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Bmi1 Regulates Self-Renewal and Tumorigenicity of Cancer Stem Cells in Non-Small Cell Lung Cancer

Author: ZhouZuo
Tutor: HuYiDe
School: Third Military Medical University
Course: Oncology
Keywords: Bmil RNA interference Intratumoral injection Cancer stem cells Non - small cell lung cancer
CLC: R734.2
Type: Master's thesis
Year: 2011
Downloads: 74
Quote: 0
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Non-small cell lung cancer (NSCLC) is a common malignant tumors of the threat to human health, retaining its position as the first of the various types of cancer morbidity and mortality. The cancer stem cell theory holds that tumor heterogeneity, there is a small amount of cells with the characteristics of stem cells capable of unlimited self-renewal and produce daughter cells identical with the previous generation, and has a variety of differentiation potential and highly proliferative capacity, resulting in tumor cells of different phenotypes. These cells in tumor formation, plays an important role in the development of metastasis and recurrence. In recent years, a large number of studies have shown that, in addition to other than leukemia, breast cancer, kidney cancer, glioma, lung tumor stem cells. A group of cells with the characteristics of stem cells in the lung tissue of normal mice, accounting for about 0.4% of the total number of cells is called the the bronchoalveolar lavage stem cells (bronchoalveolar stem cells, BASCs), surface markers Sca-1 / of CD34. Another study found part in human lung cancer tissue the CD133 phenotype of cells in immunodeficient mice in vivo tumor's ability to self-renew and to proliferate indefinitely in serum-free medium containing EGF and bFGF, injection of 1 × 104 CD133 cells could form consistent with the primary tumor phenotype transplanted tumors in mice with immunodeficiency. The discovery of cancer stem cells, for non-small cell lung tumor formation and further study of the mechanisms of chemotherapy resistance, as well as to improve the survival rate of patients with lung cancer, has brought new hope. In the current study, three enriched tumor stem cells, mainly from the two major aspects of the features of the cancer stem cell biology and its surface-specific markers for the identification and screening, including: (1) the use of flow cytometry or immunomagnetic bead separation surface marker-positive cells, CD133 and CD44 can be used to isolate cancer stem cells in solid tumors, but has not yet found what kind of markers specifically expressed in cancer stem cells; (2) the use of flow cytometry separation \Application. (3) the use of cloning ability of the cancer stem cells, the serum-free suspension culture \Stem cells in the tumor tissue were very low, the use of side population cell sorting or suspension culture cells obtained limited number, difficult to be met to conduct follow-up experiments required number of cells. Currently considered the resistance of lung cancer stem cells with high expression of stem cell ABC transporters (ATP-binding cassette transporters, ABC transporters), ABCG2 drug pump protein is the most important of which explored various tissue-derived SP cells were found to have high expression of ABCG2, and found to increase the expression of ABCG2 by transgenic technology can make the cells were SP phenotype. Therefore ABCG2 is considered to be one of the hallmarks of cancer stem cells. Resistance of lung cancer stem cells, the experiment through the establishment of a lung cancer cell line A549 mouse tumor models, while giving the mice low doses of paclitaxel load enrichment lung cancer stem cells in the body. The enrichment of lung cancer stem cells, further study of lung cancer stem cell self-renewal and proliferation mechanisms. Bmil belongs to multi-comb gene family (polycomb - of members of the genes (PcG)), is related to the cell cycle and proliferation transcription repressor. The tumor suppressor gene INK4a/ARF simultaneously encodes two proteins p16INK4a and p14ARF, these two proteins are cell cycle regulators, respectively, through the cyclinD-CDK4-Rb-E2F and MDM2-p53 pathways regulating cell cycle the original cancer gene Bmil INK4a factor / ARF upstream regulator. Many studies have shown that, in a variety of epithelial cell-derived malignancies including non-small cell lung cancer, there is a high expression of Bmil, and its expression is significantly negatively correlated with the prognosis of lung cancer patients. Recent studies have shown that the Bmil also have an important role in a variety of stem cell proliferation and self-renewal, especially in memory of cancer stem cells in the expression of an increase in the phenomenon. But Bmil whether to maintain the function of the lung cancer stem cells also have an important role, has yet to see the study reported. Body tumor injection of lentivirus-mediated's Bmil-shRNA, the experimental use of RNA interference technology to cut non-small cell lung transplant tumor lung cancer stem cells of Bmil the expression found accompanied by the expression of Bmil the decline, lung cancer stem cells in vitro update capability and