gln and Ala499Val polymorphisms were not risk factors for increasing colorectal cancer.

Ortho-silicic Acid Prevents Glucocorticoid-Induced Femoral Head Necrosis by Promoting Akt Phosphorylation to Inhibit Endoplasmic Reticulum Stress-Mediated Apoptosis and Enhance Angiogenesis and Osteogenesis.

Glucocorticoid-induced osteonecrosis of the femoral head (SONFH) is the most prevalent form of secondary osteonecrosis affecting the femoral head. Glucocorticoids can cause damage to both vascular endothelial cells and osteoblasts. Previous studies have demonstrated that silicon can improve the resistance of vascular endothelial cells to oxidative stress and positively impact bone health. However, the impact of silicon on SONFH has yet to be investigated. We examined the influence of ortho-silicic acid (OSA, Si(OH)4) on the apoptosis and proliferation of vascular endothelial cells after glucocorticoid induction. Additionally, we evaluated the expression of apoptosis-related genes such as cleaved-caspase-3, Bcl-2 and Bax. The impact of glucocorticoids and OSA on the function of vascular endothelial cells was evaluated through wound healing, transwell and angiogenesis assays. Osteogenic function was subsequently evaluated through alizarin red staining, alkaline phosphatase staining and expression levels of osteogenic genes like RUNX2 and ALP. Moreover, we investigated the potential role of OSA in vivo using the SONFH animal model. At concentrations below 100 µM, OSA exhibits no toxicity on vascular endothelial cells and effectively reverses glucocorticoid-induced apoptosis in these cells. OSA increases the resilience of vascular endothelial cells against oxidative stress and enhances osteoblast differentiation. Our study revealed that glucocorticoids activate endoplasmic reticulum stress, a process that mediates the apoptosis of vascular endothelial cells. OSA ameliorated the endoplasmic reticulum stress associated with glucocorticoids through the increased expression of p-Akt levels. In vivo, OSA treatment effectively improved SONFH by enhancing vascular endothelial cell function and promoting osteogenic differentiation. OSA counteracted the adverse effects of glucocorticoids both in vitro and in vivo, demonstrating a beneficial therapeutic effect on SONFH.

XRCC1 Arg194Trp and Arg280His polymorphisms in bladder cancer susceptibility: a meta-analysis.

The XRCC1 Arg194Trp and Arg280His polymorphisms were likely to be involved with the development of bladder cancer. However, there had been inconsistent reports of association. This meta-analysis of literatures was performed to draw a more precise estimation of the relationship. We systematically searched PubMed, Embase, and Web of Science for relevant articles with a time limit of April 25, 2013. Summary odds ratios (ORs) with 95% confidence intervals (CIs) were used to assess the strength of association between the two polymorphisms and bladder cancer susceptibility using a random-effects model. This meta-analysis including 14 case-control studies evaluated the associations between the two XRCC1 polymorphisms and bladder cancer susceptibility. Overall, for Arg194Trp, significant associations were found in TT versus CC (OR=1.78, 95% CI=1.12-2.82) and the recessive model (OR=1.71, 95% CI=1.11-2.65); for Arg280His, significant associations were also found in AG versus GG (OR=1.63, 95% CI=1.24-2.13) and the dominant model (OR=1.39, 95% CI=1.07-1.82). When stratified by ethnicity, in Asian population, significant associations were found for Arg194Trp polymorphism in TT versus CC (OR=2.99, 95% CI=1.48-6.06), the dominant model (OR=1.33, 95% CI=1.03-1.72) and the recessive model (OR=2.72, 95% CI=1.36-5.45), and for Arg280His in GA versus GG (OR=2.13, 95% CI=1.63-2.97), but no significant associations were found in no-Asian population. This meta-analysis suggested that XRCC1 Arg194Trp and Arg280His polymorphisms were risk factors for increasing bladder cancer in Asian population.

Prognostic significance of cyclooxygenase-2 in osteosarcoma: a meta-analysis.

