Corrigendum: The role of NR4A1 in the pathophysiology of osteosarcoma: A comprehensive bioinformatics analysis of the single-cell RNA sequencing dataset.

[This corrects the article DOI: 10.3389/fonc.2022.879288.].

The Role of NR4A1 in the Pathophysiology of Osteosarcoma: A Comprehensive Bioinformatics Analysis of the Single-Cell RNA Sequencing Dataset.

Osteosarcoma is a kind of aggressive human malignancy, and the prognosis of the patients with osteosarcoma remains low. Studies have demonstrated that the tumor microenvironment plays a key role in regulating osteosarcoma progression. Recent studies have also shown that scRNA-seq plays an essential role in understanding the tumor heterogeneity and distinct subpopulations of tumors. In order to further understand the scRNA-seq data of osteosarcoma tissues, the present study further analyzed the scRNA-seq dataset (GSE152048) and explored the potential role of nuclear receptor-related genes in the pathophysiology of osteosarcoma. In our analysis, we identified 11 cell types in all the osteosarcoma tissues and nuclear receptors (NRs) were distributed in all types of cells. Further stratification analysis showed that NRs were mainly detected in "TIL" and "Osteoblastic" of the metastasis osteosarcoma, in "TIL", "Myoblast", "Endothelial", and "Myeloid" of the primary osteosarcoma, and in "Chondroblastic", "Osteoblast", and "Pericyte" of the recurrent osteosarcoma. The NRs were also differentially expressed in different cell types among the metastasis, primary, and recurrent osteosarcoma. Furthermore, several NRs such as NR4A2, NR4A1, and NR3C1 have been found to be differentially expressed in most types of DEGs among metastasis, primary, and recurrent osteosarcoma. A high expression of NR4A1 in the osteosarcoma tissues was significantly correlated with a shorter 5-year overall survival of patients with osteosarcoma. On the other hand, there was no significant association between NR4A2 expression and the 5-year overall survival of patients with osteosarcoma. The expression of NR4A1 was significantly higher in the metastasis osteosarcoma tissues than in the primary osteosarcoma tissues as validated from GSE32981 and GSE154540. The expression of NR4A1 was significantly higher in osteosarcoma tissues from patients with poor chemosensitivity than that from patients with good chemosensitivity as validated from GSE154540. Further analysis of the scRNA-seq data revealed that the percentage of osteoblasts with a high NR4A1 expression was higher in the recurrent osteosarcoma tissues than that with a low NR4A1 expression. In conclusion, the present study may suggest that NR4A1 may be an important prognostic biomarker for osteosarcoma progression. However, further validation studies should be performed to confirm our findings.

Expression and effects of leukemia inhibitory factor on nucleus pulposus degeneration.

Leukemia inhibitory factor (LIF) is a multifunctional cytokine. The present study aimed to determine the expression and effects of LIF on nucleus pulposus generation. Degenerated nucleus pulposus samples were obtained from animal models and patients with lumbar intervertebral disc herniation. Degradation scores of intervertebral discs were evaluated via magnetic resonance imaging (MRI) and histology, and the protein expression levels of LIF were detected. Furthermore, cultured primary human degenerated nucleus pulposus cells (DNPCs) were stimulated with various concentrations of recombinant human LIF protein (rhLIF), and aggrecan and collagen type II α1 (COL2α1) protein expression levels were detected by western blotting. In addition, aggrecan expression was determined by toluidine blue staining. The effects of rhLIF on proliferation and apoptosis of DNPCs were evaluated by Cell Counting Kit­8 and flow cytometry, respectively. The results revealed that the degradation scores of intervertebral discs were significantly associated with modeling time, as determined by MRI and histology. In addition, the protein expression levels of LIF were initially increased in patients with lumbar disc herniation and in rabbit models, particularly in the 2­week modeling group; however, its expression decreased with the progression of disc degeneration. Notably, LIF expression in each modeling group was higher than that in the control and 0 week modeling group. The in vitro study revealed that the protein expression levels of aggrecan and COL2α1 were significantly increased in response to rhLIF, in a dose­dependent manner, and statistical differences were identified between the treatment groups and control group. The results of toluidine blue staining were consistent with this finding. Although rhLIF had no effect on proliferation, it inhibited apoptosis of DNPCs in a concentration­dependent manner. In conclusion, LIF was upregulated during the process of intervertebral disc degeneration, and may promote the expression of extracellular matrix components. It may also be hypothesized that LIF acts as a potential protective factor by inhibiting apoptosis of DNPCs without affecting cell proliferation.

