Multi-omics Analysis to Identify Key Immune Genes for Osteoporosis based on Machine Learning and Single-cell Analysis.

OBJECTIVE: Osteoporosis is a severe bone disease with a complex pathogenesis involving various immune processes. With the in-depth understanding of bone immune mechanisms, discovering new therapeutic targets is crucial for the prevention and treatment of osteoporosis. This study aims to explore novel bone immune markers related to osteoporosis based on single-cell and transcriptome data, utilizing bioinformatics and machine learning methods, in order to provide novel strategies for the diagnosis and treatment of the disease. METHODS: Single cell and transcriptome data sets were acquired from Gene Expression Omnibus (GEO). The data was then subjected to cell communication analysis, pseudotime analysis, and high dimensional WGCNA (hdWGCNA) analysis to identify key immune cell subpopulations and module genes. Subsequently, ConsensusClusterPlus analysis was performed on the key module genes to identify different diseased subgroups in the osteoporosis (OP) training set samples. The immune characteristics between subgroups were evaluated using Cibersort, EPIC, and MCP counter algorithms. OP's hub genes were screened using 10 machine learning algorithms and 113 algorithm combinations. The relationship between hub genes and immunity and pathways was established by evaluating the immune and pathway scores of the training set samples through the ESTIMATE, MCP-counter, and ssGSEA algorithms. Real-time fluorescence quantitative PCR (RT-qPCR) testing was conducted on serum samples collected from osteoporosis patients and healthy adults. RESULTS: In OP samples, the proportions of bone marrow-derived mesenchymal stem cells (BM-MSCs) and neutrophils increased significantly by 6.73% (from 24.01% to 30.74%) and 6.36% (from 26.82% to 33.18%), respectively. We found 16 intersection genes and four hub genes (DND1, HIRA, SH3GLB2, and F7). RT-qPCR results showed reduced expression levels of DND1, HIRA, and SH3GLB2 in clinical blood samples of OP patients. Moreover, the four hub genes showed positive correlations with neutrophils (0.65-0.90), immature B cells (0.76-0.92), and endothelial cells (0.79-0.87), while showing negative correlations with myeloid-derived suppressor cells (negative 0.54-0.73), T follicular helper cells (negative 0.71-0.86), and natural killer T cells (negative 0.75-0.85). CONCLUSION: Neutrophils play a crucial role in the occurrence and development of osteoporosis. The four hub genes potentially inhibit metabolic activities and trigger inflammation by interacting with other immune cells, thereby significantly contributing to the onset and diagnosis of OP.

Plasma proteins, circulating metabolites mediate causal inference studies on the effect of gut bacteria on the risk of osteoporosis development.

BACKGROUND: The role of gut bacteria in preventing and delaying osteoporosis has been studied. However, the causal relationship between gut bacteria, plasma proteins, circulating metabolites and osteoporosis (OP) risk has not been fully revealed. MATERIALS AND METHODS: In this study, a two-sample Mendelian randomization study (MR) approach was used to assess the causal associations between gut bacteria, plasma proteins and circulating metabolites, and osteoporosis risk using Genome Wide Association Study (GWAS) data from gut bacteria(n=8208), plasma proteins(n=2263), circulating metabolites (n=123), and osteoporosis (3203 cases and 16380452 controls). Inverse-variance weighted (IVW) was used as the main analytical method to estimate the MR causal effect and to perform directional sensitivity analysis of causality. Finally, the mediating effect values for the influence of gut flora on OP pathogenesis through circulating metabolites were calculated by univariate MR analysis, and multivariate MR analysis. Next, we evaluated the effect of Phosphatidylcholine on the osteogenic function of bone marrow mesenchymal stem cells (BMSCs) through relevant experiments, including Edu detection of cell proliferation, alkaline phosphatase (ALP) staining, Alizarin red staining to evaluate osteogenic function, qPCR and WB detection of osteogenic differentiation related gene expression. RESULTS: A total of 9 gut microbial taxa, 15 plasma proteins and eight circulating metabolites were analysed for significant causal associations with the development of OP. Significant causal effects of 7 on gut bacteria, plasma proteins and circulating metabolites were analysed by univariate MR analysis and these results were used as exposure factors for subsequent multivariate MR. Multivariate MR analyses yielded a significant effect of circulating metabolites Phosphatidylcholine and other cholines on OP (P<0.05). Further mediation effect analysis showed that the mediation effect of Bifidobacteriaceae affecting OP through the circulating metabolite Phosphatidylcholine and other cholines was -0.0224, with a 95 % confidence interval for the mediation effect that did not include 0, and the complete mediation effect was significant. Phosphatidylcholine can promote BMSCs proliferation and osteogenesis. CONCLUSION: Our study demonstrated significant causal associations of gut bacteria, plasma proteins and circulating metabolites on OP, and that Bifidobacteriaceae affect OP through the circulating metabolites Phosphatidylcholine and other cholines. Phosphatidylcholine affects the osteogenic ability of BMSCs. Further exploration of potential microbiota-associated mechanisms of bone metabolism may offer new avenues for osteoporosis prevention and treatment of osteoporosis.

