RESUMO
BACKGROUND: To investigate the potential role of Long Non-coding RNAs (lncRNAs) in the progression of osteosarcoma. METHODS: The candidate lncRNAs were screened with RNA-seq and confirmed with quantitative real-time PCR. Using MTS, transwell assay, and flow cytometric analysis, the effects of overexpressed lnc-SELPLG-2:1 on cell functions were determined. Immunohistochemical staining, fluorescence in situ hybridization, and luciferase reporter assay were used to evaluate the potential mechanism of lnc-SELPLG-2:1 in vivo and in vitro using a tumor model. Moreover, the effects of overexpression of hsa-miR-10a-5p on the functions of SaOS2 cells were determined using functional cell analysis. A response test was used to confirm the mechanism by which lnc-SELPLG-2:1 sponge hsa-miR-10a-5p promotes the expression of BTRC to regulate osteosarcoma. RESULTS: Lnc-SELPLG-2:1 was highly expressed in osteosarcoma compared to normal cells and bone and marrow samples. Inhibition of lnc-SELPLG-2:1 accelerated cell apoptosis and suppressed cell proliferation, migration, and invasion, whereas lnc-SELPLG-2:1 overexpression had the opposite effect. Moreover, inhibiting lnc-SELPLG-2:1 in an in vivo model decreased tumor size and suppressed the expression of cell migration-related proteins. The prediction, dual luciferase assay, and response test results indicated that hsa-miR-10-5p and BTRC were involved in the lnc-SELPLG-2:1 cascade. Unlike lnc-SELPLG-2:1, hsa-hsa-miR-10a-5p had opposite expression and function. Competitive binding of lnc-SELPLG-2:1 to hsa-hsa-miR-10a-5p prevented BTRC from miRNA-mediated degradation, thereby activating the expression of VIM, MMP9, and MMP2, promoting osteosarcoma cell proliferation, migration, and invasion, and inhibiting apoptosis. CONCLUSION: Lnc-SELPLG-2:1 is an oncogenesis activator in osteosarcoma, and its functions are performed via hsa-miR-10a-5p /BTRC cascade.
Assuntos
Neoplasias Ósseas , MicroRNAs , Osteossarcoma , RNA Longo não Codificante , Neoplasias Ósseas/patologia , Carcinogênese/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Humanos , Hibridização in Situ Fluorescente , Metaloproteinase 2 da Matriz , Metaloproteinase 9 da Matriz , Glicoproteínas de Membrana , MicroRNAs/genética , MicroRNAs/metabolismo , Osteossarcoma/patologia , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismoRESUMO
As total joint replacement is widely applied for severe arthropathy, peri-prosthetic aseptic loosening as one of the main causes of implant failure has drawn wide attention. Wear particles such as titanium particles (TiPs) derived from prosthesis can initiate macrophages inflammation and sequentially activate osteoclasts, which results in bone resorption and osteolysis for long-term. Therefore, inhibiting wear particles induced macrophages inflammation is considered as a promising therapy for AL. In this research, we found that the inhibition of p110δ, a member of class IA PI3Ks family, could significantly dampen the TiPs-induced secretion of TNFα and IL-6. By the transfection of siRNA targeting p110δ, we confirmed that p110δ was responsible for TNFα and IL-6 trafficking out of Golgi complex without affecting their expression in TiPs-treated macrophages. As the upstream transcription-repressor of p110δ, Krüppel-like factor 4 (KLF4), targeted by miR-92a, could also attenuate TiPs-induced inflammation by mediating NF-κB pathway and M1/M2 polarization. To further ascertain the roles of KLF4/p110δ, TiPs-induced mice cranial osteolysis model was established and vivo experiments validated that KLF4-knockdown could exacerbate TiPs-induced osteolysis, which was strikingly ameliorated by knockdown of p110δ. In summary, our study suggests the key role of miR-92a/KLF4/p110δ signal in TiPs-induced macrophages inflammation and osteolysis.
