Ginsenoside Rg1 relieves rat intervertebral disc degeneration and inhibits IL-1ß-induced nucleus pulposus cell apoptosis and inflammation via NF-κB signaling pathway.

The study aimed to investigate the effect of ginsenoside Rg1 on intervertebral disc degeneration (IVDD) in rats and IL-1ß-induced nucleus pulposus (NP) cells, and explore its underlying mechanism. Forty IVDD rat models were divided into the IVDD group, low-dose (L-Rg1) group (intraperitoneal injection of 20 mg/kg/d ginsenoside Rg1), medium-dose (M-Rg1) group (intraperitoneal injection of 40 mg/kg/d ginsenoside Rg1), and high-dose (H-Rg1) group (intraperitoneal injection of 80 mg/kg/d ginsenoside Rg1). The pathological change was observed by HE and safranin O-fast green staining. The expression of IL-1ß, IL-6, TNF-α, MMP3, aggrecan, and collagen II was detected. The expression of NF-κB p65 in IVD tissues was detected. Rat NP cells were induced by IL-1ß to simulate IVDD environment and divided into the control group, IL-1ß group, and 20, 50, and 100 µmol/L Rg1 groups. The cell proliferation activity, the apoptosis, and the expression of IL-6, TNF-α, MMP3, aggrecan, collagen II, and NF-κB pathway-related protein were detected. In IVDD rats, ginsenoside Rg1 improved the pathology of IVD tissues; suppressed the expression of IL-1ß, IL-6, TNF-α, aggrecan, and collagen II; and inhibited the expression of p-p65/p65 and nuclear translocation of p65, to alleviate the IVDD progression. In the IL-1ß-induced NP cells, ginsenoside Rg1 also improved the cell proliferation and inhibited the apoptosis and the expression of IL-6, TNF-α, aggrecan, collagen II, p-p65/p65, and IκK in a dose-dependent manner. Ginsenoside Rg1 alleviated IVDD in rats and inhibited apoptosis, inflammatory response, and ECM degradation in IL-1ß-induced NP cells. And Rg1 may exert its effect via inhibiting the activation of NF-κB signaling pathway.

Clinical applications of skin traction technique with adjustable tension in treatment of large area skin defects.

OBJECTIVE: To explore the clinical applications of the adjustable skin traction technique in the treatment of large area skin defects. RESEARCH DESIGN: A prospective study. BACKGROUND: The skin is the largest organ of the human body and skin tissue exposed to external environment which makes it vulnerable to damage. There are many reasons for skin defects such as trauma, infection, burns, scars, tumors resection, inflammation, pigmented nevus, etc. Skin traction is the application of pulling force to the trunk or extremities for immobilization, fracture reduction and deformity correction. This technique accurately controls skin expansion which is safe, convenient and accelerates wound healing. METHODS: A prospective study was conducted on 80 patients suffered from large area skin defects in the department of orthopedics, the first affiliated hospital of Zhengzhou University from September 2019 to January 2023. There were 40 patients in the experimental group who underwent skin traction. In contrast, 40 people in the control group underwent skin flaps or skin grafts without skin traction. The inclusion criteria include large area skin defects, normal peripheral skin & blood supply, normal vital organs, no severe coagulation dysfunction etc. Male & female with and without skin traction are 22 & 18 and 25 & 15 respectively. The skin traction device used was a hook and single rod type. The skin defect area was approximately 15 cm × 9-43 cm × 10 cm. RESULTS: Postoperatively, the experimental group with traction showed 2 cases of skin infection, 1 case of skin necrosis and 3 cases of inflammation recurrence. In contrast, the control group without traction showed 8 cases of skin infection, 6 cases of skin necrosis and 10 cases of inflammation recurrence. Skin infection (P = 0.04), skin necrosis (P = 0.02) and inflammatory response (P = 0.03) represented significant differences between two groups. There was also a significant difference in hospitalization costs (P = 0.001). CONCLUSION: Skin traction has huge clinical applications including a shorter hospital stay, faster wound healing, lower hospitalization cost, high satisfaction rate, and a fair skin appearance after surgery. It is an effective method of treating skin and musculoskeletal defects.