Published studies researching the prognostic significance of cyclooxygenase-2 (COX-2) expression in patients with osteosarcoma are inconclusive and heterogeneous. We conducted a meta-analysis to assess its prognostic value more precisely. The pooled odds ratios (ORs) or hazard ratios (HRs) with corresponding 95 % confidence intervals (CIs) were calculated to evaluate the effects. Fourteen studies with 735 osteosarcoma patients were included to estimate the relationship between COX-2 and metastasis of tumor, clinical stage, and 3-year overall survival. High expressions of COX-2 predicted neoplasm metastasis (OR = 1.891, 95 % CI 1.276-2.803, P = 0.002), advanced clinical stage (OR = 1.801, 95 % CI 1.257-2.581, P = 0.001). In addition, high COX-2 expression tended to be associated with a poor 3-year survival (HR = 1.741, 95 % CI 0.762-3.979, P = 0.188), but the difference was not significant. Therefore, this meta-analysis demonstrated that high COX-2 expression might be an unfavorable prognostic effect in osteosarcoma.

Visible trephine-based foraminoplasty in PTED leads to asymmetrical stress changes and instability in the surgical and adjacent segments: a finite element analysis.

This study aimed to construct a multi-segment lumbar finite element model (FEM) of PTED surgery to analyze the changes in stress and ROM after visible trephine-based foraminoplasty. The CT scans of a 35-year-old healthy male were used to develop a multi-segment lumbar FEM with Mimic, Geomagic Studio, Hypermesh and MSC.Patran. Different foraminoplasty was performed on the model, and these were grouped into normal group (A), the ventral resection group (B), the apex resection group (C), the ventral + apex + isthmus resection group (D), and the SAP + isthmus + lateral recess resection group (E). A vertical load of 500N and a torque of 10N·M were applied to the upper surface of the L3 vertebral body to simulate the biomechanical characteristics under the motion of flexion, extension, lateral bending, and rotation. The von Mises stress maps of the intervertebral f, vertebral body, facet joints, and the ROM of the L3-S1 intervertebral disk were calculated and analyzed. The changes of peak stress on the vertebral body for each group were not significant in the same motion state. Significant stress differences were observed in the L4/5 intervertebral disks, while no obvious stress changes were observed for the L3/4 and L5/S1 intervertebral disks. The stress of the L3/4 and L5/S1 facet joints decreased after L4/5 foraminoplasty, while the stress of L4/5 facet joints displayed an overall increasing trend. Significant asymmetrical stress changes of bilateral facet joints were observed in all three segments, particularly during bilateral rotation movements. The ROM of L3-S1 gradually increased from Group A to Group E, especially during flexion, left lateral bending, and right rotation, with the highest elevation observed for the L45 ROM. Our FEM indicated that enlarged resection and exposure of the articular surface could lead to significant asymmetrical stress changes in the bilateral facet joints and ROM instability of the surgical and adjacent segments. These findings suggested that unnecessary and excessive resection should be avoided in PTED to reduce the incidence of low back pain and the risk of postsurgical degeneration.

Ortho-silicic Acid Plays a Protective Role in Glucocorticoid-Induced Osteoporosis via the Akt/Bad Signal Pathway In Vitro and In Vivo.

Glucocorticoid-induced osteoporosis (GIOP) has been the most common form of secondary osteoporosis. Glucocorticoids (GCs) can induce osteocyte and osteoblast apoptosis. Plenty of research has verified that silicon intake would positively affect bone. However, the effects of silicon on GIOP are not investigated. In this study, we assessed the impact of ortho-silicic acid (OSA) on Dex-induced apoptosis of osteocytes by cell apoptosis assays. The apoptosis-related genes, cleaved-caspase-3, Bcl-2, and Bax, were detected by western blotting. Then, we evaluated the possible role of OSA on osteogenesis and osteoclastogenesis with Dex using Alizarin red staining and tartrate-resistant acid phosphatase (TRAP) staining. We also detected the related genes by quantitative reverse-transcription polymerase chain reaction (qRT-PCR) and western blotting. We then established the GIOP mouse model to evaluate the potential role of OSA in vivo. We found that OSA showed no cytotoxic on osteocytes below 50 µM and prevented MLO-Y4 from Dex-induced apoptosis. We also found that OSA promoted osteogenesis and inhibited osteoclastogenesis with Dex. OSA had a protective effect on GIOP mice via the Akt signal pathway in vivo. In the end, we verified the Akt/Bad signal pathway in vitro, which showed the same results. Our finding demonstrated that OSA could protect osteocytes from apoptosis induced by GCs both in vitro and in vivo. Also, it promoted osteogenesis and inhibited osteoclastogenesis with the exitance of Dex. In conclusion, OSA has the potential value as a therapeutic agent for GIOP.