RESUMO
Aseptic loosening (AL), secondary to particle-caused periprosthetic osteolysis, is one of the main reasons of artificial joint failure. Suppressing the macrophage inflammatory response caused by wear particles extends the life of prosthesis, and the long noncoding RNAs (lncRNAs) may play a predominant part in it. Here, titanium particles' (TiPs') stimulation increases both the cytoplasmic and nuclear levels of lncRNA Neat1 in bone marrow derived macrophages (BMDMs), which further induces the inflammatory response. Mechanically, Neat1 facilitates Bruton's tyrosine kinase (BTK) transcription by reducing the transcriptional factor KLF4, which further activates the NF-κB pathway, NLRP3 inflammation, and M1 polarization in BMDMs. Cytoplasmic Neat1 also works as an miRNA sponge in miR-188-5p-regulated BTK expression in the post-transcriptional stage. In vivo, Neat1 downregulation can reduce the TiP-induced pro-inflammatory factors and reverse the osteolysis induced by BTK overexpression. In addition, the PLGA-based microparticles loaded with si-Neat1 are developed for the treatment of the mouse calvarial osteolysis model via local injection, presenting satisfactory anti-osteolysis efficacy. These findings indicate that Neat1 is a key regulator of AL. STATEMENT OF SIGNIFICANCE: Due to released particles, aseptic loosening (AL) is the most common reason for prosthesis failure and surgical revision and represents a substantial economic burden worldwide. Herein, we reported that lncRNA Neat1 is a key regulator in regulating wear particles-induced osteolysis by activating NF-κB pathway, NLRP3 inflammation and M1 polarization via BTK, and the underlying mechanisms of Neat1-BTK interaction were further portrayed. For potential clinical application, the microparticles are developed for effective si-Neat1 delivery, leading to a dramatically enhanced effect for the treatment of osteolysis, which might be a novel strategy to extend the life of the implant.
Assuntos
Prótese Articular , Osteólise , RNA Longo não Codificante , Animais , Inflamação/metabolismo , Prótese Articular/efeitos adversos , Macrófagos/metabolismo , Camundongos , NF-kappa B/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR , Osteólise/induzido quimicamente , Osteólise/tratamento farmacológico , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Titânio/efeitos adversosRESUMO
OBJECTIVE: To investigate the role of hsa_circ_0008934 in osteosarcoma and the molecular mechanism involved in the regulation of the occurrence and development of osteosarcoma METHODS: Differentially expressed circRNAs in the osteosarcoma cell lines SaOS2 and MG63 and in the normal human osteoblast cell line hFOB1.19 were identified via next-generation RNA sequencing. The expression and circular morphology of hsa_circ_0008934 were analyzed via quantitative real-time polymerase chain reaction (qRT-PCR) and RT-PCR analysis, respectively. Proliferation, apoptosis, cell cycle progression, migration, and invasion of SaOS2 and MG63 cells with hsa_circ_0008934 silencing or overexpression were assessed using the MTS method, colony formation assay, flow cytometry, and the transwell system, respectively. The subcellular distribution of hsa_circ_0008934 was revealed via fluorescence in situ hybridization. The binding of hsa_circ_0008934 with microRNAs was confirmed using the dual-luciferase reporter assay. The oncogenic roles of hsa_circ_0008934 in osteosarcoma were determined using an in vivo tumorigenesis assay with nude mice. qRT-PCR, western blotting, TUNEL assay, and immunohistochemistry (IHC) were used to detect the tumorigenicity of hsa_circ_0008934 in osteosarcoma cells. RESULTS: Many circRNAs were differentially expressed in SaOS2 and MG63 cells than in hFOB1.19 cells. Hsa_circ_0008934 expression was significantly elevated in SaOS2 and MG63 cells. Hsa_circ_0008934 silencing significantly reduced proliferation, enhanced apoptosis, blocked cell cycle progression, and impaired migration and invasion capacities of SaOS2 cells. Opposite cellular alterations were achieved by overexpressing hsa_circ_0008934 in MG63 cells. Hsa_circ_0008934 was mainly distributed in the cytosol and positively regulated E2F3 expression in osteosarcoma cells. In addition, it directly bound with miR-145-5p to repress E2F3 expression and enhanced the tumorigenesis of MG63 cells in nude mice. qRT-PCR revealed that the intracellular injection of hsa_circ_0008934 lentivirus resulted in hsa_circ_0008934 overexpression and miR-145-5p downregulation. Western blotting confirmed that E2F3 was upregulated. Moreover, the TUNEL assay showed that hsa_circ_0008934 overexpression inhibited the apoptosis of tumor cells. IHC detection revealed that the hsa_circ_0008934 overexpression could promote the expression of Ki67 and PCNA. CONCLUSION: Elevated hsa_circ_0008934 expression promotes the proliferation and migration of osteosarcoma cells by sponging miR-145-5p to enhance E2F3 expression.