A Reverse Suture Anchor Technique for Arthroscopic Medial Meniscus Root Repair.

Injuries of the posterior root of the medial meniscus can be accompanied by damage to the anterior cruciate ligament or often occur independently in cases of degenerative meniscal injury in older individuals. Anchor suture repair can achieve favorable biomechanical effects and clinical outcomes. However, anchor placement is technically challenging and requires a posterior medial approach, which increases the risk of iatrogenic injury. To address these issues, we have utilized the reverse anchor technique to repair the posterior root of the medial meniscus. This technique offers advantages such as reduced surgical time, simplified operation, and reduced risk of the "bungee effect" and iatrogenic injury.

Modified elastic intramedullary nail internal fixation for distal tibial metaphyseal junction fracture.

Objective: To investigate the application of modified elastic intramedullary nail and the outcomes between modified elastic stable intramedullary nailing and traditional elastic stable intramedullary nailing in children with distal tibial metaphyseal junction fracture. Methods: A retrospective study was conducted. From January 2018 to January 2021, a total of 36 children with distal tibial metaphyseal junction fracture were treated in our hospital. All of them were treated with closed reduction and elastic stable intramedullary nailing internal fixation. A total of 18 children were treated by modified elastic stable intramedullary nailing and 18 children were treated by traditional elastic stable intramedullary nailing. Postoperative imaging, clinical efficacy, and complications were analyzed. Results: The mean follow-up time was 20 (15-36) months in modified group and 22 (16-33) months in traditional group. There were no complications such as infection, loss of reduction, and unequal length of lower limbs in modified group while loss of reduction occurred in two cases in traditional group. In these two cases of loss of reduction, we preformed manual reduction and replacement of long leg casts, and there was no loss of reduction, and the patient achieved a good prognosis. In the last follow-up, American Orthopaedic Foot & Ankle Society score was used. In modified group, excellent outcome achieved in 17 cases, good outcome achieved in 1 case, and satisfactory therapeutic effect was achieved. In traditional elastic stable intramedullary nailing group, excellent outcome achieved in 14 cases, and good outcome achieved in 4 cases. There was no statistical difference in the scores between the two groups. Conclusion: It was concluded that modified elastic stable intramedullary nailing fixation is a safe and effective treatment.

Arthroscopic Lateral Meniscus Root Repair With Reverse Suture Anchor Technique.

The stability of the knee joint is crucially dependent on the integrity of the lateral meniscus posterior root, which is often accompanied by anterior cruciate ligament injury. Anchor suture repair for lateral meniscus posterior root injury not only achieves better biomechanical effects but also ensures favorable prognosis. However, the placement of anchors often requires the establishment of a posterior approach, and the insertion of an anchor is a technical challenge. In light of this, we have applied the technique of reverse anchor fixation for repairing the lateral meniscus posterior root, which not only simplifies the procedure but also effectively reduces the "bungee effect."

Exosomal miR-331-3p derived from chemoresistant osteosarcoma cells induces chemoresistance through autophagy.