Downregulation of RSK2 influences the biological activities of human osteosarcoma cells through inactivating AKT/mTOR signaling pathways.

RSK2 (90 kDa ribosomal S6 kinase) is a downstream effector of the Ras/ERK (extracellular signal-regulated kinase) signaling pathway that has major functions in cell biological activities, including regulating nuclear signaling, cell cycle progression, cell proliferation, cell growth, protein synthesis, cell migration and cell survival, and is expressed in most types of human malignant tumors, including lung cancer, prostate and breast tumors, skin cancer and osteosarcomas (OS). RSK2 was found to be essential for osteosarcoma formation. To investigate whether RSK2 is expressed at high levels in human osteosarcome tissues and whether its expression is correlated with the aggressive biological behavior of osteosarcoma cell line (OCLs), we assessed the association between RSK2 expression and OS cell progression, as well as the effects of RSK2 inhibition on the biological activities of osteosarcoma cells. We performed immunohistochemistry to analyze the expression of RSK2 in specimens from 30 humans with osteosarcoma, and 15 normal tissues. RSK2 gene expression levels in 30 specimens with osteosarcoma were significantly higher than those of normal tissues. We performed RNA interference on three OCLs to evaluate cell apoptosis, cell growth, cell proliferation, cell motility, chemosensitivity and oncogenicity. After transfection with RSK2 shRNA, increased cell apoptosis, cell growth inhibition, cell cycle progression, weaker cell proliferation, cell migration and weaker tumor formation were observed in all OCLs. These results suggested that RSK2 expression may mediate the biological activities of OS cells and RSK2 may be an effective therapeutic target for the treatment of osteosarcomas. The AKT/mTOR, MAPK/ERK/c-Fos and Bcl2/Bax pathways were analysed to clarify the mechanisms involved.

Down-regulation of S100A9 inhibits osteosarcoma cell growth through inactivating MAPK and NF-κB signaling pathways.

BACKGROUND: Osteosarcoma (OS) is well-known for poor prognosis due to its high incidence of proliferation and metastasis. Researches have provided valuable insights into the tumorigenesis of S100A9 in some cancers. We aimed to understand the expression level, functions and mechanisms of S100A9 in human osteosarcoma for the first time. METHODS: The expression of S100A9 protein was detected in 120 human osteosarcoma tissues and 40 normal human bone tissues using tissue microarrays analysis. The knockdown of S100A9 induced by RNA interference (RNAi) method in three osteosarcoma cell lines (U2OS, 143B, MG63) was applied to analyze the effects of S100A9 on cell proliferation, cell cycle distribution, migration, invasion and xenotransplanted tumors. Moreover, MAPK-ERK1/2, MAPK-p38, NF-κB-p65, NF-κB-p50, p21, p27, CDK2 and CDK4 were tested. RESULTS: The expression of S100A9 was increased in human osteosarcoma issues and was positively correlated with clinical classification and survival rate. Down-regulation of S100A9 inhibited OS cellular proliferation, migration, invasion and cell cycle S phase in vitro and suppressed tumor formation in vivo with the reduction on PCNA and Ki67 proliferation index. Our data also demonstrated that knockdown of S100A9 repressed the protein levels of phospho-ERK1/2, phospho-p50, phospho-p65 except phospho-p38, and prompted up-regulation of p21 and p27 leading to inactivation of cyclin dependent kinase 2(CDK2) and cyclin dependent kinase 4(CDK4). CONCLUSIONS: S100A9 might be a significant role for predicting osteosarcoma prognosis and down-regulation of S100A9 could be used as a potential target for gene therapy.

SIRT1 alleviates senescence of degenerative human intervertebral disc cartilage endo-plate cells via the p53/p21 pathway.

Cartilage end plates (CEP) degeneration plays an integral role in intervertebral disc (IVD) degeneration resulting from nutrient diffusion disorders. Although cell senescence resulting from oxidative stress is known to contribute to degeneration, no studies concerning the role of senescence in CEP degeneration have been conducted. SIRT1 is a longevity gene that plays a pivotal role in many cellular functions, including cell senescence. Therefore, the aim of this study was to investigate whether senescence is more prominent in human degenerative CEP and whether SIRT1-regulated CEP cells senescence in degenerative IVD as well as identify the signaling pathways that control that cell fate decision. In this study, the cell senescence phenotype was found to be more prominent in the CEP cells obtained from disc degenerative disease (DDD) patients than in the CEP cells obtained from age-matched lumbar vertebral fractures (LVF) patients. In addition, the results indicated that p53/p21 pathway plays an important role in the senescence of CEP cells in vivo and vitro. Furthermore, SIRT1 was found to be capable of alleviating the oxidative stress-induced senescence of CEP cells in humans via p53/p21 pathway. Thus, the information presented in this study could be used to further investigate the underlying mechanisms of CEP.

