Selective Autophagy Receptor NBR1 Retards Nucleus Pulposus Cell Senescence by Directing the Clearance of SRBD1.

Intervertebral disc degeneration (IDD) is a prevalent degenerative disorder that closely linked to aging. Numerous studies have indicated the crucial involvement of autophagy in the development of IDD. However, the non-selective nature of autophagy substrates poses great limitations on the application of autophagy-related medications. This study aims to enhance our comprehension of autophagy in the development of IDD and investigate a novel therapeutic approach from the perspective of selective autophagy receptor NBR1. Proteomics and immunoprecipitation and mass spectrometry analysis, combined with in vivo and in vitro experimental verification were performed. NBR1 is found to be reduced in IDD, and NBR1 retards cellular senescence and senescence-associated secretory phenotype (SASP) of nucleus pulposus cells (NPCs), primarily through its autophagy-dependent function. Mechanistically, NBR1 knockdown leads to the accumulation of S1 RNA-binding domain-containing protein 1 (SRBD1), which triggers cellular senescence via AKT1/p53 and RB/p16 pathways, and promotes SASP via NF-κß pathway in NPCs. Our findings reveal the function and mechanism of selective autophagy receptor NBR1 in regulating NPCs senescence and degeneration. Targeting NBR1 to facilitate the clearance of detrimental substances holds the potential to provide novel insights for IDD treatment.

Osteoimmunity-Regulating Biomimetically Hierarchical Scaffold for Augmented Bone Regeneration.

Engineering a proper immune response following biomaterial implantation is essential to bone tissue regeneration. Herein, a biomimetically hierarchical scaffold composed of deferoxamine@poly(ε-caprolactone) nanoparticles (DFO@PCL NPs), manganese carbonyl (MnCO) nanosheets, gelatin methacryloyl hydrogel, and a polylactide/hydroxyapatite (HA) matrix is fabricated to augment bone repair by facilitating the balance of the immune system and bone metabolism. First, a 3D printed stiff scaffold with a well-organized gradient structure mimics the cortical and cancellous bone tissues; meanwhile, an inside infusion of a soft hydrogel further endows the scaffold with characteristics of the extracellular matrix. A Fenton-like reaction between MnCO and endogenous hydrogen peroxide generated at the implant-tissue site triggers continuous release of carbon monoxide and Mn2+ , thus significantly lessening inflammatory response by upregulating the M2 phenotype of macrophages, which also secretes vascular endothelial growth factor to induce vascular formation. Through activating the hypoxia-inducible factor-1α pathway, Mn2+ and DFO@PCL NP further promote angiogenesis. Moreover, DFO inhibits osteoclast differentiation and synergistically collaborates with the osteoinductive activity of HA. Based on amounts of data in vitro and in vivo, strong immunomodulatory, intensive angiogenic, weak osteoclastogenic, and superior osteogenic abilities of such an osteoimmunity-regulating scaffold present a profound effect on improving bone regeneration, which puts forward a worthy base and positive enlightenment for large-scale bone defect repair.

Injectable self-healing hydrogel with siRNA delivery property for sustained STING silencing and enhanced therapy of intervertebral disc degeneration.

Inflammatory responses of nucleus pulposus (NP) can induce imbalanced anabolism and catabolism of extracellular matrix, and the cytosolic dsDNA accumulation and STING-NF-κB pathway activation found in NP inflammation are considered as fairly important cause of intervertebral disc (IVD) degeneration. Herein, we constructed a siSTING delivery hydrogel of aldehyde hyaluronic acid (HA-CHO) and poly(amidoamine) PAMAM/siRNA complex to intervene the abnormal STING signal for IVD degeneration treatment, where the formation of dynamic Schiff base bonds in the system (siSTING@HPgel) was able to overcome the shortcomings such as low cellular uptake, short half-life, and rapid degradation of siRNA-based strategy. PAMAM not only formed complexes with siRNA to promote siRNA transfection, but also served as dynamic crosslinker to construct hydrogel, and the injectable and self-healing hydrogel efficiently and steadily silenced STING expression in NP cells. Finally, the siSTING@HPgel significantly eased IVD inflammation and slowed IVD degeneration by prolonging STING knockdown in puncture-induced IVD degeneration rat model, revealing that STING pathway was a therapeutic target for IVD degeneration and such novel hydrogel had great potential for being applied to many other diseases for gene delivery.

