| Glioma is the most common and incurable intracranial tumor. Its pathogenesis and effective therapeutic modalities still have to be studied. As the knowledge of tumor biology and molecular genetics increased, it has been shown that the development of gliomas, just like the tumors in other sites of the body, is involved in the processes of uncontrolled cell proliferation, cell dedifferentiation and dysregulation of cell apoptosis. However, up to date, it is still not clear which are the initiating molecular events in detail. In addition, gene products exert their function through signaling pathways and there are links and crosstalks even forming a complex network among different signaling pathways in different tissues. Accordingly, it is more important to study the abnomal activities of gene transduction pathways or the crosstalks among the different gene transduction pathways than to study a single gene activity. To optimize treatment strategies and to develop novel therapeutic approaches for gliomas, a more precise understanding of the cellular and molecular basis of gliomas is necessary, so it is important to continue seeking more genes which may play a key role in the gliomagenesis.In our previous work, the mRNA expression profiles of 63 samples of different pathological types of human gliomas as well as 5 samples of human normal brain tissues were selectively analyzed by Atlas Human Cancer Array 1.2. It had been observed that some members of Notch pathway were differentially expressed. Meanwhile, we also found inhibition of proliferation, cell differentiation and induction of cell apoptosis when the Notch 1 expression was knocked down by RNAi technology. It also had been demonstrated that the growth of xenograft tumors in nude mice step down. This evidence indicated that overexpression of Notch 1 can cause glioma. But, according to some reports, elevated expression of Notch2 was detected in contrast with negative expression of Notchl in medulloblastomas. Notchl and Notch2 have different expression in astrocytomas and medulloblastomas. For further studying the aberration and the role of Notchl and Notch2 in gliomas, we began with examining the expression of Notchl,2 in a large number of glioma specimens in the present study and aimed at better understanding of the expression of the major genes in the Notch signaling pathway and interruption of Notch pathway in the prevention of progression of gliomas. Moreover, the established subcutaneous xenograft gliomas in nude mice were treated with Notch2 cDNA for further defining the role of Notch2 in the progression of gliomas.The present study was divided into four parts.The first part of this study focused on the expression of Notch 1/2 in 60 samples of gliomas and 10 samples of medulloblastomas,6 samples of normal brain tissues in tissue array and their correlation with degree of malignancy of gliomas by immunohistochemcal staining. In addition, freshly resected samples including 34 samples of gliomas with different grades,4 samples of medulloblastomas and 2 samples of normal brain tissues were examined by Western Blotting and Realtime PCR. Immunohistochemcal staining was also used for PCNA, and cyclinD1 expression and their correlation was analyzed. Based on these findings, a eukaryotic expression vector of Notch2 was constructed for studying its biological effect on glioma cell growth. Notch 1 mRNA expression increased correspondingly to the ascending order of tumor grade but Notch2 mRNA expression can hardly be detected by Realtime PCR in gliomas. In immunohistochemical study, the protein expression of Notchl, PCNA and cyclinDl also increased with the degree of malignancy of tumors, and there was statistically significant difference between their expression in low-grade and high-grade tumors. In addition, the expression of Notchl, PCNA, and cyclinDl was correlated positively with each other. Such results imply that aberrant Notch pathways might be important for the progression of malignant gliomas, either overexpression of Notchl or depletion of Notch2 may contribute to tumor proliferation and invasion, which are the important phenotypes of malignant gliomas.In the second part of this study, Notch2 construct was transfected to U251 and A172 malignant glioma cells. The positive clones were selected by G418 and identified by RT-PCR, immunofluorence and Western blot analysis. For observing the phenotypic changes of the cells transfected with Notch2, The cell proliferation was determined by MTT assay and cell cycle was