Interference Of Notch2Inhibits Progression Of Gliomas

Glioma is the most common malignant tumor of the central nervous system, with a strong aggressive and progressive. At present the main method is mainly used in the clinical treatment of glioma are surgical resection, radiotherapy and chemotherapy combination therapy. But because of the growth characteristics of glioma that has blurred boundaries with surrounding brain tissues, complete surgical excision is difficult, and the clinical effects of radiotherapy and chemotherapy are limited due to its tolerance to therapy. The median survival times are short, especially for high-grade gliomas. A high relapse rate occurred after treatment with poor prognosis, high mortality, and median survival less than one year. It is a serious threat to the lives of patients affects human health. Studying its pathogenesis, looking for new and effective treatment is one of the main goals of clinical research. Cell cycle regulation and apoptosis are closely related to the occurrence and development of gliomas. There are a lot of signal transduction systems which adjust the balance of the internal environment. The immunotherapy and gene therapy at the molecular level are the new research directions in treatment of glioma recent years. Inhibiting the abnormal activation of signaling pathways and regulating cell cycle are one of the effective ways to promote tumor cell apoptosis and to inhibit tumor cell proliferation. Notch signaling pathway is ubiquitous in both invertebrates and vertebrates with highly conservatism, playing an important role in cell proliferation, differentiation and development. Relationship between Notch signaling pathway and cancer has become one of the hot study spots in recent years. It has many interactions with the tumor pathways, shows abnormal expression of Notch receptors and ligands in a variety of tumor, which closely relates to occurrence and development of tumor. While the role of Notch expression are not the same in different types of tumors, even in the same tumor at different stages of development. It can be expressed as carcinogenic or inhibitive to tumor. Notch receptors, including Notch1-4, each of which having different biology functions in tumor, even opposite functions. Therefore, it is necessary to study each receptor alone. Notch signaling and tumorigenesis on causality is inconclusive. Targeting studies of Notch signaling pathway in tumorigenesis by using vitro and animal tests to explore the Notch signaling pathway and mechanism of action of tumor formation is a commonly used mode for providing new ideas and methods for cancer diagnosis and treatment.RNA interference is an effective means to study gene function, facilitates for effective target for drug discovery rapidly and comfimed by experimental techniques such as cell culture in vitro. Short hairpin RNA (shRNA) is a non-coding small RNA designed to be capable of forming a hairpin sequence which can be expressed in the nucleus itself inhibiting the expression of specific genes by endogenous RNA interference and silencing targeted genes long time stability. The plasmid vector is one of the main carriers of shRNA which can help aim shRNA to go through physiological barriers and to complete self-replication in nucleus to develop stabilizing and lasting interference effects. ShRNA plasmid construct and transfected into tumor cells is an important means to tumor study at the molecular level. Nude is deficient mutant mice with congenital thymus commonly used in oncology research as animal models. It is widely used in cancer research as for its characteristics of immune deficiency and easy observation.Recent studies have shown that Notch signals also play a role in human gliomas. However, the occurrence of Notch signaling in glioma and the development of the specific role to play in gliomas is not clear. In this study, we did the immunohistochemical tests of Notch2protein in32glioma specimens and20brain tissue samples to investigate the significance of Notch2in human brain glioma. The Notch2-shRNA interference plasmid were transfected into human glioma cell lines U251to study the transfection efficiency, cell cycle, cell proliferation and cell apoptosis in Notch2knockout glioma cell line after transfection by Real-time quantitative PCR, western blot, MTT and flow cytometry (FCM). After that we cultivated a stable Notch2knockout cell line and applied the cell line to build the nude mice tumor transplant model to observe the role of Notch2in tumor formation in vivo and provide reliable clinical theory basis in the cancer treatment. This study is divided into three parts.Part1:The study of the expression of Notch2protein and its significance in human gliomasObjective:To study the expression and significance of Notch2in human gliomas.Methods:immunohistochemistry was used to examine the expression