BACKGROUND: Osteosarcoma is a common malignant bone tumor, and chemotherapy can effectively improve the prognosis. MicroRNA-331 (MiR-331) is associated with poor cancer outcomes. However, the role of miR-331 in osteosarcoma remains to be explored. METHODS: Drug-resistant osteosarcoma cells were cultured, and their exosomes were purified. The secretion and uptake of exosomes by drug-resistant osteosarcoma and osteosarcoma cells were confirmed using a fluorescence tracking assay and Transwell experiments. The effects of drug-resistant exosomes on cell proliferation were determined using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. siRNA-Drosha and neutral sphingomyelinase inhibitor GW4869 were used to determine the transfer of miRNAs. qRT-PCR and western blotting were used to detect the role of autophagy in the regulation of drug-resistant cell-derived exosomal miR-331-3p. RESULTS: Exosomal miR-331-3p levels in drug-resistant cells were higher than in exosomes from osteosarcoma cells. The exosomes secreted by the drug-resistant osteosarcoma cells could be absorbed by osteosarcoma cells, leading to acquired drug resistance in previously non-resistance cells. Inhibition of miRNAs resulted in reduced transmission of drug resistance transmission by exosomes. Exosomes from drug-resistant osteosarcoma cells transfected with siRNA-Drosha or treated by GW4869 could not enhance the proliferation of MG63 and HOS cells. Finally, miR-331-3p in the exosomes secreted by drug-resistant osteosarcoma cells could induce autophagy of osteosarcoma cells, allowing them to acquire drug resistance. The inhibition of miR-331-3p decreased drug resistance of osteosarcoma cells. CONCLUSION: Exosomes secreted from chemoresistant osteosarcoma cells promote drug resistance through miR-331-3p and autophagy. Inhibition of miR-331-3p could be used to alleviate drug resistance in osteosarcoma.

Exosomal miR-429 derived from adipose-derived stem cells ameliorated chondral injury in osteoarthritis via autophagy by targeting FEZ2.

BACKGROUND: Osteoarthritis (OA) is the most prevalent musculoskeletal disease, imposing a significant public health burden. Exosomes might be an effective means of treating OA. PURPOSE: To investigate the role of exosomes from adipose tissue-derived stromal cells (ADSCs) in OA. We explored whether exosomes from ADSCs could be absorbed by OA chondrocytes, whether there were differences in miR-429 expression in the exosomes of ADSCs and chondrocytes, and whether ADSC exosomal miR-429 could enhance chondrocyte proliferation to exert therapeutic effects in OA. STUDY DESIGN: Controlled laboratory study. METHODS: ADSCs were isolated and cultured from 4-week-old Sprague-Dawley rats. ADSCs and chondrocytes were identified by flow cytometry assay and fluorescent staining, respectively. The exosomes were extracted and identified. Exosome transport was verified by cell staining and co-culture. Beclin 1, collagen II, LC3-II/I, miR-429, and FEZ2 mRNA and protein expression were investigated with real-time PCR and western blotting, respectively. Chondrocyte proliferation was investigated with Cell Counting Kit-8 (CCK-8) assay. The association between miR-429 and FEZ2 was verified with luciferase assay. A rat OA model was established and rat knee joint cartilage tissue was examined with hematoxylin-eosin and toluidine blue staining. RESULTS: Both ADSCs and chondrocytes secreted exosomes and ADSC-derived exosomes could be absorbed by the chondrocytes. ADCS exosomes contained higher miR-429 levels than chondrocyte exosomes. The luciferase assay demonstrated that miR-429 directly targeted FEZ2. Compared with the OA group, miR-429 promoted chondrocyte proliferation while FEZ2 decreased it. miR-429 promoted autophagy by targeting FEZ2 to ameliorate cartilage injury. In vivo, miR-429 promoted autophagy to alleviate OA by targeting FEZ2. CONCLUSION: ADSC exosomes could be beneficial for OA and could be absorbed by chondrocytes to promote chondrocyte proliferation through miR-429. miR-429 ameliorated cartilage injury in OA by targeting FEZ2 and promoting autophagy.

MiR-217 regulates autophagy through OPG/RANKL/RANK in giant cell tumors.

