RESUMO
PURPOSE: To identify molecular subtypes of oxidative stress-related genes in head and neck squamous cell carcinoma (HNSCC) and to construct a scoring model of oxidative stress-related genes. METHODS: R language based scRNA-seq and bulk RNA-seq analyses were used to identify molecular isoforms of oxidative stress-related genes in HNSCC. An oxidative stress-related gene scoring (OSRS) model was constructed, which were verified through online data and immunohistochemical staining of clinical samples. RESULTS: Using TCGA-HNSCC datasets, nine predictive genes for overall patient survival, rarely reported in previous similar studies, were screened. AREG and CES1 were identified as prognostic risk factors. CSTA, FDCSP, JCHAIN, IFFO2, PGLYRP4, SPOCK2 and SPINK6 were identified as prognostic factors. Collectively, all genes formed a prognostic risk signature model for oxidative stress in HNSCC, which were validated in GSE41613, GSE103322 and PRJEB23709 datasets. Immunohistochemical staining of SPINK6 in nasopharyngeal cancer samples validated the gene panel. Subsequent analysis indicated that subgroups of the oxidative stress prognostic signature played important roles during cellular communication, the immune microenvironment, the differential activation of transcription factors, oxidative stress and immunotherapeutic responses. CONCLUSIONS: The risk model might predict HNSCC prognosis and immunotherapeutic responses.
Assuntos
Neoplasias de Cabeça e Pescoço , Neoplasias Nasofaríngeas , Humanos , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/terapia , Prognóstico , Imunoterapia , Estresse Oxidativo/genética , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/terapia , Microambiente Tumoral/genética , Proteoglicanas , Inibidores de Serinopeptidase do Tipo KazalRESUMO
Bcl-2-associated athanogene 1 (Bag-1) is a multifunctional and antiapoptotic protein that binds to the antiapoptosis regulator Bcl-2 and promotes cell survival. To investigate the protective function of Bag-1, we examined the effects of Bag-1L, one isoform of Bag-1, in an in vitro cell culture model of lung ischemia-reperfusion injury (LIRI) generated by treatment of A549 cells with hypoxia/reoxygenation. Overexpression of full-length Bag-1L increased the viability of A549 cells and reduced cell apoptosis in response to 6 h of hypoxia/reoxygenation treatment. Furthermore, Bag-1L overexpression enhanced the heat shock protein 70 (HSP70) and Bcl-2 protein levels, increased the phosphorylation of AKT, decreased Bax and cleaved caspase-3 levels, and was able to overcome cell cycle arrest. These effects were not observed in A549 cells overexpressing a truncated form of Bag-1L lacking the "Bag domain," denoted Bag-1Lâ³C. The "Bag domain" is the C-terminal 47 amino acids. Taken together, the results of this study suggest that Bag-1L overexpression can protect against oxidative stress and apoptosis in an in vitro LIRI model, with a dependence on the Bag domain.
Assuntos
Apoptose/efeitos dos fármacos , Proteínas de Ligação a DNA/farmacologia , Pulmão/metabolismo , Substâncias Protetoras/farmacologia , Traumatismo por Reperfusão/tratamento farmacológico , Fatores de Transcrição/farmacologia , Células A549 , Adenoviridae , Ciclo Celular , Sobrevivência Celular , Proteínas de Ligação a DNA/genética , Expressão Gênica , Proteínas de Choque Térmico HSP70/metabolismo , Humanos , Hipóxia , Isoformas de Proteínas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Fatores de Transcrição/genéticaRESUMO
Lung ischemia-reperfusion can cause acute lung injury, which is closely associated with apoptosis. Heat shock protein 70 (HSP70) is an anti-apoptotic protein that promotes cell survival under a variety of different stress conditions. However, the role and mechanism of HSP70 in lung ischemia-reperfusion injury is yet to be fully elucidated. In the present study, an in vitro hypoxia/reoxygenation model of A549 cells was established to simulate lung ischemia-reperfusion and HSP70 was silenced by transfecting A549 cells with an shRNA sequence targeting HSP70. Western blotting, reverse transcription-quantitative polymerase chain reaction, Cell Counting kit-8 and flow cytometry were used to detect protein levels, RNA expression, cell activity and apoptosis. The results revealed that silencing HSP70 reduced cell viability, aggravated apoptosis, increased lactate dehydrogenase levels and induced a G2/M blockade in a hypoxia-reoxygenation A549 cell model. Furthermore, silencing HSP70 decreased the phosphorylation levels of protein kinase B (AKT) and extracellular signal-regulated kinase (ERK); however, the total AKT and ERK levels did not change significantly. Pretreating A549 cells with the AKT pathway inhibitor, LY294002 and the ERK pathway inhibitor, U0216 led to a decrease in HSP70 expression. These results indicate that silencing HSP70 may aggravate apoptosis in hypoxia-reoxygenation cell models, potentially via the mitogen-activated protein kinase/ERK and phosphoinositide 3-kinase/AKT signaling pathways.
RESUMO
PURPOSE: B-cell lymphoma-2 (Bcl-2) associated athanogene 1 (Bag-1) is a multifunctional protein, and Bag -1 overexpression is associated with progression, metastasis, and drug resistance in lung cancer. This study assessed the effects of Bag-1 siRNA on sensitization of cisplatin on non-small cell lung cancer (NSCLC) cells. MATERIAL AND METHODS: NSCLC A549 cell line was transfected with Bag-1 or negative control siRNA and then treated with cisplatin for cell viability, CCK-8, LDH, and flow cytometry assays. The Ca2+ levels were analyzed using Fluo-3/AM fluorescence staining, and the protein levels were assessed using Western blot analysis. RESULTS: Bag-1 siRNA significantly knocked down Bag-1 expression and inhibited cell invasion versus the negative control siRNA, while Bag-1 silence sensitized cisplatin to induce A549 cells to apoptosis by induction of cell cycle G1 arrest. At protein level, Bag-1 silence reduced the expression ratio of Bcl-2 to Bcl-2 associated X protein (Bax), downregulated activity of the PI3K/AKT and mitogen-activated protein kinase (MAPK) pathways, and potently upregulated the calcium signaling-mediated pathway. CONCLUSION: This study demonstrated that Bag-1 silencing sensitized A549 to cisplatin to enhance A549 cell apoptosis by modified multiple gene pathways. Further study will evaluate the usefulness of Bag-1 siRNA as a potential targeting therapy for NSCLC.