RESUMO
BACKGROUND: The study examines the function of hypoxia-mediated down-regulation of microRNAs (miRNAs) (mir-30c, mir-135a, and mir-27a) in the process of bladder cancer immune escape. METHODS: Quantitative Real-time PCR (qRT-PCR) was carried out to determine gene expression levels of Drosha and Dicer under hypoxia treatment, while western blotting and flow cytometry were used to determine protein expression. Seven reported miRNAs were identified via qRT-PCR assay. Flow cytometry detection of CD3/CD4/CD8-positive expression and statistics. Enzyme-linked immunosorbent assay (ELISA) detected cellular immune factors content. Cell apoptosis was checked via flow cytometry assay. Luciferase report assay and western blot assays were both used to verify the relationship between miRNAs and Casitas B-lineage lymphoma proto-oncogene b (Cbl-b). The animal model was established and Hematoxylin-eosin (HE) staining, TdT-mediated dUTP Nick-End Labeling (TUNEL) staining, and immunohistochemistry (IHC) assays were separately used to verify the conclusions. RESULTS: The CD3 + /CD4 + expression was increased in the hypoxia group, while CD3 + /CD8 + expression, the cellular immune factors content Interleukin-2 (IL-2) and Tumor Necrosis Factor-α (TNFα) along with the cell apoptosis were suppressed. The protein expression of Cbl-b was found to be up-regulated in the hypoxia group. After constructing the overexpression/ knockdown of Cbl-b in peripheral blood mononuclear cell (PBMC), Cbl-b has been found to promote tumor immune escape in bladder cancer. Furthermore, Cbl-b had been identified as the co-targets of mir-30c, mir-135a, and mir-27a and down-regulation of miRNA biogenesis promotes Cbl-b expression and deactivating T cells in vitro/in vivo. CONCLUSION: Hypoxia-mediated down-regulation of miRNAs' biogenesis promotes tumor immune escape in bladder cancer, which could bring much more advance to the medical research on tumors.
Assuntos
Regulação para Baixo/imunologia , MicroRNAs/metabolismo , Evasão Tumoral/imunologia , Hipóxia Tumoral/imunologia , Neoplasias da Bexiga Urinária/imunologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Apoptose , Complexo CD3/metabolismo , Antígenos CD4/metabolismo , Antígenos CD8/metabolismo , RNA Helicases DEAD-box/genética , Modelos Animais de Doenças , Regulação Neoplásica da Expressão Gênica , Humanos , Interleucina-2/metabolismo , Leucócitos Mononucleares/metabolismo , Masculino , Camundongos Endogâmicos C57BL , MicroRNAs/biossíntese , Estudos Prospectivos , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-cbl/genética , Distribuição Aleatória , Ribonuclease III/genética , Fator de Necrose Tumoral alfa/metabolismo , Regulação para Cima , Neoplasias da Bexiga Urinária/metabolismoRESUMO
Radiotherapy is the treatment of choice for many cancer patients. Residual tumor leads to local recurrence after a period of an equilibrium created between proliferating, quiescent and dying cancer cells. The tumor microenvironment is a main obstacle for the efficacy of radiotherapy, as impaired blood flow leads to hypoxia, acidity and reduced accessibility of radiosensitizers. Eradication of remnant disease is an intractable clinical quest. After more than a century of research, anti-tumor immunity has gained a dominant position in oncology research and therapy. Immune cells play a significant role in the eradication of tumors during and after the completion of radiotherapy. The tumor equilibrium reached in the irradiated tumor may shift towards cancer cell eradication if the immune response is appropriately modulated. In the modern immunotherapy era, clinical trials are urged to standardize immunotherapy schemes that could be safely applied to improve clearance of the post-radiotherapy remnant disease.