BACKGROUND: Increasing evidence suggests that microRNAs (miRNAs) play a crucial role in cancer development and progression. Our previous study showed remarkably lower levels of miR-217 in GCT cells and tissues, and miR-217 re-expression inhibited the occurrence and development of GCT in vitro; however, the associated mechanisms remain unknown. Thus, this study aimed to explore the mechanisms underlying the proliferation inhibitory effect of miR-217 in GCT cells. METHODS: The proliferative potential of the GCT cells was measured with an MTT assay and BrdU straining. Changes in GCT cell migration and invasion was assessed by a transwell assay. Finally, Western blot and RT-PCR assays were employed to evaluate OPG/RANKL/RANK signaling pathway-related protein expression. RESULTS: The excessive upregulation of miR-217 markedly suppressed GCT cell proliferation and tumorigenesis both in vitro and in vivo. miR-217 overexpression could inhibit the OPG/RANKL/RANK signaling pathway in vitro and in vivo. Furthermore, ALP activity was significantly decreased in GCT cells following miR-217 treatment. Importantly, miR-217 could inhibit autophagy-related protein expression and autophagosome/autolysosome formation in GCT cells and tissues. CONCLUSION: These results suggest that miR-217 upregulation could inhibit the occurrence and development of GCT by blocking autophagy. These findings offer an effective therapeutic target to improve the survival rates of patients with CGT in the future.

The Impact of Economic Policy Uncertainty on Regional Total-Factor Energy Productivity in China.

This paper investigates the effect and possible mechanism of implicit macroeconomic policy uncertainty on regional energy productivity in China. This study takes the unexpected output of environmental pollution from energy consumption into account and uses the DEA-SBM method to measure the regional total-factor energy productivity (RTFEP) of prefecture cities in China from 2003 to 2017. Based on the economic policy uncertainty (EPU) index constructed by Baker et al. this paper estimates the effects of EPU on RTFEP and finds that there is a significant negative relationship between them. Specifically, for every unit increase in EPU, RTFEP will decrease by 5.7%. This paper further examines the mechanism of EPU on RTFEP from the perspective of the market and the government and finds that EPU exerts a restraining effect on RTFEP by affecting the consumption structure of the energy market and government intervention in the economy. In addition, the results show that there is heterogeneity in the impact of EPU on RTFEP which varies between resource-based cities at different development stages and different dominant resources. Finally, this paper proposes to cope with the negative impact of EPU on RTFEP by optimizing the energy consumption structure, regulating government investment areas, and transforming the economic development mode.

Interfacial Anchored Sesame Ball-like Ag/C To Guide Lithium Even Plating and Stripping Behavior.

Lithium (Li) metal is a candidate anode for the next generation of high-energy density secondary batteries. Unfortunately, Li metal anodes (LMAs) are extremely reactive with electrolytes to accumulate uncontrolled dendrites and to generate unwanted parasitic electrochemical reactions. Much attention has been focused on carbon materials to address these issues. Ulteriorly, the failure mechanism investigation of lithiophilic sites on carbon materials has been not taken seriously. Herein, we design a new type of sesame ball-like carbon sphere (AgNPs@CS, an average diameter of ∼700 nm) with uniformly interfacial anchored silver nanoparticles (AgNPs), which is used as the dendrite-free Li metal anode host. This anchored structure significantly enhances reversible and chemical affinity of Li, effectively inhibiting "dead Li". In addition, the protective effect of the carbon layer can avoid the damage of lithiophilic AgNPs in the carbon matrix. With a plating/striping capacity of 2 mA h cm-2, the AgNPs@CS electrode can be cycled over 2400 h at 0.5 mA cm-2. When the stripping voltage increases to 1 V, the AgNPs@CS electrode also enables excellent cycling stability to achieve over 260 cycles (1 mA cm-2, 1 mA h cm-2) and 130 cycles (2 mA cm-2, 1 mA h cm-2). This material by electrochemical characterization highlights the efficacy of this facile method for developing dendrite-free LMAs.

Strategies and challenges of carbon materials in the practical applications of lithium metal anode: a review.

Lithium (Li) metal is strongly considered to be the ultimate anode for next-generation high-energy-density rechargeable batteries due to its lowest electrochemical potential and highest specific capacity. However, Li metal anode has limitations, involving inevitable dendritic Li growth, nonuniform Li deposition, enormous volume expansion, and ultimate electrode pulverization, which lead to rapid decrease in Coulombic efficiency and short circuits, significantly hindering its practical use. Various strategies have been proposed to solve uncontrollable dendritic Li growth and parasitic electrochemical reactions. Carbon materials and their composites with excellent structure tunability and properties have been explored to solve these issues and have shown great potential applications in Li metal anodes. This review presents various protective strategies of Li metal anode based on carbon materials. The rational design of carbon materials with specific functionalities in Li metal anode protecting solutions and manufacturing methods of composite electrodes with metallic Li and carbon materials are discussed in detail. In addition, a comprehensive understanding of the challenges and our outlook on the future development of carbon materials for stabilizing Li metal anodes in practical applications are discussed and prospected.