Duhuo Jisheng Decoction suppresses apoptosis and mitochondrial dysfunction in human nucleus pulposus cells by miR-494/SIRT3/mitophagy signal axis.

BACKGROUND: Increasing evidence suggests that mitophagy is responsible for the pathogenesis of intervertebral disk (IVD) degeneration. Previous studies have shown that Duhuo Jisheng Decoction (DHJSD), a classic Fangji of traditional Chinese medicine, can delay IVD degeneration; however, its specific mechanism of action is unknown. In this study, we investigated the mechanism by which DHJSD treatment prevented IVD degeneration in IL-1ß-treated human nucleus pulposus (NP) cells in vitro. METHODS: Cell Counting Kit-8 was performed to explore the effects of DHJSD on the viability of NP cells exposed to IL-1ß. The mechanism by which DHJSD delays IVD degeneration was explored using luciferase reporter assay, RT-qPCR, western blotting, TUNEL assay, mitophagy detection assay, Mito-SOX, Mitotracker and in situ hybridization. RESULTS: We observed that DHJSD enhanced the viability of NP cells treated with IL-1ß in a concentration-time dependent approach. Moreover, DHJSD lessened IL-1ß-induced NP apoptosis and mitochondrial dysfunction and activated mitophagy in NP cells treated with IL-1ß. Mitophagy suppressor cyclosporin A reversed the beneficial impacts of DHJSD in NP cells. In addition, the differential expression of miR-494 regulated IL-1ß-induced NP apoptosis and mitochondrial dysfunction, and the protective impact of miR-494 on NP cells treated with IL-1ß was achieved by mitophagy activation, which was regulated by its target gene, sirtuin 3 (SIRT3). Finally, we observed that DHJSD treatment could effectively delay IL-1ß-induced NP apoptosis by affecting the miR-494/SIRT3/mitophagy signal axis. CONCLUSIONS: These results show that the miR-494/SIRT3/mitophagy signaling pathway is responsible for the apoptosis and mitochondrial dysfunction of NP cells and that DHJSD may exert protective effects against IVD degeneration by regulating the miR-494/SIRT3/mitophagy signal axis.

Circular RNA_0003800 exacerbates IL-1ß-induced chondrocyte injury via miR-197-3p/SOX5 axis.

BACKGROUND: Osteoarthritis (OA) is a serious degenerative disease of articular cartilage, which has a great impact on the quality of life of patients. Circular RNA (circRNA) plays an important role in OA progression. Our study aims to explore the role and mechanism of circ_0003800 in OA. METHODS: Circ_0003800, microRNA-197-3p (miR-197-3p) and SRY-box transcription factor 5 (SOX5) contents were measured by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot. Cell Counting Kit-8 (CCK8), 5-ethynyl-2'-deoxyuridine (EdU), flow cytometry, western blot and enzyme-linked immunosorbent assay (ELISA) were deployed to evaluate cell proliferation, apoptosis, extracellular matrix (ECM) degradation, inflammatory response and oxidative stress. Interaction of miR-197-3p and circ_0003800 or SOX5 was evidenced by dual-luciferase reporter system, RNA immunoprecipitation (RIP) and RNA pull down assays. RESULTS: OA tissues and model cells had higher abundance of circ_0003800 and SOX5, while miR-197-3p content was lower. Functionally, circ_0003800 knockdown alleviated IL-1ß-mediated injury in C28/I2 cells. Mechanistically, circ_0003800 could sponge miR-197-3p, and miR-197-3p could target SOX5. Besides, in-miR-197-3p reversed the suppressive effect of circ_0003800 downregulation on IL-1ß-induced C28/I2 cell injury, and SOX5 overexpression could also diminish the inhibitory effect of miR-197-3p on IL-1ß-induced C28/I2 cell injury. CONCLUSION: Circ_0003800 exacerbates IL-1ß-induced chondrocyte injury via miR-197-3p/SOX5 axis.