of Notch2protein in22primary human gliomas and20normal cerebral tissuses. The correlation between expression of Notch2protein and pathological grades of gliomas were analyzed.Results:The expression level of Notch2protein in human gliomas was considerably higher than those in normal cerebral tissuses (P<0.001). The expression level of Notch2protein in human gliomas in grade Ⅰ～Ⅱ was significantly lower than those in grade III-IV (P<0.01).Conclusion:The Notch2protein is closely correlated with carcinogenesis and progression of human gliomas, which provide new thread and aceodance in gliomas clinical diagnosis and prognosis. Part2:Effects of shRNA-Notch2stable transfection on the proliferation, apoptosis and cell cycle of U251glioma cell lineObjective:The study was designed to investigate the long-term effect of stable expression shRNA-Notch2on proliferation, cell cycle and apoptosis of U251cells.Methods:The shRNA expressed vector that expresses the specific small hairpin RNA targeting Notch2mRNA and independence sequence was transfected into U251cells, the stably expressing shRNA cells were selected and continuously cultured. Then, the Notch2mRNA, Notch2signaling proteins, cellular proliferation activity as well as the cell cycle and apoptotic states were observed by RT-PCR, Westenblot, MTT assay and flow cytometry respectively.Results:Compared with the nontransfection group and the Negative-shRNA interference group, the protein expressions of Notch2, NICD, cyclinDl and MCM2in interference group were significantly decreased (P<0.01), and the protein expressions of p21and p27were significantly increased (P<0.01). In Notch2-shRNA interference group the cell proliferation was slow down begin at the5th day, the apoptosis rate in Notch2-shRNA interference group(13.22±1.90)%was significantly higher than the nonsence sequence group and Negative-shRNA interference group (2.62±0.26)%å'Œ(2.47±0.36)%(P<0.01). The cells in G1phase the Notch2-shRNA interference group was significantly higher than the Negative- shRNA interference group and nontransfection group (P=0.000<0.001, P=0.000<0.001,). The cells in S phase (22.37±1.90%) the Notch2-shRNA interference group was significantly lower than the Negative-shRNA interference group and nontransfection group (P=0.000<0.001, P=0.000<0.001,).Conclusion:Notch2is correlated to the proliferation, apoptosis and cell cycle of U251glioma cell line. Inhibiting of expression Notch2can inhibit the proliferation and induce the apoptosis of tumor cell. The cell cycle is blocked in G1to S. The function above may be developed by down regulation of cyclinDl and MCM2protein expression and up regulation of p21and p27protein expression. Part3:Studies of Notch2-shRNA stably transfected U251cell line subcutaneous transplanted in nude mouse.Objective:To observe the impact of stable transfection of Notch2-shRNA eukaryotic expression vector targeting Notch gene on tumorigenesis and angiogenesis of U251gliomas cells in nude mice in vivo.Methods:U251cells transfected stably with shRNA eukaryotic expression vector targeting Notch2gene, and U251cells transfected stably with blank vector, were inoculated respectively into subcutaneous tissue in flank of nude mice to establish xenograft models of human brain glioma. Each group contents10mice. The tumor growth status was observed and tumor volume was measured termly, and the tumor growth curve was drawn. The4animals in each group were killed and the tumor weight was investigated60days after inoculation and tumor inhibition rate was calculated. Protein expression of Notch2was investigated by immunohistochemistry method. Results:Both tumor volume and growth velocity in Notch2-shRNA transfection group decreased significantly compared to the nontransfection group and the Negative-shRNA transfection group, Tumor volume of nude mice in Notch2-shRNA transfection group was (2.72±0.21)mm2, significantly smaller than (9.23±0.17) mm2and (9.91±0.77) mm2in the nontransfection group and the Negative-shRNA transfection group (PO.001), and the tumor weight of nude mice in Notch2-shRNA transfection group was (0.46±0.04)g, significantly smaller than (1.32±0.03)g and (1.35±0.11) g in the nontransfection transfection group and the Negative-shRNA transfection group (P<0.001). In Notch2-shRNA transfection group, the tumor inhibition rate was65.74%. Notch2protein expression was down regulated markedly in Notch2-shRNA transfection group than other groups (P<0.01). The live time of nude mice in Notch2-shRNA transfection group was (114.33±9.05)d, significantly longer than those in the Negative-shRNA transfection group and nontransfection were [(70.83±10.50) d,(71.67±8.82)d](P<0.001).Conclusion:The specific shRNA targeting Notch2gene can inhibit significantly tumorigenesis and angiogenesis of U251cells in vivo. For this, Notch gene may act as a valuable target for gene therapy of glioma.