Lithiophilic onion-like carbon spheres as lithium metal uniform deposition host.

Lithium metal is considered as a promising anode material for next-generation secondary batteries, owing to its high theoretical specific capacity (3860 mA h g-1). Nevertheless, the practical application of Li in lithium metal batteries (LMBs) is hampered by inhomogeneous Li deposition and irreversible "dead Li", which lead to low coulombic efficiency (CE) and safe hazards. Designing unique lithiophilic structure is an efficient strategy to control Li uniformly plating /stripping. Here, we report the silver (Ag) nanoparticles coated with nitrogen-doped onion-like carbon microspheres (Ag@NCS) as a host to reduce the nucleation overpotential of Li for dendrite-free LBMs. The Ag@NCS were prepared by a simple one-step injection pyrolysis. The lithiophilic Ag is demonstrated to be priority selective deposition of Li in the carbon cage. Meanwhile, the onion-like structure benefits to uniform lithium nucleation and dendrite-free lithium during cycling. Impressively, we successfully captured lithium metal on different hosts at atomic scale, further proving that Ag@NCS can effectively and uniformly deposit Li. Besides, Ag@NCS show a superiorly electrochemical performance with a low nucleation overpotential (∼1 mV), high CE and stable cycling performance (over 400 cycles at 0.5 mA cm-2) compared to the Ag-free onion-like carbon in LMBs. Even under harsh conditions (1 mA cm-2, 4 mA h cm-2), Ag@NCS still present superior cycling stability for more than 150 cycles. Furthermore, a full cell composed of LiFePO4 cathode exhibits significantly improved voltage hysteresis with low voltage polarization. This work provides a new choice and route for the design and preparation of lithiophilic host materials.

A General Multi-Interface Strategy toward Densified Carbon Materials with Enhanced Comprehensive Electrochemical Performance for Li/Na-Ion Batteries.

Fast charging rate and large energy storage are key requirements for lithium-ion batteries (LIBs) in electric vehicles. Developing electrode materials with high volumetric and gravimetric capacity that could be operated at a high rate is the most challenging problem. In this work, a general multi-interface strategy toward densified carbon materials with enhanced comprehensive electrochemical performance for Li/Na-ion batteries is proposed. The mixture of graphene oxide and sucrose solution is sprayed into a water/oil system and furtherly carbonized to get graphene/hard carbon spheres (GHSs). In this material, abundant ingenious internal interfaces between the crystalline graphene and the carbon matrix are created inside the hard carbon spheres. The constructed interfaces can not only work as a pathway for the escape of volatile gas generated during sucrose pyrolysis to prevent the formation of abundant pores, which leads high packing density of 0.910 g cm-3 and low surface area of 13.3 m2  g-1 , but can also provide a conductive "highway" for ions and electrons. When used as the anode material for both LIBs and sodium-ion batteries (SIBs), the GHS shows the high gravimetric/volumetric reversible capacities, high-rate performance, and low temperature properties simultaneously, implying the great potential application in practical LIBs and SIBs.

Repair of rotator cuff tears in patients aged 75 years and older: Does it make sense? A systematic review.

Background: Rotator cuff injuries are common, and morbidity increases with age. The asymptomatic full-thickness tear rate is 40% in the over 75-year-old population. Purpose: This study aimed to systematically review the literature on the outcomes of rotator cuff repair among >75 years old patients. Study design: Systematic review. Methods: A systematic review of the literature was performed following the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) guidelines. A literature search was performed in the electronic databases of PubMed, Medline, Embase, and The Cochrane Library. Studies in English evaluating repair of full-thickness rotator cuff tears in patients aged >75 years were included. Results: Six studies were reviewed, including 311 patients (313 shoulders) treated with arthroscopic and/or open rotator cuff repair. Sixty-one patients were lost to follow-up, leaving 252 shoulders with outcome data. Patients in this age group demonstrated a significant improvement in the clinical and functional scores after rotator cuff repair, with a high satisfaction rate. The mean American Shoulder and Elbow Surgeons scores improved from 43.8 (range, 42.0-45.5) preoperatively to 85.3 (range, 84.0 to 86.5) postoperatively, and the mean Constant scores improved from 45.4 (range, 34.7-55.5) to 78.6 (range, 67.0-91.6). Pain, evaluated in all studies by the visual analog scale for pain, showed a significant improvement at the last follow-up compared with the mean preoperative score. Furthermore, range of motion and return to daily activities and sports gained marked improvements. Conclusion: Rotator cuff repair in patients aged >75 years could achieve high clinical success rates with good outcomes and pain relief. Although patients in this age group are at a high risk of retear, rotator cuff repair may offer a good option with significant functional and clinical improvement.