Knockdown of Circ_0037658 Alleviates IL-1ß-Induced Osteoarthritis Progression by Serving as a Sponge of miR-665 to Regulate ADAMTS5.

Background: Osteoarthritis (OA) is a chronic musculoskeletal degeneration disease which brings great pain to patients and a tremendous burden on the world's medical resources. Previous reports have indicated that circular RNAs (circRNAs) are involved in the pathogenesis of OA. The purpose of this study was to explore the role and mechanism of circ_0037658 in the OA cell model. Methods: The content of interleukin-6 (IL-6) and tumor necrosis factor α (TNF-α) was measured using enzyme-linked immunosorbent assay (ELISA). Cell proliferation ability and apoptosis were detected using Cell Counting Kit-8 (CCK-8), 5-ethynyl-2'-deoxyuridine (EDU), and flow cytometry assays. Western blot assay was used to measure the protein levels of Bcl-2-related X protein (Bax), cleaved-caspase-3, MMP13, Aggrecan, and ADAMTS5. The expression of circ_0037658, microRNA-665 (miR-665), and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) 5 was detected using real-time quantitative polymerase chain reaction (RT-qPCR). Dual-luciferase reporter assay and RNA Immunoprecipitation (RIP) assay were manipulated to analyze the relationships of circ_0037658, miR-665, and ADAMTS5. Results: Human chondrocytes (CHON-001 cells) were treated with interleukin-1ß (IL-1ß) to establish an OA cell model. Circ_0037658 and ADAMTS5 levels were increased, and miR-665 was decreased in OA cartilage samples and IL-1ß-treated chondrocyte cells. Moreover, circ_0037658 silencing promoted proliferation and impaired inflammation, apoptosis, and ECM degradation in IL-1ß-treated CHON-001 cells. Mechanically, circ_0037658 acted as a sponge for miR-665 to regulate ADAMTS5 expression. Conclusion: Circ_0037658 knockdown relieved IL-1ß-triggered chondrocyte injury via regulating the miR-665/ADAMTS5 axis, promising an underlying therapeutic strategy for OA.

Effect of Hypoxia-Inducible Factor-1α on Osteogenesis of Titanium Dioxide Nanotube Bone Marrow Mesenchymal Stem Cells with Different Diameters Under Periodic Tensile Stress.

Bone marrow mesenchymal stem cells (BMSC) have the ability to multi polarize with multiple tropisms and participate in tissue remodeling. This study assessed the effect of titanium dioxide nanotubes with different diameters on ossification of BMSC cells and HIF-1α expression in BMSC ossification. Titanium dioxide nanotubes with different diameters were prepared and then the following groups were set according to the size of pressure; Ti group, NT10 group, NT30 group, and NT60 group. Analysis of cell morphology was done by fluorescence microscope, while adhesion and proliferation were assessed by MTT assay. Moreover, ALP activity, collagen secretion and outer matrix mineralization and expression of HIF-1α, VEGF, and TWIST were assessed by RT-PCR and Western blot. The P3 generation of BMSC cells was successfully obtained. Three types of nanotubes were arranged regularly and contact angle showed NT60Ti>NT30>NT60 (P < 0.05). Cells from NT30 and N60 groups showed obvious expansion with pseudopodia and pseudo plates of cells. Cell adhesion showed changes in sizes of NT10>Ti>NT30>NT66. NT60 group showed lower cell proliferation and higher ALP activity and collagen secretion than the other groups. NT30 and NT60 group presented higher mineralization level, larger diameter, and higher degree of promotion. The NT30 group presented lowest content of HIF-1α (0.12 ± 0.03), VEGF (0.013 ± 0.004), and TWIST (0.014 ± 0.003). Inoculation of BMSCs on titanium dioxide nanotubes of different diameters under cyclical tensile stress environment can promote growth of BMSC cells in a diameter-dependent manner.

Expression of immune-related genes as prognostic biomarkers for the assessment of osteosarcoma clinical outcomes.