Dickkopf-1 is involved in BMP9-induced osteoblast differentiation of C3H10T1/2 mesenchymal stem cells.

Bone morphogenetic protein 9 (BMP9) is a potent inducer of osteogenic differentiation of mesenchymal stem cells. The Wnt antagonist Dickkopf-1 (Dkk1) is involved in skeletal development and bone remodeling. Here, we investigated the role of Dkk1 in BMP9-induced osteogenic differentiation of MSCs. We found that overexpression of BMP9 induced Dkk1 expression in a dose-dependent manner, which was reduced by the P38 inhibitor SB203580 but not the ERK inhibitor PD98059. Moreover, Dkk1 dramatically decreased not only BMP9-induced alkaline phosphatase (ALP) activity but also the expression of osteocalcin (OCN) and osteopontin (OPN) and matrix mineralization of C3H10T1/2 cells. Furthermore, exogenous Dkk1 expression inhibited Wnt/ß-catenin signaling induced by BMP9. Our findings indicate that Dkk1 negatively regulates BMP9-induced osteogenic differentiation through inhibition of the Wnt/ß-catenin pathway and it could be used to optimize the therapeutic use of BMP9 and for bone tissue engineering. [BMB Reports 2016; 49(3): 179-184].

Percutaneous fibrin gel injection under C-arm fluoroscopy guidance: a new minimally invasive choice for symptomatic sacral perineural cysts.

BACKGROUND: Symptomatic sacral perineural cysts are a common cause of chronic pain. Surgery is one choice for symptom relief but has a high risk of cyst recurrence and complications. As a simple and safe method to manage symptomatic sacral perineural cysts, C-arm fluoroscopy-guided fibrin gel injection may represent a new minimally invasive alternative. To evaluate the efficacy of this new method, we conducted a retrospective study of 42 patients. METHODS AND FINDINGS: From June 2009 to August 2012, a total of 42 patients with symptomatic sacral perineural cysts underwent C-arm fluoroscopy-guided percutaneous fibrin gel injection therapy. Patient outcomes in terms of improvements in pain and neurologic function were evaluated during a follow-up period of 13-39 months. The preoperative and postoperative pain severity were assessed according to a 10-cm visual analog pain scale, and imaging changes were evaluated by magnetic resonance imaging. We also assessed postoperative complications. Most patients experienced benefit from the procedure: twenty-five patients (59.5%) reported excellent recovery, eleven (26.2%) reported good recovery, three (7.1%) reported fair recovery, and three (7.1%) reported poor recovery. The overall effectiveness rate (excellent and good recoveries) was 85.7%. No serious postoperative complications were observed. CONCLUSION: Percutaneous fibrin gel injection under C-arm fluoroscopy guidance could be a simple, safe and effective treatment option for symptomatic sacral perineural cysts.

SIRT1 protects against apoptosis by promoting autophagy in degenerative human disc nucleus pulposus cells.

SIRT1 could protect degenerative human NP cells against apoptosis, and there were extensive and intimate connection between apoptosis and autophagy. Up to now, the role of autophagy in the process of human IVD degeneration is unclear. We sought to explore the relationship between autophagy and human IVD degeneration and to understand whether autophagy is involved in the protective effect of SIRT1 against apoptosis in NP cells. Our results showed that the autophagosomes number, the mRNA level of LC3 and Beclin-1, the protein expression of LC3-II/I and Beclin-1, decreased in NP from DDD. Resveratrol could increase the protein expression of LC3-II/I and Beclin-1, and reduce apoptosis in degenerative NP cells. In contrast, the protein levels of LC3-II/I and Beclin-1 were down-regulated and apoptosis level was significantly up-regulated in treatment with nicotinamide or SIRT1-siRNA transfection. Further analysis identified that the expression of cleaved Caspase3 and apoptosis incidence significantly increased with the pretreatment of bafilomycin A, whether resveratrol was added or not. These suggested that autophagy may play an important role in IVD degeneration, and SIRT1 protected degenerative human NP cells against apoptosis via promoting autophagy. These findings would aid in the development of novel therapeutic approaches for degenerative disc disease treatment.