MicroRNA­22 regulates autophagy and apoptosis in cisplatin resistance of osteosarcoma.

Osteosarcoma (OS) is a primary malignant tumor of bone tissue. Effective chemotherapy may improve the survival of patients with OS. MicroRNAs (miRs) serve significant roles in the regulatory function of tumorigenesis and chemosensitivity of different types of cancer. miR­22 has been revealed to inhibit the proliferation and migration of OS cells, as well as increasing their sensitivity to cisplatin (CDDP). The mechanisms of action behind the functions of miR­22 in OS drug resistance require investigation. Therefore, in the present study, the human OS cell lines (MG­63, U2OS, Saos2 and OS9901) and a drug­resistant cell line (MG­63/CDDP) were cultured. Cell proliferation, apoptosis and autophagy assays were performed to investigate the proliferation, apoptosis and autophagy of cell lines transfected with miR­22 mimic. Reverse transcription­quantitative polymerase chain reaction and western blot analysis were performed to investigate the expression levels of associated genes. The results revealed that miR­22 inhibited the proliferation of MG­63 cells and MG­63/CDDP cells, and enhanced the anti­proliferative ability of CDDP. miR­22 induced apoptosis and inhibited autophagy of MG­63 cells and MG­63/CDDP cells. Apoptosis­related genes, including caspase­3 and Bcl­2­associated X protein were upregulated, while B­cell lymphoma­2 was downregulated in both cell lines transfected with the miR­22 mimic. Autophagy protein 5, beclin1 and microtubules­associated protein 1 light chain 3 were downregulated in both cell lines transfected with miR­22 mimic. Furthermore, the in vitro and in vivo expression levels of metadherin (MTDH) in the OS/OS­CDDP­resistant models were downregulated following transfection with the miR­22 mimic. Therefore, the results of the present study suggested that miR­22 promoted CDDP sensitivity by inhibiting autophagy and inducing apoptosis in OS cells, while MTDH may serve a positive role in inducing CDDP resistance of OS cells.

MicroRNA­22 mediates the cisplatin resistance of osteosarcoma cells by inhibiting autophagy via the PI3K/Akt/mTOR pathway.

Osteosarcoma (OS) is the most common primary malignant tumor of the bone affecting children and adolescents. Chemotherapy is now considered as a standard component of OS treatment, not only for children, but also for adults. However, chemoresistance continues to pose a challenge to therapy. Inhibition of autophagy has been demonstrated to decrease chemoresistance in OS. Moreover, microRNA­22 (miR­22) inhibits autophagy, leading to an improvement in the sensitivity of cisplatin (CDDP) in OS. The aim of the present study was therefore to investigate whether miR­22 could mediate the CDDP resistance of OS cells by inhibiting autophagy via the phosphoinositide 3­kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway. Cell proliferation assay, LC3 flow cytometry assay and monodansylcadaverine staining in MG63 cells and CDDP resistance cells (MG63/CDDP) were performed to explore to role of miR­22 and CDDP in OS chemoresistance. Inoculation of tumor cells in an in vivo model, reverse transcription­quantitative PCR (RT­qPCR) assay, western blot analysis, and immunohistochemistry analysis were performed to investigate the role of miR­22 and CDDP in the PI3K/Akt/mTOR pathway as it is affected by autophagy. The results revealed that miR­22 inhibited the proliferation of MG63 and MG63/CDDP cells, and enhanced the anti­proliferative ability of CDDP in vivo and in vitro. miR­22 mediated the CDDP resistance of OS cells by inhibiting autophagy and decreasing CDDP­induced autophagy via downregulation of the expression of PI3K, Akt, and mTOR at the mRNA level, and the expression of PI3K, phosphorylated (p)­Akt, and p­mTOR at the protein level. It was also convincingly demonstrated that miR­22 mediates the CDDP resistance of OS by inhibiting autophagy via the PI3K/Akt/mTOR pathway. Furthermore, in the MG63 cells that were affected by CDDP, the role of miR­22 was shown to be similar to that of the investigated inhibitor of PI3K (wortmannin) in terms of regulating the PI3K/Akt/mTOR pathway, and wortmannin could also promote the effect of miR­22. Interestingly, CDDP was demonstrated to induce autophagy by inhibiting the PI3K/Akt/mTOR pathway, whereas the pathway was upregulated in the state of chemoresistance. In conclusion, downregulation of the PI3K/Akt/mTOR pathway was shown to assist in the process of preventing chemoresistance.

