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.
Subject(s)
Humans , Carcinoma , Cell Cycle , Cell Division , Cell Line, Tumor , Cyclin-Dependent Kinase Inhibitor p16 , Metabolism , Gene Silencing , Hyaluronan Receptors , Metabolism , Lentivirus , Nasopharyngeal Carcinoma , Nasopharyngeal Neoplasms , Genetics , Pathology , Neoplastic Stem Cells , Cell Biology , Polycomb Repressive Complex 1 , Genetics , RNA, Messenger , RNA, Small Interfering , Tumor Suppressor Protein p14ARF , Metabolism , Tumor Suppressor Protein p53 , MetabolismABSTRACT
OBJECTIVE@#To detect the effect of Celecoxib on the proliferation and apoptosis of human nasopharyngeal carcinoma cell line CNE-2.@*METHOD@#The growth inhibition rate of CNE-2 by Celecoxib was evaluated with MTT method. Apoptosis related morphology changes were observed with transmission electron microscopy (TEM). The cell cycle and apoptosis were measured with flow cytometric method (FCM). Apoptotic index (AI) was counted by the TDT-mediated dUTP-biotin nick end-labeling (TUNEL) assay.@*RESULT@#The growth of CNE-2 cell was inhibited by celecoxib in a dose-and time-dependent manner. Apoptosis with nuclear chromatin condensation, cell shrinkage, periplasm loss and the formation of apoptotic bodies was observed with TEM. Apoptotic rates of CNE-2 cells treated with 80 and 100 micromol/L celecoxib were (10.47+/-0.18)% and (20.17+/-0.55)% respectively, significantly higher than those of the control group (1.57+/-0.27)% with FCM. The percentage of G0/G1 phase cells increased, whereas the S and G2/M phases cells decreased in a dose-dependent manner after the treatment. TUNEL assay showed that the apoptosis ratio (AI) of CNE-2 treated with Celecoxib was higher than control group (P<0.01).@*CONCLUSION@#Celecoxib can inhibit the growth of human nasopharyngeal carcinoma cell line CNE-2 and induce the cell apoptosis, which may be related to blocking the cell cycle progress of CNE-2 cells.
Subject(s)
Humans , Apoptosis , Celecoxib , Cell Line, Tumor , Cell Proliferation , Cyclooxygenase 2 Inhibitors , Pharmacology , Nasopharyngeal Neoplasms , Pathology , Pyrazoles , Pharmacology , Sulfonamides , PharmacologyABSTRACT
Objective:To detect the effect of Celecoxib on the proliferation and apoptosis of human nasopha-ryngeal carcinoma cell line CNE-2. Method:The growth inhibition rate of CNE-2 by Celecoxib was evaluated with MTT method. Apoptosis related morphology changes were observed with transmission electron microscopy (TEM). The cell cycle and apoptosis were measured with flow cytometric method (FCM). Apoptotic index ( AI) was counted by the TDT-mediated dUTP-biotin nick end-labeling (TUNED assay. Result: The growth of CNE-2 cell was inhibited by celecoxib in a dose-and time-dependent manner. Apoptosis with nuclear chromatin condensa-tion, cell shrinkage, periplast loss and the formation of apoptotic bodies was observed with TEM. Apoptotic rates of CNE-2 cells treated with 80 and 100 μmol/L celecoxib were (10. 47±0. 18)% and (20. 17±0. 55)% respective-ly, significantly higher than those of the control group (1. 57±0. 27)% with FCM. The percentage of G_0/G_1 phase cells increased, whereas the S and G_2/M phases cells decreased in a dose-dependent manner after the treatment. TUNEL assay showed that the apoptosis ratio( AI) of CNE-2 treated with Celecoxib was higher than control group (P<0. 01). Conclusion:Celecoxib can inhibit the growth of human nasopharyngeal carcinoma cell line CNE-2 and induce the cell apoptosis, which may be related to blocking the cell cycle progress of CNE-2 cells.