- gene silencing enhances H9 T lymphocyte-mediated killing of human laryngeal squamous cancer Hep-2 cells.
10.12122/j.issn.1673-4254.2019.05.09
- Author:
Saiming CHEN
1
;
Zhiqun LI
1
;
Limin ZHOU
2
;
Yunxia ZHANG
2
Author Information
1. Department of Otolaryngology-Head and Neck Surgery, First Affiliated Hospital of Hainan Medical College.
2. Scientific Experimental Center of Hainan Medical College, Haikou 570102, China.
- Publication Type:Journal Article
- Keywords:
gene silencing;
human laryngeal squamous cell carcinoma;
immune killing;
ubiquitin ligase
- MeSH:
Carcinoma, Squamous Cell;
genetics;
therapy;
Gene Silencing;
Humans;
Laryngeal Neoplasms;
genetics;
therapy;
Lymphocyte Activation;
RNA, Small Interfering;
T-Lymphocytes
- From:
Journal of Southern Medical University
2019;39(5):554-560
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVE:To investigate the effect of sputum ubiquitin ligase (Cbl-b) gene known-down on the cytotoxicity of H9 T lymphocytes against human laryngeal squamous cancer Hep-2 cells and explore the underlying mechanism.
METHODS:CD4 T lymphocytes isolated from 12 patients with laryngeal squamous carcinoma and 12 healthy individuals were examined for Cbl-b mRNA expressions using RT-PCR. H9 T lymphocytes cultured in 96-well plates were transfected with Cbl-b siRNA via liposomes followed by treatment with an anti-IL-2 monoclonal antibody, with H9 T lymphocytes transfected with a scrambled sequence as the negative control. The expressions of Cbl-b mRNA and protein in the cells were detected using real-time fluorescent quantitative PCR and Western blotting, respectively. The killing effect of the treated T lymphocytes against Hep-2 cells was assessed using the cell counting kit (CCK-8). The positive expression rates of CD69 and CD25 on the surface of H9 T lymphocytes were determined using flow cytometry, and the levels of interleukin-2 (IL-2) and interferon-gamma (INF-γ) in the culture supernatants of H9 T lymphocytes were detected with ELISA.
RESULTS:The CD4 T lymphocytes from patients with laryngeal squamous carcinoma showed significantly increased Cbl-b mRNA level compared with those from healthy individuals ( < 0.05). Transfection of H9 T lymphocytes with Cbl-b siRNA significantly reduced the expression levels of Cbl-b mRNA and protein ( < 0.05), which were not significantly affected by subsequent treatment of the cells with the anti-IL-2 antibody (>0.05). At different target-effector ratios, the Cbl-b siRNA-transfected cells showed significantly higher Hep-2 cell killing rates and higher positivity rates of CD69 and CD25 expressions than the blank and negative control cells and the cells with both Cbl-b siRNA transfection and anti-IL-2 treatment ( < 0.05). Cbl-b silencing in H9 T lymphocytes resulted in significantly increased levels of IL-2 and INF-γ in the supernatant as compared with those in the blank and negative control groups ( < 0.05).
CONCLUSIONS:Cbl-b gene silencing effectively enhances the killing effect of H9 T lymphocytes against Hep-2 cells probably as the result of enhanced IL-2 secretion and T lymphocyte activation.