Cancer immunotherapy is a promising therapeutic approach, but the prognostic value of immune-related genes in osteosarcoma (OS) is unknown. Here, Target-OS RNA-seq data were analyzed to detect differentially expressed genes (DEGs) between OS subgroups, followed by functional enrichment analysis. Cox proportional risk regression was performed for each immune-related gene, and a risk score model to predict the prognosis of patients with OS was constructed. The risk scores were calculated using the risk signature to divide the training set into high-risk and low-risk groups, and validation was performed with GSE21257. We identified two immune-associated clusters, C1 and C2. C1 was closely related to immunity, and the immune score was significantly higher in C1 than in C2. Furthermore, we validated 6 immune cell hub genes related to the prognosis of OS: CD8A, KIR2DL1, CD79A, APBB1IP, GAL, and PLD3. Survival analysis revealed that the prognosis of the high-risk group was significantly worse than that of the low-risk group. We also explored whether the 6-gene prognostic risk model was effective for survival prediction. In conclusion, the constructed a risk score model based on immune-related genes and the survival of patients with OS could be a potential tool for targeted therapy.

Circular RNA hsa_circ_0032463 Acts as the Tumor Promoter in Osteosarcoma by Regulating the MicroRNA 498/LEF1 Axis.

Several studies have examined the relationship between osteosarcoma (OS) and microRNAs (miRNAs). However, only a few researchers have investigated the underlying mechanism of circular RNAs (circRNAs) in OS development. Our paper aimed to assess how hsa_circ_0032463 (abbreviated "circ_0032463" here) initiates and regulates OS progression. We detected circ_0032463 expression in OS tissues and cell lines by using reverse transcription-quantitative PCR (RT-qPCR) analysis and then investigated the interaction between circ_0032463, miRNA 489 (miR-498), and LEF1 using RNA pulldown, RNA immunoprecipitation (RIP), and luciferase assays. The effect of the circ_0032463/miR-498/LEF1 axis on the migration, proliferation, and apoptosis levels of OS cells was explored using CCK-8, bromodeoxyuridine (BrdU), wound healing, and fluorescein isothiocyanate (FITC) assays. Our findings revealed that circ_0032463 expression was upregulated in OS tissues and cell lines. We also found that circ_0032463 interacted with miR-498, thereby reducing the expression of miR-498 in OS cells. Experimental results indicated that miR-498 could directly target LEF1 in OS cells and that circ_0032463 could abrogate the tumor-inhibitory effect of miR-498 by upregulating LEF1 in OS. More specifically, by binding to miR-498 and inhibiting LEF1 expression, circ_0032463 promoted the migration and proliferation abilities of OS cells and suppressed the apoptosis ability of OS cells. Overall, this research suggested that circ_0032463 could promote OS development by regulating the miR-498/LEF1 axis.

Punicalagin attenuates osteoarthritis progression via regulating Foxo1/Prg4/HIF3α axis.

BACKGROUND: Punicalagin (PUN) is a common anti-inflammatory polyphenol. However, the function and mechanism of PUN in osteoarthritis remains unknown. METHODS: Chondrocytes were isolated from rats, and confirmed by toluidine blue staining and immunofluorescence. Chondrocytes were challenged by lipopolysaccharide (LPS), and rat osteoarthritis model was established by Hulth method. The secretion of inflammatory factors, cell viability and apoptosis were tested via enzyme linked immunosorbent assay (ELISA), MTT and flow cytometry. The levels of forkhead box O1 (Foxo1), proteoglycan 4 (Prg4), hypoxia-inducible factor-3α (HIF3α), autophagy-related genes or extracellular matrix (ECM)-related proteins were examined via quantitative reverse transcription polymerase chain reaction (qRT-PCR), western blot or immunohistochemistry. The cartilage tissue damage was assessed via hematoxylin-eosin (HE) staining, toluidine blue staining and terminal dexynucleotidyl transferase (TdT)-mediated dUTP nick and labeling (TUNEL) staining. RESULTS: LPS triggered inflammatory injury in chondrocytes. PUN promoted autophagy to mitigate LPS-induced inflammatory injury. Foxo1 silence attenuated the effect of PUN on LPS-mediated autophagy inhibition and inflammatory injury. Promotion of Prg4/HIF3α axis abolished the influence of Foxo1 knockdown on LPS-mediated chondrocytes injury. PUN mitigated the inflammatory injury in rat osteoarthritis model by promoting autophagy and inhibiting inflammation and ECM degradation via Foxo1/Prg4/HIF3α axis. CONCLUSION: PUN attenuates LPS-induced chondrocyte injury and osteoarthritis progression by regulating Foxo1/Prg4/HIF3α axis.

