Effect of gene silencing of Bmi-1 on proliferation regulation of CD44+ nasopharyngeal carcinoma cancer stem-like cells.
- Author:
Xinhua XU
;
Yang LIU
;
Daojun LI
;
Jin SU
;
Juan HU
;
Mingqian LU
;
Fang YI
;
Jinghua RENG
;
Weihong CHEN
- Publication Type:Journal Article
- MeSH:
Carcinoma;
Cell Cycle;
Cell Division;
Cell Line, Tumor;
Cyclin-Dependent Kinase Inhibitor p16;
metabolism;
Gene Silencing;
Humans;
Hyaluronan Receptors;
metabolism;
Lentivirus;
Nasopharyngeal Carcinoma;
Nasopharyngeal Neoplasms;
genetics;
pathology;
Neoplastic Stem Cells;
cytology;
Polycomb Repressive Complex 1;
genetics;
RNA, Messenger;
RNA, Small Interfering;
Tumor Suppressor Protein p14ARF;
metabolism;
Tumor Suppressor Protein p53;
metabolism
- From:
Journal of Clinical Otorhinolaryngology Head and Neck Surgery
2015;29(10):941-947
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVE:To investigate the effect of gene silencing of Bmi-1 on proliferation regulation of CD44+ nasopharyngeal carcinoma cancer stem-like cells (CSC-LCs).
METHOD:The sequence-specific short hairpin RNA lentivirus targeting at human Bmi-1 gene (LV-Bmi-1shRNA) was constructed and was used to infect CD44+ nasopharyngeal carcinoma cells which were sorted by flow cytometry. A lentiviral which included a random sequence was also designed to serve as a negative control. We employed fluorescence microscope and flow cytometry to detect infection efficiency; real-time PCR was used to detect Bmi-1 and its downstream gene while each protein expression level was confirmed by western blotting protocol; CCK-8 proliferation assay was applied to measure proliferation capacity; tumor spheroid assay was used to evaluate the self-renewal capacity. Colony formation assay was used to measure cell colony formation capability; flow cytometry analyzed cell cycle distribution.
RESULT:The constructed LV-Bmi-1shRNA successfully infected into the CD44+ nasopharyngeal carcinoma cells. The infection efficiency could reach above 95%; LV-Bmi-lshRNA effectively inhibited Bmi-1 mRNA and protein expression, while the downstream gene p16INK4a and p14ARF mRNA as well as protein expression level were upregulated (P < 0.05). Notablely, the proliferation, colony formation, self-renewal capabilities of the experimental group decreased significantly (P < 0.05). In addition, the cell cycle arrested at the G0-G1 phase.
CONCLUSION:Gene silencing of Bmi-1 inhibited the proliferation, colony formation and self-renewal capabilities of the CD44+ nasopharyngeal carcinoma CSC-LCs, inhibited the cell cycle processes, which may mediate through Bmi1-p16INK4a/p14ARF-p53 pathway. Our experimental results indicated that Bmi-1 gene may play an important role in the maintenance of the stem cell-like characteristics of CD44+ nasopharyngeal carcinoma cells. Bmi-1 gene may be a potential new target for the treatment of nasopharyng al carcinoma in the future.