Influence of MicroRNA-141 on Inhibition of the Proliferation of Bone Marrow Mesenchymal Stem Cells in Steroid-Induced Osteonecrosis via SOX11.

OBJECTIVE: To investigate whether miR-141 and the sex determination region of Y chromosome box 11 (SOX11) play roles in steroid-induced avascular necrosis of the femoral head (SANFH), and to explore whether miR-141 could target SOX11 to influence the proliferation of bone marrow mesenchymal stem cells (BMSC). METHODS: Bone marrow mesenchymal stem cells (BMSC) were isolated and cultured from 4-week-old Sprague Dawley rats. A flow cytometry assay was performed to identify BMSC. BMSC were divided into two groups: a control group and a dexamethasone (DEX) group. BMSC were transfected by miR-141 mimic, miR-141 inhibitor, and SOX11. Real-time polymerase chain reaction (PCR) assay was performed to investigate the mRNA expression of miR-141 and SOX11. The results were used to determine the effect of transfection and to verify the expression in each group and the association between miR-141 and SOX11. Luciferase reporter assay revealed the targeted binding site between miR-141 and the 3'-untranslated region of SOX11 mRNA. MTT assays were performed to investigate the proliferation of BMSC in the miR-141 mimic, miR-141 inhibitor, and SOX11 groups. RESULT: The results of the flow cytometry assay suggested that cells were positive for CD29 and CD90 while negative for CD45. This meant that the isolated and cultured cells were not hematopoietic stem cells. In addition, cell transfection was successful based on the expression of miR-141 and SOX11. According to the results of real-time PCR assay, the mRNA expression of miR-141 in SANFH was upregulated (4.117 ± 0.042 vs 1 ± 0.027, P < 0.001), while SOX11 was downregulated (0.611 ± 0.055 vs 1 ± 0.027, P < 0.001) compared with the control group. Based on the results of the luciferase experiment, MiR-141 could directly target the expression of SOX11. Inhibition of miR-141 could upregulate the expression of SOX11 (2.623 ± 0.220 vs 1 ± 0.095, P < 0.001) according to the results of a real-time PCR assay. MiR-141 inhibited the proliferation of BMSC (0.618 ± 0.092 vs 1.004 ± 0.082, P < 0.001), while suppression of miR-141 increased the proliferation of BMSC (0.960 ± 0.095 vs 0.742 ± 0.091, P < 0.001). Furthermore, according to the results of the MTT assay, SOX11 promoted the proliferation of BMSC (1.064 ± 0.093 vs 0.747 ± 0.090, P < 0.001). CONCLUSION: MiR-141 inhibited the proliferation of BMSC in SANFH by targeting SOX11. Inhibition of miR-141 upregulated the expression of SOX11 and promoted the proliferation of BMSC. MiR-141 and SOX11 could be new targets for investigating the mechanism of SANFH.

MMP-8 single-nucleotide polymorphisms are related to ankylosing spondylitis in Chinese Han population.