Down-regulation of circITCH promotes osteosarcoma development and resistance to doxorubicin via the miR-524/RASSF6 axis.

BACKGROUND: Osteosarcoma (OS) is a malignant bone cancer, in which circular RNAs (circRNAs) act as important modulators. The present study aimed to explore the functional role of circRNA itchy E3 ubiquitin protein ligase (circITCH) in the development and doxorubicin (DXR) resistance of OS and the possible mechanistic pathway. METHODS: A quantitative real-time polymerase chain reaction or western blot assays were exploited to analyze the expression of circITCH, miR-524 and Ras association domain family member 6 (RASSF6). Cell viability and half-maximal inhibitory concentration (IC50 ) value of DXR were monitored using a cell counting kit-8 assay. Cell migration, invasion and apoptosis were determined via a transwell assay and flow cytometry. The target interaction among circITCH, miR-524 and RASSF6 was validated by dual-luciferase reporter and RNA immunoprecipitation assays. A xenograft model of MG-63/DXR cells stably expressing circITCH in nude mice was established for assessing the role of circITCH in vivo. RESULTS: Down-regulation of circITCH and RASSF6, as well as the up-regulation of miR-524, was revealed in OS by investigating 40 paired OS tissue and normal tissue samples. Overexpression of circITCH lowered the cell viability, IC50 value of DXR, migration and invasion, whereas it facilitated apoptosis of OS cells. circITCH sponged miR-524 to up-regulate RASSF6, causing OS progression inhibition and DXR resistance reduction. Additionally, circITCH up-regulation reduced tumor growth in vivo. CONCLUSIONS: Transduction with circITCH represses OS progression and promotes DXR sensitivity by the miR-524/RASSF6 axis, providing a new perspective for therapeutic intervention.

Cartilage tissue miR-214-3p regulates the TrkB/ShcB pathway paracrine VEGF to promote endothelial cell migration and angiogenesis.

BACKGROUND: This study was designed to explore the mechanisms through which chondrocytes regulated endothelial cell migration and angiogenesis in osteoarthritis (OA). METHODS: The expressions of related genes of OA were detected by Western blot and real-time quantitative PCR. Chondrocytes were co-cultured with endothelial cells, and migration as well as angiogenesis rates, and vascular endothelial growth factor (VEGF) secretion of the cells were detected. The relationship between miRNA and TrkB were analyzed by bioinformatics analysis, RNA immunoprecipitation and dual-luciferase assays. The effects of miRNA on the histopathology of the OA mice were determined. RESULTS: The expressions of NGF, TrkA, TrkB, and ShcB were increased significantly in OA patients. IL-1ß promoted the expressions of TrkA, TrkB, and ShcB in chondrocytes and inhibited the expressions of chondrogenic differentiation markers, but shTrkB partially reversed IL-1ß-mediated chondrogenic differentiation. Overexpression of TrkB promoted cell migration, angiogenesis, and VEGF levels, while silencing ShcB reversed the regulation of TrkB. Moreover, chondrocytes miR-214-3p regulated endothelial cell migration and angiogenesis by targeting TrkB paracrine VEGF to activate PI3K/Akt pathway proteins. In addition, overexpressed miR-214-3p improved collagenase-induced cartilage and synovial damage in OA mice. CONCLUSION: The activation of TrkB/ShcB signaling pathway paracrine VEGF is mediated by miR-214-3p in chondrocytes and it regulates endothelial cell migration and angiogenesis in the development of OA.

LncRNA X inactive-specific transcript promotes osteoclast differentiation through Tgif2 by acting as a ceRNA of miR-590-3p in a murine model.