Aurora-B knockdown inhibits osteosarcoma metastasis by inducing autophagy via the mTOR/ULK1 pathway.

BACKGROUND: Autophagy plays an essential role in metastasis of malignancies. Although our studies showed that Aurora-B facilitate pulmonary metastasis in OS, the mechanism of Aurora-B kinase on autophagy and metastasis in OS has not been explored. METHODS: Clinical-pathological parameters and follow-up information was collected in OS patients. Immunohistochemical staining was performed to detect Aurora-B and LC3 protein in OS tissues. Short hairpin RNA transfection was used to silence Aurora-B in OS cells. Real-time quantitative PCR (RT-qPCR) was performed to detect Aurora-B mRNA expression in OS cells. Aurora-B and autophagy related protein were measured by Western blot. Transmission electron microscopy and laser scanning confocal microscopy were performed to observe the formation of autophagosomes and autolysosomes. Migratory and invasive ability of OS cells were measured by Wound healing and transwell assays. Orthotopic xenograft model was used to evaluate the effect of autophagy mediated by Aurora-B inhibition on pulmonary metastasis of OS. RESULTS: The elevated expression of Aurora-B protein in OS tissues negatively associated with the overall survival of OS patients. Further investigation has found that Aurora-B expression was negatively correlative with autophagy related protein LC3 in OS patient tissues. Knockdown Aurora-B stimulates autophagy and inhibits migratory and invasive ability of OS cells. Mechanistically, Aurora-B knockdown suppressed the mTOR/ULK1 signaling pathway and reactivation of the mTOR/ULK1 pathway decreased autophagy level. Furthermore, the inhibition effect of silencing Aurora-B on migration and invasion of OS was reversed by chloroquine and mTOR activator in vitro and vivo. CONCLUSIONS: Our results suggest that silencing of Aurora-B stimulate autophagy via decreasing mTOR/ULK1 and result in inhibiting OS metastasis. Targeted Aurora-B/mTOR/ULK1 pathway may be a promising treatment strategy for OS patients.

[Aurora kinase-B silencing promotes apoptosis of osteosarcoma 143B cells by ULK1 phosphorylation-induced autophagy].

OBJECTIVE: To investigate the effect of Aurora kinase B (AURKB) silencing-induced autophagy on apoptosis of osteosarcoma 143B cells and the underlying molecular mechanisms. METHODS: Human osteosarcoma 143B cells were transfected with Lv/shAURKB or the negative control vector Lv/shScrambled followed by treatment with chloroquine (CQ) for 24 h. Western blotting was performed to detect the protein expression levels of AURKB, P62, LC3, cleaved caspase-3, Bcl-2, and P-ULK1Ser555. Transmission electron microscopy and LC3 dual-label fluorescence method were used to trace the autophagosomes in 143B cells to assess cell autophagy, and the cell apoptosis was detected using flow cytometry and TUNEL assay. Co-immunoprecipitation assay was used to detect the interaction between AURKB and ULK1. RESULTS: The ratio of autophagy-related proteins LC3 II/I and the number of autophagosomes were significantly increased in 143B cells after transfection with Lv/shAURKB (P < 0.05), which significantly increased the expression of cleaved caspase-3 and reduced the expression of Bcl-2 (P < 0.05). Combined treatment of the cells with Lv/shAURKB and the autophagy inhibitor chloroquine obviously restored the expressions of caspase-3 and Bcl-2 (P < 0.05). Transfection with Lv/shAURKB significantly increased the apoptosis rate of 143B cells (P < 0.05), and this effect was significantly antagonized by combined treatment with chloroquine (P < 0.05). AURKB silencing strongly activated the phosphorylation of the autophagy-initiating protein ULK1Ser555 in 143B cells (P < 0.05). The results of co-immunoprecipitation assay confirmed when AURKB was immunoprecipitated, ULK1 also precipitated. CONCLUSIONS: Silencing AURKB can induce autophagy by activating ULK1Ser555 phosphorylation to promote apoptosis in 143B cells.

