Department of Urology, The First Affiliated Hospital of Anhui Medical University, No.218 Jixi Road, Hefei, 230000, China.
Institute of Urology, Anhui Medical University, Hefei, 230000, China.
Clin Transl Oncol. 2021 Aug;23(8):1678-1687. doi: 10.1007/s12094-021-02569-x. Epub 2021 Feb 24.
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.
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.
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.
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.
本研究探讨了低氧介导的 microRNAs（miRNAs）（mir-30c、mir-135a 和 mir-27a）下调在膀胱癌免疫逃逸过程中的作用。
采用定量实时 PCR（qRT-PCR）检测低氧处理下 Drosha 和 Dicer 的基因表达水平，采用 Western blot 和流式细胞术检测蛋白表达。通过 qRT-PCR 检测鉴定了 7 个报道的 miRNA。流式细胞术检测 CD3/CD4/CD8 阳性表达并进行统计学分析。酶联免疫吸附试验（ELISA）检测细胞免疫因子含量。流式细胞术检测细胞凋亡。采用荧光素酶报告基因检测和 Western blot 检测验证 miRNAs 与 Casitas B-lineage lymphoma proto-oncogene b（Cbl-b）之间的关系。建立动物模型，分别采用苏木精-伊红（HE）染色、TdT 介导的 dUTP 缺口末端标记（TUNEL）染色和免疫组织化学（IHC）检测验证结论。
低氧组 CD3+/CD4+表达增加，而 CD3+/CD8+表达、细胞免疫因子含量白细胞介素-2（IL-2）和肿瘤坏死因子-α（TNFα）以及细胞凋亡受到抑制。低氧组 Cbl-b 蛋白表达上调。构建外周血单个核细胞（PBMC）中 Cbl-b 的过表达/敲低后，发现 Cbl-b 促进膀胱癌肿瘤免疫逃逸。此外，Cbl-b 被鉴定为 mir-30c、mir-135a 和 mir-27a 的共同靶标，miRNA 生物发生的下调促进 Cbl-b 表达并在体外/体内使 T 细胞失活。
低氧介导的 miRNA 生物发生下调促进膀胱癌肿瘤免疫逃逸，可为肿瘤的医学研究带来更多进展。