tumorigenic ability in vivo in immunodeficient mice, are significantly decreased. Also detected downstream regulation of gene Bmil one p16INK4A expression, found with the reduction of Bmil, p16INK4a upregulation of lung cancer stem cells. The reveals the Bmil lung cancer stem cell self-renewal, an important role in the proliferation process, P16INK4a may be one of the Bmil target genes play a role, which laid the foundation for the proliferation of the molecular mechanism for further study on lung cancer stem cell renewal,. Objective: To establish the A549 cells in NOD / SCID mice subcutaneously transplanted tumor to give chemotherapy load intratumoral enriched in lung adenocarcinoma stem cells using RNA interference technology to down lung adenocarcinoma stem cells by the intratumoral injection lentiviral mediated Bmil-shRNA The expression within Bmil observed Bmil in lung cancer stem cell self-renewal, proliferation. Method: 1. Enrichment of stem cells of the lung. A549 cells were injected into NOD / SCID mice subcutaneously, and the establishment of tumor-bearing mouse model. By giving the mice by intraperitoneal injection of low-dose paclitaxel and tumor blocks cells in vivo passaging, the third generation of transplanted tumor cells A549-3rd. A549-3rd cells by flow cytometry CD133 cell ratio, real-time RT-PCR and Western blotting detection A549-3rd cells Bmil and ABCG2 expression levels. 2.RNA interference to inhibit the expression of Bmil lung cancer stem cells. NC-containing human Bmil-shRNA and negative control shRNA lentiviral expression vector by the Shanghai Ji Kaiji design synthesis and identification of chemical technology company, the vectors expressing the green fluorescent protein GFP. The tumor-bearing mice were randomly divided into three groups, were given intratumoral injection vsh-Bmil vsh-NC or PBS. Vivo fluorescence microscopy GFP expression in the transplanted tumors to determine the efficiency of transfection. real-time PCR and Western blotting detection of tumors in vivo Bmil by p16INK4a in expression levels. Self-renewal and tumorigenicity in vivo detection. Limiting dilution method and serum-free suspension culture, determination of A549 cells in A549-3rd cells in A549-4th cells in A549-4th-Bmil cells and A549-4th-NC cells of \capacity. The method of using NOD / SCID mice were injected subcutaneously compare the ability of the cells in each group in vivo tumorigenicity. Results: 1. Stem cell enrichment effect analysis. 7 days after the suspension culture in serum-free medium, only a (1.2% ± 0.4%) of A549 cells to form stem cell spheres \capacity 12-fold (P = 0.01). A549-3rd cells formed spheroids in a serum-free medium after 8-10 subcultures still form a \stem cells ball. \2 × 105 of A549 and A549-3rd cells were injected into 6 NOD / SCID mice subcutaneously, A549 cells in which a mouse subcutaneous tumor, and 2 × 105 A549-3rd cells 5 in which Subcutaneous tumor. Flow cytometry A549-3rd cells CD133 cells ratio is about 69.0%, compared to 2.3% of the A549 cells was significantly higher. A549-3rd than A549 the cells within Bmil and ABCG2 expression was significantly increased, with a statistically significant difference (P = 0.02). 2.RNA interference inhibition of lung cancer stem cells Bmil expression and regulation of gene expression changes. Intratumoral injection of lentiviral vector of 2 times, the second time after 72 hours, in vivo fluorescence microscopy observed GFP expression in the tumor in vivo. A549-4th-Bmil cells by measuring intratumoral injection vsh-Bmil more injections A549-4th-NC the vsh-NC or PBS intratumoral cells or A549-4th cells Bmil expression decreased significantly (P = 0.02 ), the expression of p16INK4a increases, a statistically significant difference (P = 0.04). Self-renewal and tumorigenicity in vivo changes. Cultured for 7 days in serum-free medium (0.9% ± 0.2%) of the A549-4th-Bmil cells, (13.9% ± 1.1%) of the A549-4th-NC cells (14.7% ± 1.3%) of the A549- 4th cells to form a ball of cells. A549-4th-Bmil ability of cells into a ball 14 times lower than the other two groups (P = 0.01). Three groups of 2 × 105 cells were injected subcutaneously into 6 NOD / SCID mice subcutaneously, A549-4th-NC subcutaneous tumor cells in 5 mice A549-4th cell tumorigenicity in six mice subcutaneously, and the A549-4th-Bmil cells only in mice subcutaneous tumor, significantly decreased the ability of tumor formation. Conclusion: 1. A549-3rd cells in the body chemotherapy load by giving the capacity of self-renewal and tumorigenic ability than A549 cells significantly improved of CD133 cells increased the proportion of ABCG2 expression increased, indicating that the low-dose chemotherapy by a certain time, The third-generation cell A549-3rd enriched with a large number of self-renewal, proliferative capacity of lung adenocarcinoma stem cells. 2. Using RNA interference technology makes the expression of Bmil Bmil cells was significantly inhibited. Due to decline in Bmil of expression, of p16INK4a expression increased lung adenocarcinoma stem cells in the \and regulation mechanisms may include the inhibition of p16INK4a gene.

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