Aurora-B Promotes Osteosarcoma Cell Growth and Metastasis Through Activation of the NPM1/ERK/NF-κß/MMPs Axis.

PURPOSE: Osteosarcoma (OS) is the most common primary malignant tumor of the bone in young adolescents and children. We explored the underlying mechanism of Aurora-B in promoting OS cell proliferation and metastasis. PATIENT AND METHODS: Bioinformatics was employed to predict the substrate of Aurora-B. IHC and Western blot were used to confirm the correlation between Aurora-B and NPM1. ERK/NF-κß pathway-related proteins were detected by Western blot and immunofluorescence (IF). CCK8, wound healing, transwell, and Tunel assays were used to identify the cell proliferation, migration and apoptosis potential. Spontaneous metastasis xenografts were established to confirm the role of Aurora-B and NPM1. RESULTS: Aurora-B promotes NPM1 phosphorylation on Ser125. The phosphorylation of NPM1Ser125 induced by Aurora-B activates the ERK/NF-κß signaling. Further study revealed that Aurora-B promotes proliferation, migration and inhibits apoptosis via phosphorylating NPM1 in vitro and in vivo. CONCLUSION: Aurora-B promotes OS malignancy via phosphorylating NPM1Ser125 and activating ERK/NF-κß signaling.

LHX2 promotes malignancy and inhibits autophagy via mTOR in osteosarcoma and is negatively regulated by miR-129-5p.

The transcript factor LHX2 is dysregulated in many cancers but its role in osteosarcoma (OS) remains unclear. In this study, we confirm that LHX2 is up-regulated in osteosarcoma, and that its silencing inhibits OS malignancy and induces autophagy via mTOR signaling. We further demonstrate that miR-129-5p negatively regulates LHX2 and suppresses the malignant phenotypes of OS. LHX2 overexpression could restore the malignant phenotypes. In conclusion, LHX2 regulates tumorigenesis and autophagy via mTOR in OS and is negatively regulated by miR-129-5p. Targeting the miR-129-5p/LHX2/mTOR axis therefore represents a novel therapeutic strategy for OS treatment.

Anoikis resistant mediated by FASN promoted growth and metastasis of osteosarcoma.

The pulmonary metastasis of osteosarcoma (OS) occurs commonly, which resulted from anoikis resistant (AR) of tumor cells as reported by previous studies, but the exact roles of AR in osteosarcoma were not fully studied. Our previous investigations showed fatty acid synthase (FASN) was relating to clinical features of patients with OS. In this study, we aim to explore the functions of FASN in the AR OS cells in vitro and in vivo and study the downstream effectors of FASN. In the present study, we used our established cell model to study the AR. We revealed that AR promoted cell proliferation and migration as determined by colony formation assay and transwell assay. In addition, AR assisted tumor growth in vivo. In the AR cells, the expression of FASN was higher. Thus, we constructed lentiviruses to silence or overexpress FASN in four cell lines to study functions of FASN. Silence of FASN reduced cell colonies and migration while overexpression of FASN increased colonies and migration in suspended cells. Loss of functions of FASN induced cell apoptosis in suspended OS cells while gain of function of FASN suppressed apoptosis as determined by flow cytometry. We found the levels of p-ERK1/2 and Bcl-xL declined when FASN was silenced while they increased when FASN was overexpressed. In addition, results showed that the levels of FASN and its potential related molecules (p-ERK1/2 and Bcl-xL) increased in 143B-AR and MG-63-AR cells. In vivo study showed that inhibition of FASN decreased pulmonary metastasis of OS. In conclusion, we showed that anoikis resistant and FASN as two interactional factors facilitated the progress of osteosarcoma.