Investigation Of BMI1Gene In Laryngeal Squamous Cell Carcinoma And Laryngeal Cancer Stem Cells

PART I Identification of BMI1gene in human laryngeal squamous cell carcinomaObjects To investigate the expression of BMI1gene in human laryngeal squamous cell carcinoma specimens and the relationship between clinical stages or locations of laryngeal carcinoma and BMI1expression.Methods Collected human laryngeal squamous cell carcinoma specimens and normal paracancerous tissues. Immunofluorescence qualitatively determined the expression of BMI1gene in the laryngeal carcinoma specimens. Real-time PCR and Western Blot compared the BMI1expression between laryngeal carcinoma specimens and paracancerous tissues from molecular and protein level, and the relationship between BMIl expression and the clinical stages or locations.Results Collected20laryngeal squamous cell carcinoma specimens and20paracancerous tissues, including6samples of stagel-2and14samples of stage3-4; or4glottic samples and16supraglottic samples. Immunofluorescence of BMI1gene in laryngeal carcinoma specimens showed that BMI1gene was commonly expressed. Real-time PCR revealed that the expression of BMIl in tumor samples was significantly higher than in paracancerous tissues (p<0.01). The BMI1expression in stages3-4tumors was approximately twice that in stages1-2tumors (p<0.01), but there was non-significant difference in BMIl expression between glottic and supraglottic tumors. The results of Western blot were consistent with those of real-time PCR.Conclusion BMIl gene significantly expresses in human suqmous cell carcinomas. The levels of the BMI1expression are related to the clinical stages, but have no significant relationship with the tumor locations. Part Ⅱ The effects of BMI1gene on human laryngeal Hep-2cell lineObjects To compare the Hep-2cells and RNA-interference cells and investigate the effects of BMI1gene on human laryngeal Hep-2cell line.Methods Established lentiviral vector and shRNA interference knocked down the BMI1expression in laryngeal Hep-2cell line. Conventionally cultured Hep-2and RNAi cells in96-wells plates, observed the growth on1,3,5and7day, and determined the proliferative capability with CCK-8cell counting method. Using the limited dilution method, only one cell was allowed in each well and cultured for2weeks. Observed the colony formation capability and evaluated the percentage of the areas of the colonies with a fixed measuring frame. Stained Hep-2and RNAi cells with TUNEL and FITC-Annexin V, and compared the apoptosis rates. Stained both cells with PI and compared the cell cycles through the analysis of flow cytometry. After cultured for24hours, both cells were exposed to irradiation with the doses of2,4and8Gy and after48hours, calculated the ultraviolet absorbance and calculated the inhibition rate with CCK-8method. After24hours culturing, both cells were treated with3μg/mL,6μg/mL and12μg/mL Cisplatin Injection. After cultured for24hours, compared the inhibition rates of both cells with CCK-8method. Injected1×105Hep-2and RNAi cells to the axillary fossa of10NOD/SCID mice, observed the tumor formation after8weeks and compared the tumorigenicity of both cells.Results shRNA knocked down the expression of BMI1gene. The interference rate was83.75%. After being cultured for1week and evaluated the ultraviolet absorbance, the absorbance of RNAi cells was significantly lower than Hep-2cells from the third day and had the trend of ceasing growth. Single Hep-2and RNAi cell was cultured in96-well plate for2weeks, observed and calculated the percentage of area with a fixed measuring frame. We found that the RNAi cell-formed spheres were obviously smaller and the percentage of area was significantly lower (p<0.01). Both cells were stained with TUNEL and observed and we found that RNAi cells showed stronger apoptosis capability. FITC-Annexin V staining and flow cytometric analysis were also used and the results also revealed that RNAi cells were easier to apoptosis (p<0.01). PI staining and flow cytometric analysis were used to determine the cell cycle and the results showed that there were more RNAi cells accumulating in G0/G1phase, and the cells in S and G2/M phases were much less than Hep-2cells. After irradiation with the doses of2Gy,4Gy and8Gy, the inhibition rates of RNAi cells were significantly increased. After treated with3μg/mL,6μg/mL and12μg/mL Cisplatin Injection, the inhibition rates of RNAi cells were higher than Hep-2cells with the increasing doses of cisplatin. The results of in vivo tumorigenicity assay indicated that the size of the RNAi cell-formed tumors was obviously smaller in size and lighter in weight (p<0.01).Conclusion BMIl knockdown is able to inhibit the proliferative capability of Hep-2cells, promote apoptosis, accumulate cells in G0/G1phase, decrease tolerance to radiochemotherapy and inhibit the tumorigenicity in vivo. Part Ⅲ Identification of BMIl gene in cancer stem cells of human laryngeal cancer Hep-2cell lineObjects To investigate BMI1and CD133expressions in laryngeal cancer specimens and the expression of BMI1in CD133+laryngeal cancer stem cells.Methods BMI1and CD133were used to perform immunofluorescence in laryngeal cancer samples and their expressions were observed under fluorescence microscope and laser confocal microscope. Flow cyctometric analysis determined the rates of CD133+laryngeal cancer stem cells in both Hep-2and RNAi cells, and fluorescence-activated cell sorting was also performed. Observed the growth of the cells after sorting. Real-time PCR quantitatively evaluated the CD133expressions in both group of cells, and the expressions of another two stem cell genes Notch2and PTEN were also evaluated.Results BMIl was coexpressed with CD133in laryngeal cancer samples. CD133was expressed on the cell membrane and BMI1was mainly expressed in the nuclear of CD133+cells. The results of Real-time PCR revealed that the BMI1expression in CD133+cells was significantly higher than in CD133-cells (p<0.01), however, there was non-statistical significance between the expressions of Notch2and PTEN in CD133+cells and CD133-cells.Conclusion BMI1and CD133are coexpressed in laryngeal squamous cell carcinoma and BMI1is mainly expressed in CD133+laryngeal cancer stem cells and has influences on them. Notch2and PTEN have non-specific effects on CD133+laryngeal cancer stem cells. Part IV The functions of BMI1gene on the cancer stem cells of human laryngeal cancer Hep-2cell lineObjects To investigate the effects of BMI1gene on CD133+laryngeal cancer stem cells and the influences on related genes and signal pathways.Methods Flow cytometric analysis determined the percentage of CD133+laryngeal cancer stem cells in both Hep-2and RNAi cells, and fluorescence activated sorting was also performed. Cultured the CD133+cancer stem cells after sorting, and compared the capability of sphere-forming. Real-time PCR evaluated the CD133relative expression in Hep-2and RNAi cells and the expressions of related gene p53, p16and cyclinDl, in order to analyze the influences on the signal pathways.Results The results of flow cytometric analysis revealed that RNAi cells contained much less CD133+cells than Hep-2cells (p<0.01). Observed the sorted cells and found the growth rate and the capability of sphere-forming of Hep2-CD133+cells were both obviously stronger than RNAi-CD133+cells. The results of Real-time PCR showed that the expression of CD133in RNAi cells was significantly decreased (p<0.01). More, in RNAi cells, the expressions of p53and p16were markedly increased (p<0.05) and the cyclinDl expression was distinctly decreased (p<0.01).Conclusion BMI1silencing leads to the decrease of CD133+laryngeal cancer stem cells, consequently repressing proliferation of tumor cells, promoting apoptosis and reducing tolerance to radiochemotherapy. More, BMI1plays critical roles in the regulation of INK4A/ARF/p53signal pathway.