Combined Use of Tranexamic Acid and Rivaroxaban in Posterior/Transforaminal Lumbar Interbody Fusion Surgeries Safely Reduces Blood Loss and Incidence of Thrombosis: Evidence From a Prospective, Randomized, Double-Blind, Placebo-Controlled Study.

STUDY DESIGN: A prospective, randomized, double-blind, placebo-controlled study. OBJECTIVES: There are few studies examining the balance between preventing venous thrombus embolism (VTE) and reducing blood loss in posterior/transforaminal lumbar interbody fusion (PLIF/TLIF) surgeries. This study aimed to evaluate the efficacy and safety of the combine application of TXA and rivaroxaban in patients undergoing PLIF/TLIF and explore relevant factors related to blood loss and VTE. METHODS: Patients in group A which was the control group received 0.9% NaCl solution intravenously. Group B was treated by an intravenous injection of 2 g tranexamic acid (TXA) and the local use of 1 g intraoperatively. Group C was treated the same as group B intraoperatively, and they received 10 mg rivaroxaban qd treatment postoperatively. Eligible patients with an Autar score ≤ 10 were randomly assigned to group A or group B. Patients with an Autar score >10 were allocated into group C. RESULTS: The intraoperative blood loss and postoperative drainage were lower in groups B and C than in group A (P < .001). The blood transfusion rate in group B was lower than that in group A (P < .001), while the incidence of VTE in group C was lower (P < .001). Four factors were found to be positively correlated with obvious total blood loss (P < .05). The data showed that 5 factors were correlated with the development of a thrombus (P < .1). CONCLUSIONS: The combination of TXA and rivaroxaban in PLIF/TLIF patients is safe and effective in reducing D-dimer levels associated with VTE and reducing blood loss.

Development and validation of a N6 methylation regulator-related gene signature for prognostic and immune response prediction in non-small cell lung cancer.

N6 methylation (m6A) has been reported to play an important role in tumor progression. Non-small cell lung cancer (NSCLC) is the predominant pathological type of lung cancer with a high mortality rate. The purpose of this study was to develop and validate a N6 methylation regulator-related gene signature for assessing prognosis and response to immunotherapy in NSCLC. Data from The Cancer Genome Atlas was used as the training cohort. Data from Gene Expression Omnibus and Xena served as the two validation cohorts. We performed Cox regression, last absolute shrinkage and selection operator, receiver operating characteristic curves and Kaplan-Meier survival analysis to generate and validate a prognostic signature based on m6A regulator-related genes. We explored the association between the signature and tumor microenvironment including genomic mutation, immune cell infiltration and tumor mutation burden. We also analyzed the association between the signature and immunotherapy. Finally, among the genes that constituted the signature, GGA2 was the only favorable factor for NSCLC prognosis. Molecular experiments were used to explore GGA2 function in NSCLC. We generated a prognostic signature based on seven m6A regulator-related genes (GGA2, CD70, BMP2, GPX8, YWHAZ, NOG and TEAD4). And the data from three cohorts showed that the signature could effectively assess prognosis in NSCLC. Patients with high risk scores had the higher mutational load and lower immune infiltration levels and were more likely to not respond to immunotherapy. The experiments revealed overexpression of GGA2 inhibited proliferation and motility of NSCLC cells. Mechanically, GGA2 downregulated METTL3 expression and thus reduced m6A abundance in NSCLC. This study developed and validated a prognostic signature based on m6A regulator-related genes, providing useful insights for the management of NSCLC. And GGA2 may be a target of m6A regulation.

M2 macrophage-derived exosomal miR-486-5p influences the differentiation potential of bone marrow mesenchymal stem cells and osteoporosis.

