| Laryngeal carcinoma is a common malignant tumor of the head and neck, and its incidence is increasing year by year, especially in the northeastern parts of china. Comprehensive treatment of head and neck cancer has become a trend, in which chemotherapy plays an important part. Despite advances in the treatment options for patients with laryngeal carcinoma, survival rates for most cancer patients have improved only slightly, a subset of patients at each stage of the disease fails to respond to treatment, or their cancers recur.In recent years, there has been considerable evidence that tumors are histologically heterogeneous and contain multiple diverse cell populations. The capability to maintain tumor formation and growth is exclusively due to a small subset of tumor cells termed cancer stem cells. The existence of cancer stem cells was first proven in the context of acute myeloid leukemia. This principle was further extended to solid tumors, such as breast cancer, brain tumors, lung cancer, and pancreatic cancer. Recently, we and other groups have identified a small population of cancer stem cells in Hep-2 cells and head and neck squamous cell carcinoma. CD133 has been identified as a candidate marker for cancer stem cells in Hep-2 cells. The in vivo tumorigenic potential of CD133+ Hep-2 cells was assayed by subcutaneous injection in immunocompromised mice.The cancer stem cells have the ability of self-renewal, differentiation and tumorigenic capacity, Bim-1 plays an important role in self-renewal. A growing body of evidence has demonstrated that cancer stem cells derived from a variety of solid tumors can display substantial resistance to classical chemo-and radiation therapies. However, the role of these cancer stem cells in drug resistance of laryngeal carcinoma still remains unclear.The purpose of this study was to investigate the chemoresistance of cancer stem cells in laryngeal carcinoma, and employing the RNA interference technique to silence gene expression of Bmi-1 in CD133+ cancer stem cells to study the effect of the proliferation, apoptosis, invasion and chemo-sensitization in vitro.Part I:Study on chemoresistance of CD133+ cancer stem cells in Hep-2 cellsObjective:To investigate the chemoresistance of cancer stem cells in Hep-2 cells, and detect the expression mRNA and protein levels of Bmi-1 in CD133+ cells and CD133- cells. Methods:①The response of Hep-2 cells to different chemotherapeutic agents was investigated and the expression of CD133 was studied.②Nude mice tumor xenograft model were developed, immunochemistries and flow cytometry analyzed the expression of CD133 after 5-FU treatment.③Fluorescence-activated cell sorting analysis was used to identify CD133. and the CD133+subset of cells was separated and analyzed chemotherapy resistance. After treatment of 5-FU 24 hours, colony formation assays were studied and cells were injected subeutaneously into axillary fossa of node mice to measure the tumor forming ability. The mice were monitored for palpable tumor formation and were sacrificed after 4 weeks to assess tumor formation rate, volume and weight in vivo.④Semi-quantities RT-PCR and Western-blot analyses were used to detect the expression levels of Bmi-1 in the different subpopulation cells. Results:It was concluded that chemotherapy enriched the CD133+ subpopulation 2-4 fold, relative to the untreated cells.1.55%±0.28% of Hep-2 cells were observed to be CD133+ cells. Flow cytometric analysis revealed that after the treatment with these chemotherapeutic agents, the expression of CD133 was up to 5.16%±0.86%, 4.94%±0.58%.3.66%±0.59%. After 5-FU treatmen the expression of CD133 were 6.7%±1.6% relative to the untreated mice 2.6%±0.96% by nude mice tumor xenograft model. CD133+ cancer stem cells were more resistant to chemotherapy, the proliferation capability and tumor forming ability were no difference after chemotherapy. Semi-quantitative RT-PCR and Western-blot analyses provided strong evidence that Bmi-1 expression in CD133+ cells different from CD133 cells remarkably. Conclusions:Taken together, it was confirmed that CD133+ cancer stem cells were chemoresistance and Bmi-1 were highly expressed in CD133+ cells.PartⅡ:The effects of silencing Bmi-1 gene by RNA interference on the biological phenotype and enhancement of chemosensitivity in transfected CD 133+ cancer stem cells of Hep-2 cellsObjective:To explore the effects of silencing Bmi-1 gene on the proliferation, apoptosis, invasion and chemosensitivity in transfected CD133+ cells. Methods: Bmi-1-siRNA plasmids were transfected into CD133+ cancer stem cells. The expression of Bmi-1 was evaluated by reverse transcriptase polymerase chain reaction (RT-PCR) and Western-blot. Experiments were divided into:Control group (CD133+ Hep-2 cells), Negative control group (siRNA-scramble transfection group), Experimental group (Bmi-1 siRNA transfection group). The expression of p16 was determined by RT-PCR and western blot. Cell Counting Kit-8 (CCK-8) growth curve, colony forming assay were used to measure the proliferation capcity. Flow cytometry was used to analyze cell apoptosis, and the early apoptosis rates were observed by DAPI staining. The abilities of invasion were detected by transwell chamber assay. The chemosensitivity were determined by CCK-8. Results:Compared with the control groups and negative control group, Bmi-1 protein and mRNA expression were significantly decreased in CD133+ cells transfected with double-promoter pFIV-Hl/U6 siRNA. The pFIV-H1/U6 Bmi-1 siRNA-703 expression Plasmid had the most eficient inhibitory effect, followed by pFIV-H1/U6 Bmi-1 siRNA-483 and pFIV-H1/U6 Bmi-1 siRNA-176. RT-PCR and Western-blot revealed that the P16 expression were higher in experimental group compared with blank control group and negative control group (P<0.01). Compared with blank control group and Negative control group, Cell proliferation curve revealed that cell growth was significantly inhibited in experimental group (P<0.01). FCM demonstrated that experimental group enhanced apoptosis of cells compared with blank control group and negative control group, (P<0.01). The inhibition of invasion was observed compared with blank control group and negative control group by fewer penetrating cells by transwell chamber assay (P<0.05). The chemosensitivity to 5-FU was enhanced in experimental group compared with blank control group and negative control group. Conclusions: By transfecting with the expression plasmid pFIV-H1/U6 Bmi-1 siRNA-703, the expression of P16 can be up-expressed, the cell proliferation, invasion ability of CD 133+ cancer stem cells can be inhibited obviously, apoptosis was enhanced, the chemosensitivity was enhanced.
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