detected by flowcytometry, cell apoptosis was detecteded with Annexin V staining and cell invasion was evaluated by Transwell test. Moreover, the molecules regulating the cell proliferation, invasion and apoptosis were examined by immunofluorence staining and Western blot analysis. The major members of PI3K/AKT pathway were also detected by Western blotting so as to explore the link or crosstalk between Notch and AKT pathways. Meanwhile, cancer stem cell marker CD 133 and Nestin also had been detected to show the relationship between Notch signaling and cancer stem cells. As compared to control and empty vector transfected cells group, the proliferation and invasion activity were inhibited and apoptotic cells increased in U251 cells transfected with Notch2 constructs, the cell cycle analysis showed the lowering SPF and arrest of cells in G0/G1 phase. At the same time, the expression of p-AKT was downregulated and cancer stem cell marker expression including CD 133 and Nestin also decreased.In the third part of the present study, in vivo experiment was carried out. Nude mice with well-established subcutaneous explanted U251 gliomas were divided into six groups:1) control group:gliomas treated with PBS; 2) empty vecter group; gliomas treated with empty vectors; 3) Notch2 group:gliomas treated with Notch2-lipofectamine complex; 4) nonsense siRNA group; 5) Notchl siRNA group: gliomas treated with Notchl siRNA; 6) Notch2+Notchl siRNA group:gliomas treated with Notch2 plasmid and Notchl siRNA synchronously. Each group consists of eight nude mice. Tumor volume was measured regularly in subcutaneous glioma models. Notchl Notch2、Bcl-2、Caspase-3、MMP2、MMP9、PCNA、Cyclin D1、AKT、p-AKT、p53 were detected by immunohistochemistry, CD133、Nestin were detected by Western blotting in tumors of each group. Apoptosis was also detected with TUNEL method. Compared to control and empty vecter group, the tumors in mice treated with Notch2 constructs grew slowly and the tumor volumes were much smaller than those in control group. Also, as compared to control and nonsense siRNA treated group, the tumors in mice treated with siRNA targeting Notchl grew slowly and the rumor volumes were much smaller than those in control group. By immunohistochemical and Western blotting study, the expression of Bcl-2、MMP2、MMP9、PCNA、Cyclin D1、AKT、p-AKT、CD133、Nestin of tumors treated with Notchl siRNA and Notch2 constructs were decreased. A lot of apoptotic cells could be found in tumors with treatment of Notchl siRNA and Notch2 construct group, only a little apoptotic cells were found in control and nonsense transfected group as well. But combinations of these two treatments are not more efficient than using them singly. It maybe suggested that there is not synergetic and superposable between these two treatments.The fourth part included construction of Lentiviral expression vector of RBP-J banding sequence in Notch signaling pathway, Lentiviral construct infecting to glioma cell line, sorting out Notch activity cell population by flow cytometry and studying the relationship between Notch signaling and tumor stem cells. Entry plasmid pRBS-EGFP was constructed by insert the RBP-J banding sequence RBS and EGFP to plasmid vector.The entry plasmids and lentiviral vector were recombinated and lentiviral expression plasmids pLenti-RBS-EGFP was constructed. Lentiviral expression plasmids and packaging plasmids were transfect to 293T cells to produce Lenti-RBS-EGFP virus. Glioblastoma cell line was infected by Lenti-RBS-EGFP and flow cytometry was used to sort the EGFP+, EGFP-cells. According to the result, the cell population with Notch activity expressed higher tumor stem cells marker CD 133 and Nestin as compared to cell population without Notch activity. Cell proliferation marker Ki67 was increased and apoptosis factor caspase-3 was reduced. Colony-forming assays suggest that cell population with active Notch signaling posesses a stronger ability of tumorigenisis.From the present study, it has been demonstrated that Notch 1 was overexpressed and Notch2 was deleted in gliomas. Transfection of Notch2 gene reversed the malignant phenotypes of glioma cells while inhibition of Notch 1 gene expression by RNAi technology had the similar effect. So Notch signaling pathway may play an important and complex role in the gliomagenesis and Notch can be the candidate for gene therapy of human gliomas. The relationship between Notch and cancer stem cell should be further investigated. |
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