Ankylosing spondylitis (AS) is an extreme form of inflammatory arthritis which always leads to bony fusion of vertebral and chronic pain of back. A lot of genes including interleukin, matrix metalloproteinases (MMPs), and endoplasmic reticulum aminopeptidase were found associated with AS. MMP family members were involved in the autoimmune disease and orthopedic diseases such as rheumatoid arthritis and osteoarthritis, while few studies concentrated on the correlation between single-nucleotide polymorphisms (SNPs) in MMP and AS. In addition, there is no report on the relationship between MMP-8 and AS. To investigate the association between SNPs in MMP-8 and AS, we recruited 268 patients with AS and 654 healthy people to conduct a case-control study. Five SNPs including rs3740938, rs2012390, rs1940475, rs11225394, and rs11225395 of MMP-8 gene were genotyped. It was found rs3740938 of MMP-8 was associated with an increased risk of AS under the dominant model and additive model after adjustment for gender and age by performing logistic regression analysis (odds ratio [OR] = 1.49, 95% confidence interval [CI] = 1.02-2.18, P = .038; OR = 1.37, 95% CI = 1.01-1.87, P = .042, respectively). Moreover, haplotype "GGTCA" was associated with an increased risk of AS without adjustment for age and gender (OR = 1.75, 95% CI = 1.05-2.92, P = .032), while no positive result was found after adjustment for age and gender. Based on our results, our study indicates significant association between SNPs of MMP-8 and AS risk in a Chinese Han population and these results provide the first evidence that MMP-8 is correlated with AS.

Dexamethasone-induced production of reactive oxygen species promotes apoptosis via endoplasmic reticulum stress and autophagy in MC3T3-E1 cells.

Apoptosis of osteoblasts, triggered by prolonged or excessive use of glucocorticoids (GCs), has been identified as a dominant contributor to the development of osteoporosis and osteonecrosis. However, the molecular mechanisms underlying GC­induced apoptosis are multifaceted and remain to be fully elucidated. The present study aimed to explore the correlation between dexamethasone (DEX)­induced reactive oxygen species (ROS), autophagy and apoptosis in MC3T3­E1 osteoblast­like cells. Cell viability was assessed using a Cell Counting Kit­8 assay, and flow cytometry was performed to assess cellular apoptosis, cell cycle and ROS production. Immunofluorescence and western blot analysis were respectively used to detect autophagic vacuoles and the expression of proteins, including cyclin D kinase (CDK)2, poly[ADP ribose] polymerase, caspase­3, activating transcription factor (ATF)4, CCAAT/enhancer­binding protein homologous protein (CHOP), Beclin1, microtubule­associated proteins 1A/1B light chain (LC)3B and P62. It was revealed that DEX not only reduced cell viability, but also promoted apoptosis via the activation of endoplasmic reticulum (ER) stress. In addition, DEX induced cell cycle arrest at G0/G1 phase via inhibition of the expression of CDK2, and the production of ROS was activated. Of note, the DEX­mediated changes in viability and apoptosis were attenuated in MC3T3­E1 cells after treatment with 3­methyladenine, which is an autophagy inhibitor. Treatment with the antioxidant N­acetylcysteine abolished the effect of DEX on the proliferation, apoptosis, ER stress and autophagy of MC3T3­E1 cells. In conclusion, the present results indicated that DEX promoted the production of ROS, which enhanced apoptosis through activation of autophagy and ER stress in MC3T3-E1 cells.

Genetic variations in TERC and TERT genes are associated with lung cancer risk in a Chinese Han population.

The study was aimed to explore whether the TERT and TERC polymorphisms are associated with the lung cancer risk. Five TERC and TERT polymorphisms were genotyped from 554 lung cancer patients and 603 healthy controls. We used χ2 test, genetic model, linkage disequilibrium (LD) and haplotype analyses to evaluate the association between the polymorphisms and lung cancer risk. We found that the allele "C" of rs10936599 (TERC) and the allele "T" of rs10069690 (TERT) were associated with increased risk of lung cancer (OR = 1.32, 95% CI: 1.12-1.55, P = 0.001; OR = 1.41, 95% CI: 1.14-1.76, P = 0.002, respectively). The genotype of "CC" of rs10936599, genotype "CT" of rs10069690 and genotype "GG and "AG" of rs2853677 were also associated with increased the risk of lung cancer. In addition, rs10936599 under the dominant, recessive and log-additive models; rs10069690 under the dominant, overdominant and log-additive models; rs2853677 under the dominant and log-additive models were found to be associated with increased lung cancer risk. The SNP rs2242652 was found to be associated with an increased lung cancer risk under the dominant and overdominant models without adjustment. The haplotype "TA" of TERT was also associated with an increased risk of lung cancer. Our study indicated that rs10936599 (TERC) and rs10069690, rs2242652 and rs2853677 in TERT and haplotype "TA" of TERT were revealed as risk factors of lung cancer in a Chinese Han population. However, it required to verify our result and investigate the function genetic variants and mechanism of lung cancer.