Aim: This study aims to investigate whether long noncoding RNA (lncRNA)  X-inactive specific transcript (Xist) can regulate osteoclast differentiation in osteoporosis and the mechanism. Materials & methods: The mouse model of osteoporosis was established by ovariectomy surgery. Osteoclast differentiation from RAW264.7 cells was induced in vitro. The relationships between associated genes were assessed. Results:Xist and Tgif2 were upregulated, but miR-590-3p was downregulated in ovariectomy mouse femurs and cell models. Xist knockdown or miR-590-3p overexpression inhibited Tgif2 expression and osteoclast differentiation. Tgif2 and Xist were the targets of miR-590-3p. Increased miR-590-3p expression inhibited Tgif2 level and osteoclast differentiation, while Xist overexpression reversed these effects. Conclusion:Xist serves as a ceRNA of miR-590-3p to promote Tgif2 level; thereby, contributing to osteoclast differentiation.

Hsa_circ_0032463 acts as the tumor promoter in osteosarcoma by regulating the miR­330­3p/PNN axis.

Osteosarcoma (OS), also known as bone cancer, is a threat to the lives of millions of adolescents worldwide. Although dedicated efforts have been invested in reducing the mortality rate of this bone cancer, the research community is yet to find the exact causes of OS. Thus, the present research aimed to study the association between circular RNA circ_0032463 and OS progression. The impact of circ_0032463 on cells with OS was first evaluated using reverse transcription­quantitative PCR. This evaluation was followed by the assessment of cell proliferation, viability, apoptosis, invasion and adhesion using BrdU, Cell Counting Kit­8, flow cytometry, Transwell and cell adhesion assays, respectively. RNA pull­down, RNA immunoprecipitation chip and dual­luciferase reporter systems were utilized to investigate the relationship between circ_0032463, microRNA (miR)­330­3p and Pinin desmosome associated protein (PNN) in OS. The findings indicated that circ_0032463 and PNN were highly expressed in OS tissues and OS cell lines, and that they facilitated cell proliferation, viability, invasion and adhesion, but attenuated cell apoptosis in OS cells. The low expression of miR­330­3p suppressed OS development. It was also noted that circ_0032463 inhibited miR­330­3p to upregulate PNN expression. In conclusion, this study confirmed that by regulating the miR­330­3p/PNN axis, circular RNA circ_0032463 could function as a tumor enhancer in cells with OS.

Sex difference for the risk of amputation in diabetic patients: A systematic review and meta-analysis.

The risk of amputation is a sequelae of diabetic foot ulceration, which are significantly increased in diabetic patients and caused huge morbidly and mortality. However, whether the risk amputation in diabetic patients are differing in male and female remains inconclusive. We therefore conducted a systematic review and meta-analysis to assess the sex difference for the risk of amputation in diabetic patients. We systematically searched PubMed, EmBase, and the Cochrane library to identify eligible study from their inception up to November 2020. The diagnostic value of male patients on subsequent amputation risk were assessed by using sensitivity, specificity, positive and negative likelihood ratio (PLR and NLR), diagnostic odds ratio (DOR), and area under the receiver operating characteristic curve (AUC). Twenty-two studies recruited a total of 33,686,171 diabetic patients were selected for quantitative analysis. The risk of amputation in male diabetic patients was greater than female diabetic patients (DOR: 1.38; 95%CI: 1.13-1.70; P<0.001). The sensitivity and specificity for male diabetic patients on the risk of amputation were 0.72 (95%CI: 0.72-0.73), and 0.51 (95%CI: 0.51-0.51), respectively. Moreover, the PLR and NLR of male diabetic patients for predicting amputation were 1.13 (95%CI: 1.05-1.22), and 0.82 (0.72-0.94), respectively. Furthermore, the AUC for male diabetic patients on amputation risk was 0.56 (95%CI: 0.48-0.63). This study found male diabetic patients was associated with an increased risk of amputation than female diabetic patients, and the predictive value of sex difference on amputation risk in diabetic patients was mild.

Downregulation of long non-coding RNA UCA1 represses tumorigenesis and metastasis of osteosarcoma via miR-513b-5p/E2F5 axis.