Assuntos
Neoplasias Ósseas/metabolismo , Fator de Transcrição E2F3/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Osteossarcoma/metabolismo , RNA Circular/metabolismo , Animais , Neoplasias Ósseas/genética , Neoplasias Ósseas/patologia , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Fator de Transcrição E2F3/genética , Xenoenxertos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Osteossarcoma/genética , Osteossarcoma/patologia , RNA Circular/genéticaRESUMO
Aseptic loosening (AL) caused by wear particles released from implant surfaces is one of the main causes for the failure of artificial joints, which is initiated by macrophage inflammatory responses. Emerging evidence suggests that the member of a broad-complex, tramtrack, bric-a-brac/poxvirus and zinc finger (BTB/POZ) family as well as zinc finger and BTB domain-containing protein 20 (ZBTB20) can inhibit IκBα gene transcription, promote NF-κB activation, and initiate innate immune responses. The molecular mechanism(s) by which ZBTB20 contributes to titanium particle (TiP)-induced macrophage inflammatory responses and osteolysis has not been fully elucidated. Here, we showed that ZBTB20 increased either in the AL group's synovial membranes or in TiP-stimulated bone-marrow-derived macrophages (BMDMs) as compared to that in the control groups. Moreover, the knockdown of ZBTB20 led to the inhibition of proinflammatory factors induced by TiPs in BMDMs, such as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interferon-ß (IFN-ß). Here, we also reported that the knockdown of ZBTB20 suppressed TiP-induced NF-κB activation and M1 polarization as well as stabilized the trans Golgi network (TGN) in BMDMs. The dual-luciferase reporter assay identified the binding between the IκBα promoter and ZBTB20, and IκBα knockdown could rescue the antiinflammatory effects induced by the ZBTB20 knockdown in BMDMs. Finally, we found that sh-ZBTB20 lentivirus injection could reduce TiP-induced osteolysis in mouse calvaria, inhibiting TiP-induced proinflammatory factors and loss of bone volume/total volume (BV/TV) as well as bone mineral density (BMD). These results suggest that ZBTB20 positively regulated NF-κB activation and M1 polarization as well as the production of TGN-derived tubular carriers in BMDMs, playing a positive role in macrophage activation and mouse cranial osteolysis induced by TiPs. It may be a potential therapeutic target for the prevention of aseptic loosening of prostheses.