BACKGROUND: An imbalance between osteogenesis and adipogenesis in bone marrow mesenchymal stem cells (BMMSCs) can cause osteoporosis. Macrophage-derived exosomes (MD-Exos) and microRNAs (miRNAs) enriched in exosomes participate in the differentiation of BMMSCs. METHODS: Bioinformatics methods were used to analyze differentially expressed miRNAs. We cocultured M2 macrophages and BMMSCs to examine the biological function of exosomal microRNA-486-5p (miR-486-5p) on BMMSCs differentiation. Gain-of-function experiments related to osteogenesis were designed to investigate the effects of exosomes carrying miR-486-5p on an ovariectomized (OVX) mice model and the direct impact of miR-486-5p on BMMSCs. A dual luciferase experiment was performed to demonstrate the target gene of miR-486-5p. RESULTS: Bioinformatics analysis identified high expression of miRNA-486 in M2 macrophage-derived exosomes (M2D-Exos). The in vitro results demonstrated that M2 macrophage-derived exosomal miR-486-5p enhanced osteogenic capacity but inhibited the adipogenesis of BMMSCs. The direct effect of miR-486-5p on BMMSCs showed the same effects. Animal experiments revealed that exosomal miR-486-5p rescued bone loss of OVX mice. SMAD2 was characterized as a target gene of miR-486-5p. Pathway analysis showed that M2 macrophage-derived exosomal miR-486-5p stimulated osteogenic differentiation via the TGF-ß/SMAD2 signalling pathway. CONCLUSIONS: Taken together, M2 macrophage-derived exosomal miR-486-5p influences the differentiation potential of BMMSCs through the miR-486-5p/SMAD2/TGF-ß signalling pathway and osteoporosis.

WTAP-Mediated m6A RNA Methylation Regulates the Differentiation of Bone Marrow Mesenchymal Stem Cells via the miR-29b-3p/HDAC4 Axis.

N6-methyladenosine (m6A) methylation, a well-known modification with new epigenetic functions, has been reported to participate in the progression of osteoporosis (OP), providing novel insights into the pathogenesis of OP. However, as the key component of m6A methylation, Wilms tumor 1-associated protein (WTAP) has not been studied in OP. Here we explored the biological role and underlying mechanism of WTAP in OP and the differentiation of bone marrow mesenchymal stem cells (BMMSCs). We demonstrated that WTAP was expressed at low levels in bone specimens from patients with OP and OVX mice. Functionally, WTAP promoted osteogenic differentiation and inhibited adipogenic differentiation of BMMSCs in vitro and in vivo. In addition, microRNA-29b-3p (miR-29b-3p) was identified as a downstream target of WTAP. M6A modifications regulated by WTAP led to increased miR-29b-3p expression. WTAP interacted with the microprocessor protein DGCR8 and accelerated the maturation of pri-miR-29b-3p in an m6A-dependent manner. Target prediction and dual-luciferase reporter assays identified the direct binding sites of miR-29b-3p with histone deacetylase 4 (HDAC4). WTAP-mediated m6A modification promoted osteogenic differentiation and inhibited adipogenic differentiation of BMMSCs through the miR-29b-3p/HDAC4 axis. Furthermore, WTAP-mediated m6A methylation negatively regulates osteoclast differentiation. Collectively, our study first identified a critical role of WTAP-mediated m6A methylation in BMMSC differentiation and highlighted WTAP as a potential therapeutic target for OP treatment.

WTAP-mediated m6A modification modulates bone marrow mesenchymal stem cells differentiation potential and osteoporosis.

An imbalance in the differentiation potential of bone marrow mesenchymal stem cells (BMSCs) is an important pathogenic mechanism underlying osteoporosis (OP). N6-methyladenosine (m6A) is the most common post-transcriptional modification in eukaryotic cells. The role of the Wilms' tumor 1-associated protein (WTAP), a member of the m6A functional protein family, in regulating BMSCs differentiation remains unknown. We used patient-derived and mouse model-derived samples, qRT-PCR, western blot assays, ALP activity assay, ALP, and Alizarin Red staining to determine the changes in mRNA and protein levels of genes and proteins associated with BMSCs differentiation. Histological analysis and micro-CT were used to evaluate developmental changes in the bone. The results determined that WTAP promoted osteogenic differentiation and inhibited adipogenic differentiation of BMSCs. We used co-immunoprecipitation (co-IP), RNA immunoprecipitation (RIP), methylated RNA immunoprecipitation (MeRIP), RNA pulldown, and dual-luciferase assay to explore the direct mechanism. Mechanistically, the expression of WTAP increased during osteogenic differentiation and significantly promoted pri-miR-181a and pri-miR-181c methylation, which was recognized by YTHDC1, and increased the maturation to miR-181a and miR-181c. MiR-181a and miR-181c inhibited the mRNA expression of SFRP1, promoting the osteogenic differentiation of BMSCs. Our results demonstrated that the WTAP/YTHDC1/miR-181a and miR-181c/SFRP1 axis regulated the differentiation fate of BMSCs, suggesting that it might be a potential therapeutic target for osteoporosis.