Effect Of ShRNA-mediated Silencing Of Bmi-1Gene Expression On Biological Characteristics Of CD44~+Nasopharyngeal Carcinoma Cancer Stem-like Cells

Objective To investigate the effect of shRNA-mediated silencing of Bmi-1geneexpression on biological characteristics of CD44+nasopharyngeal carcinoma cancer stemcell-like cells (CSC-LCs) and its potential mechanism.Methods The sequence-specific short hairpin RNA lentivirus targeting at humanBmi-1gene (LV-Bmi-1shRNA) were designed, synthesized and constructed, which wereused to infect CD44+nasopharyngeal carcinoma CSC-LCs sorted by flow cytometry.Empty vector with a random sequence was set as a negative control. We employed flowcytometry to detect transfection efficiency; real-time PCR and western blot assay wereused to detect Bmi-1gene and its downstream gene p16INK4aand p14ARFmRNA andBMI-1, P16INK4a, P14ARFas well as P53protein expression level; using CCK-8proliferation assay to measure proliferation capacity; tumor spheroid assay to evaluate theself-renewal capacity; through the wound healing assay, transwell migration and invasionassays to observe cell invasion and migration capacity; colony formation assay and MTTassay were used to measure chemotherapy sensitivity with chemotherapeutic drugs5-FUand cisplatin; flow cytometry analyzed cell cycle distribution; subcutaneous inoculation oftumor cells to nude mice experiments assessed the tumorigenicity; immunohistochemicaltechniques detected the expression of CD44in tumor tissues.Results (1) In nasopharyngeal carcinoma SUNE-15-8F cell line, CD44+cellsoccupied about52.4%(32%~53%) of the total cells.(2) The constructed LV-Bmi-1shRNAsuccessfully infected into the CD44+nasopharyngeal carcinoma CSC-LCs, the infectionefficiency could reach above90%.(3) LV-Bmi-1shRNA effectively inhibited the Bmi-1mRNA and BMI-1protein expression level, while the downstream gene p16INK4aandp14ARFmRNA level as well as the P16INK4a, P14ARFand P53protein expression level wereupregulated (P<0.05).(4) The proliferation, colony formation, migration, invasion,self-renewal capabilities of the LV-Bmi-1shRNA infectioned CD44+nasopharyngealcarcinoma CSC-LCs decreased significantly. In addition, the sensitivity of the cells to5-FU and cisplatin increased, with the cell cycle arrested at the G0-G1phase (P<0.05).(5)The uninfected CD44+cells and the cells of negative control group could form tumors innude mice, and the CD44expression in tumor tissues presented positively (+++), whileCD44-nasopharyngeal carcinoma cells and LV-Bmi-1shRNA infected cells could not formtumor in nude mice. Conclusion (1) The tumorigenicity ability of CD44+nasopharyngeal carcinoma cellsin vivo is stronger than that of CD44-nasopharyngeal carcinoma cells, and the CD44+nasopharyngeal carcinoma cells own the ability to differentiate, showing characteristics ofstem cells.(2) LV-Bmi-1shRNA can effectively inhibite the Bmi-1mRNA and BMI-1protein expression.(3) LV-Bmi-1shRNA-mediated Bmi-1gene expression silencinginhibits the proliferation, colony formation, migration, invasion, self-renewal capabilitiesin vitro and tumorigenesis capacity in vivo of the CD44+nasopharyngeal carcinomaCSC-LCs, elevates the sensitivity of cell to cisplatin and5-FU, inhibits the cell cycleprocesses. Our experimental results indicate that CD44+nasopharyngeal carcinomaCSC-LCs own the capability to differentiate, and present stem cell-like properties. Besides,Bmi-1gene may play an important role in the maintenance of the stem cell-likecharacteristics of CD44+nasopharyngeal carcinoma CSC-LCs. Bmi-1gene may be apotential and new target for the treatment of nasopharyngeal carcinoma in the future.