title: [Effect of gene silencing of Bmi-1 on proliferation regulation of CD44+ nasopharyngeal carcinoma cancer stem-like cells].
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).
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.forename: Xinhua
authors.initials: X
authors.lastname: Xu
authors.forename: Yang
authors.initials: Y
authors.lastname: Liu
authors.forename: Daojun
authors.initials: D
authors.lastname: Li
authors.forename: Jin
authors.initials: J
authors.lastname: Su
authors.forename: Juan
authors.initials: J
authors.lastname: Hu
authors.forename: Mingqian
authors.initials: M
authors.lastname: Lu
authors.forename: Fang
authors.initials: F
authors.lastname: Yi
authors.forename: Jinghua
authors.initials: J
authors.lastname: Reng
authors.forename: Weihong
authors.initials: W
authors.lastname: Chen
mesh_terms: D002277:Carcinoma; D002453:Cell Cycle; D002455:Cell Division; D045744:Cell Line, Tumor; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000378:metabolism; D020868:Gene Silencing; D006801:Humans; D018960:Hyaluronan Receptors / Q000378:metabolism; D016086:Lentivirus; D000077274:Nasopharyngeal Carcinoma; D009303:Nasopharyngeal Neoplasms / Q000235:genetics / Q000473:pathology*; D014411:Neoplastic Stem Cells / Q000166:cytology*; D063150:Polycomb Repressive Complex 1 / Q000235:genetics*; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D025803:Tumor Suppressor Protein p14ARF / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism
chemical_list: C069077:BMI1 protein, human; D019941:Cyclin-Dependent Kinase Inhibitor p16; D018960:Hyaluronan Receptors; D012333:RNA, Messenger; D034741:RNA, Small Interfering; C495901:TP53 protein, human; D025803:Tumor Suppressor Protein p14ARF; D016159:Tumor Suppressor Protein p53; C583516:p12(INK4a) protein, human; D063150:Polycomb Repressive Complex 1