Assuntos
Macrófagos/efeitos dos fármacos , Falha de Prótese , Titânio/toxicidade , Fatores de Transcrição/imunologia , Idoso , Idoso de 80 Anos ou mais , Animais , Artroplastia de Quadril , Células Cultivadas , Citocinas/imunologia , Feminino , Prótese de Quadril , Humanos , Macrófagos/imunologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Nus , Pessoa de Meia-Idade , NF-kappa B/imunologia , Osteólise/induzido quimicamente , Osteólise/imunologia , Reoperação , Crânio/efeitos dos fármacos , Crânio/patologia , Membrana Sinovial/imunologia , Fatores de Transcrição/genéticaRESUMO
Total hip arthroplasty (THA) is a widely-used surgical intervention for treating patients with end-stage degenerative and inflammatory osteoarthropathy. However, wear particles from the artificial titanium joint can induce osteolysis, limiting the long-term survivorship of THA. Monocyte/macrophage lineage cells are the key players in the response to wear particles, and the proinflammatory NF-κB and phosphoinositide 3-kinase (PI3K)-AKT Ser/Thr kinase (AKT)-signaling pathways have been shown to be the most important contributors to wear particle-induced osteolysis. In contrast, ubiquitin-specific protease 14 (USP14) specifically removes the polyubiquitin chains from the nucleotide-binding and oligomerization domain (NOD)-like receptor family Caspase recruitment domain (CARD)-containing 5 (NLRC5) and thereby enhances the NLRC5-mediated inhibition of NF-κB signaling. In this study, we aimed to clarify the role of the USP14-NLRC5 pathway in wear particle-induced osteolysis in vitro and in vivo We found that NLRC5 or USP14 overexpression inhibits titanium particle-induced proinflammatory tumor necrosis factor α (TNFα) production and NF-κB pathway activation, and it also decreases M1 macrophage polarization and PI3K/AKT pathway activation. Of note, NLRC5 and USP14 overexpression attenuated titanium particle-induced cranial osteolysis in mice. In conclusion, the findings of our study indicate that the USP14-NLRC5 pathway inhibits titanium particle-induced osteolysis by suppressing the NF-κB and PI3K/AKT pathways both in vitro and in vivo.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Macrófagos , Osteólise , Transdução de Sinais/efeitos dos fármacos , Crânio , Titânio/toxicidade , Ubiquitina Tiolesterase/metabolismo , Animais , Linhagem Celular , Prótese de Quadril/efeitos adversos , Humanos , Macrófagos/metabolismo , Macrófagos/patologia , Camundongos , NF-kappa B/metabolismo , Osteólise/induzido quimicamente , Osteólise/metabolismo , Osteólise/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Crânio/metabolismo , Crânio/patologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
OBJECTIVE: To investigate the functions and mechanisms of methylprednisolone (MP) through endothelin receptor B (EDNRB) on the cell proliferation of neural progenitor cells (NPCs) to regulate spinal cord injury. METHODS: Primary NPCs were isolated from fetal mice and subjected to treatments with MP and IRL-1620 (EDNRB agonist). The cell viability was determined using the MTS assay. Total RNA was extracted from the cells, and RNA-seq was performed to screen for lncRNAs. The targets of the candidate lncRNAs were predicted by GO and KEGG analyses, and the expressions of lncRNAs were validated via qPCR. Furthermore, protein levels of the PI3K-AKT pathway were determined via Western blotting, and the expression of lncRNAs was detected after inhibiting the pathway with AKT inhibitor. RESULTS: MTS assays revealed that MP decreased the cell viability of NPCs, whereas the EDNRB agonist reversed this effect of MP. NPCs were used for RNA-seq in the following three groups: normal control (NC), MP, and MP combined with EDNRB agonist (MP + EDNRB). Our results suggested that the NONRATT030699.2, NONRATT004088.2, and NONRATT005601.2 lncRNAs might be involved in the signaling pathway that is correlated to MP and the EDNRB agonist. GO and KEGG pathway analyses revealed that this was the PI3K/AKT pathway. The relevant genes involved in the pathway were validated by Western blotting. The EDNRB agonist promoted cell proliferation mainly via the activation of the PI3K/AKT pathway; however, it suppressed the expression of p-ERK, thereby increasing the expression of cyclin D1 and attenuating the effect of MP in suppressing cell proliferation. Meanwhile, after the AKT signal pathway was inhibited, these lncRNA expressions were consistent with those in the MP + EDNRB group. CONCLUSION: MP inhibits NPC proliferation, whereas EDNRB activation reverses the effect of MP via lncRNA.