Long non-coding RNAs have the regulatory roles in different kinds of human cancers. The key point of this study was to research the functional mechanisms of urothelial carcinoma associated 1 (UCA1) in the development of osteosarcoma. Quantitative real-time PCR was adopted for the expression detection of UCA1, microRNA-513b-5p (miR-513b-5p) and E2F transcription factor 5 (E2F5). The target relation was verified via dual-luciferase reporter assay and RNA pull-down assay. Cell proliferation was evaluated using Cell Counting Kit-8 and colony formation assays. Transwell assay was applied to assess cell migration and invasion. Western blot was performed for protein examination. Xenograft experiment was used to explore the effect of UCA1 on osteosarcoma in vivo. UCA1 expression was enhanced while miR-513b-5p was refrained in osteosarcoma tissues and cells. MiR-513b-5p was a target of UCA1. Inhibition of UCA1 or overexpression of miR-513b-5p suppressed osteosarcoma cell proliferation, migration and invasion. E2F5 was identified as a downstream gene of miR-513b-5p. MiR-513b-5p inhibitor or E2F5 overexpression rescued the progression inhibition of osteosarcoma by UCA1 knockdown, and UCA1 regulated E2F5 and Cyclin E expression by targeting miR-513b-5p. Downregulation of UCA1 restrained the tumorigenesis of osteosarcoma in vivo through the miR-513b-5p/E2F5 axis. Collectively, knockdown of UCA1 inhibited tumorigenesis and metastasis of osteosarcoma via regulating the miR-513b-5p/E2F5 axis. UCA1 might be a biological indicator in the progression and treatment of osteosarcoma.

LncRNA NEAT1 regulates chondrocyte proliferation and apoptosis via targeting miR-543/PLA2G4A axis.

Osteoarthritis (OA), which is characterized by articular cartilage degeneration, shows a gradually increasing incidence with age. This study explored the molecular mechanism underlying the proliferation and apoptosis of chondrocytes during OA progression. In this study, chondrocytes were isolated from human knee cartilages. The targeted relationship among nuclear enriched abundant transcript 1 (NEAT1), microRNA-543 (miR-543) and PLA2G4A was predicted on TargetScan V7.2 and starBase and validated by performing dual-luciferase reporter assay. High-expressed NEAT1 was detected in OA cartilage and chondrocytes. NEAT1 was negatively correlated with miR-543 and was low-expressed in OA cartilage and PLA2G4A was negatively correlated with miR-543 and was high-expressed in OA cartilage. In OA chondrocytes, the overexpressed NEAT1 inhibited the expressions of p-Akt1 and Bcl-2 and upregulated that of matrix metalloprotease (MMP)-3, MMP-9, MMP-13, interleukin (IL)-6 and IL-8, but such effects of overexpressed NEAT1 were reversed by miR-543 mimic. SiRNA-NEAT1 exerted an opposite effect to NEAT1 overexpression on OA chondrocytes, but this could be reversed by miR-543 inhibitor. The effect of PLA2G4A overexpression was the opposite to miR-543 mimic on OA chondrocytes. In conclusion, NEAT1 could sponge miR-543 to induce PLA2G4A expression, inhibit chondrocyte proliferation and promote apoptosis.

ROS-Mediated Necroptosis Is Involved in Iron Overload-Induced Osteoblastic Cell Death.

Excess iron has been reported to lead to osteoblastic cell damage, which is a crucial pathogenesis of iron overload-related osteoporosis. However, the cytotoxic mechanisms have not been fully documented. In the present study, we focused on whether necroptosis contributes to iron overload-induced osteoblastic cell death and related underlying mechanisms. Here, we showed that the cytotoxicity of iron overload in osteoblastic cells was mainly due to necrosis, as evidenced by the Hoechst 33258/PI staining, Annexin-V/PI staining, and transmission electronic microscopy. Furthermore, we revealed that iron overload-induced osteoblastic necrosis might be mediated via the RIPK1/RIPK3/MLKL necroptotic pathway. In addition, we also found that iron overload was able to trigger mitochondrial permeability transition pore (mPTP) opening, which is a critical downstream event in the execution of necroptosis. The key finding of our experiment was that iron overload-induced necroptotic cell death might depend on reactive oxygen species (ROS) generation, as N-acetylcysteine effectively rescued mPTP opening and necroptotic cell death. ROS induced by iron overload promote necroptosis via a positive feedback mechanism, as on the one hand N-acetylcysteine attenuates the upregulation of RIPK1 and RIPK3 and phosphorylation of RIPK1, RIPK3, and MLKL and on the other hand Nec-1, siRIPK1, or siRIPK3 reduced ROS generation. In summary, iron overload induced necroptosis of osteoblastic cells in vitro, which is mediated, at least in part, through the RIPK1/RIPK3/MLKL pathway. We also highlight the critical role of ROS in the regulation of iron overload-induced necroptosis in osteoblastic cells.

MicroRNA-613 alleviates IL-1ß-induced injury in chondrogenic CHON-001 cells by targeting fibronectin 1.

BACKGROUND: Osteoarthritis (OA) is an aging-related chronic degenerative joint disease. A number of miRNAs have been found to be involved in the development of OA, but the role of miR-613 in OA remains unclear. Thus, this study aimed to investigate the role of miR-613 during the progression of OA. METHODS: CHON-001 cells were transfected with miR-613 agonist for 48 h, and then exposed to 10 ng/mL IL-1ß for 24 h. Cell viability, cell proliferation and cell apoptosis in CHON-001 cells were assessed by CCK-8, immunofluorescence, and flow cytometry assays, respectively. In addition, the dual luciferase reporter system assay was used to determine the interaction of miR-613 and fibronectin 1 in CHON-001 cells. RESULTS: The level of miR-613 was significantly decreased in IL-1ß-treated CHON-001 cells. Overexpression of miR-613 markedly inhibited IL-1ß-induced apoptosis in CHON-001 cells. In addition, upregulation of miR-613 obviously alleviated IL-1ß-induced inflammatory response and cartilage matrix degradation in CHON-001 cells. Meanwhile, fibronectin 1 was identified as a direct binding target of miR-613 in CHON-001 cells. Overexpression of miR-613 alleviated IL-1ß-induced injury in CHON-001 cells via downregulating the expression of fibronectin 1. Furthermore, overexpression of miR-613 alleviated cartilage degradation, and reduced OARSI scores and subchondral bone thickness in a mouse model of OA. CONCLUSION: Our data indicated that overexpression of miR-613 could inhibit IL-1ß-induced injury in CHON-001 cells via decreasing the level fibronectin 1 in vitro, and alleviate the symptoms of OA in vivo. Therefore, miR-613 might be a potential therapeutic option for the treatment of OA.

Ubiquitin-specific protease 49 attenuates IL-1ß-induced rat primary chondrocyte apoptosis by facilitating Axin deubiquitination and subsequent Wnt/ß-catenin signaling cascade inhibition.

Osteoarthritis (OA) is an age-related chronic joint degenerative disease. Interleukin 1 beta (IL-1ß) is considered a marker for the progression of OA. In this study, we found that Ubiquitin-Specific Peptidase 49 (USP49) was significantly less expressed in OA patients compared with healthy individuals. Treating primary rat chondrocytes with different concentrations of IL-1ß resulted in decreased Usp49 expression, while Usp49 overexpression could attenuate IL-1ß-induced chondrocyte apoptosis by promoting Axin deubiquitination. The deubiquitination of Axin led to the accumulation of the protein, which in turn resulted in ß-catenin degradation and Wnt/ß-catenin signaling cascade inhibition. Interestingly, we also found that [6]-gingerol, an anti-OA drug, could upregulate the protein level of Usp49 and suppress the Wnt/ß-catenin signaling cascade in primary rat chondrocytes. Taken together, our study not only demonstrates that Usp49 can negatively regulate the progression of OA by inhibiting the Wnt/ß-catenin signaling cascade, but also elucidates the underlying molecular mechanisms.