The Study Of Notch2 Protein Expersstion In Human Brain Gliomas And The Role In Glioma Cell Proliferation, Cell Cycle, Apoptosis And Tumor Formation

Gliomas are the one of the most common brain tumors in human with poor prognosis. Although recent advances in surgical techniques and postoperative radiotherapy, chemotherapy applications made the survival rate of the gliomas increased than before, but it was still lower than 15 months. Survival rate for patients with glioblastoma, the most aggressive glioma, although individually variable, has improved after diagnosis in the last 5 years due to improvements in the standard of care. Furthermore, new discoveries are being made in basic and translational research, which are likely to improve this situation. These include agents that block one or more of the disordered tumor proliferation signaling pathways, and targeted therapies such as antiangiogenic therapy with antivascular endothelial growth factor antibodies are finding their way into clinical practice. Many studies in the world made a gradually profound understanding and many progress in molecular basis and genetic changes. I believe that in the near future, a new method for comprehensive treatment in tumor-related molecular pathways critical will reduce the mortality and morbidity in patients.RNA interference (RNAi) technology has not only become a powerful tool for functional genomics, but also allows rapid drug target discovery and in vitro validation of these targets in cell culture. Furthermore, RNAi represents a promising novel therapeutic option for treating human diseases, in particular cancer. Selective gene silencing by RNAi can be achieved essentially by two nucleic acid based methods: cytoplasmic delivery of short doublestranded (ds) interfering RNA oligonucleotides (siRNA), where the gene silencing effect is only transient in nature, and possibly not suitable for all applications; or nuclear delivery of gene expression cassettes that express short hairpin RNA (shRNA), which are processed like endogenous interfering RNA and lead to stable gene downregulation. Therefore, in vivo delivery of RNAi therapeutics must use technology that enables the RNAi therapeutic to traverse biological membrane barriers and Self-replicating in vivo. Plasmid and viruses vectors derived from them carry out precisely this task and have become a major delivery system for shRNA. The RNAi technology has been proven to enable us to study the molecular mechanisms of glioma to more in-depth and specific. In recent years, some studies have found the Notch signaling exist in human brain tumors. 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 test of Notch2 protein in 37 glioma specimens and 5 brain tissue samples to investigate the significance of Notch2 in human brain glioma. The pGFR-Notch2-shRNA interference plasmid were transfected into human glioma cell lines U251 to study the transfection efficiency, cell cycle, cell proliferation and cell apoptosis in Notch2 knockout glioma cell line after transfection. After that we cultivated a stable Notch2 knockout cell line and applied the cell line to build the nude mice tumor transplant model to observe the role of Notch2 in tumor formation in vivo and provide reliable clinical theory basis in the cancer treatment. This study is divided into four parts.Part one: The study of the expression of Notch2 protein and its significance in human gliomasObjective: To study the expression of Notch2 in human gliomas and the correlation between the expression and the pathological grades of gliomas, to explore the role of Notch2 in the tumorigenesis and progression of human gliomas, accordingly to provid some proof of theory for treatment of human gliomas.Method: immunohistochemistry was used to examine the expression of Notch2 protein in 37 primary human gliomas and 5 normal cerebral tissuses.Results: The expression of Notch2 protein have been detected in human gliomas and normal cerebral tissuses and the expression level of Notch2 protein in human gliomas was considerably higher than those in normal cerebral tissuses (Z=4.69,P=0.00); The Notch2 expression was significantly positively correlated with the grade of the gliomas (spearman correlation coefficient = 0.956, P = 0.000).Conclusion: The Notch2 protein expression has been detected in both human gliomas and normal cerebral tissuses, and the expression of Notch2 in human gliomas was considerably high. The expression of Notch2 is correlation to the pathological grades of human gliomas. Notch2 protein was invoived in the tumorigenesis and progression of human gliomas.Part two: Effects of short hairpin RNA targeting Notch2 in glioma cell line U251Objective: To investigate the effection of proliferation,apoptosis and cell cycle in human glioblastoma cell line U251 by Notch2 signal.Method: pGFP-V-RS Notch2 shRNA plasmid was transfected into U251 by Lipofectamine2000. The inhibitory rate of cell proliferation was detected by MTT assay and the transfection efficiency of shRNA plasmid,apoptosis rate and cell cycle were assessed by flow cytometry (FCM).The transcription changes of cell cycle-related protein levels were measured by RT-PCR and Western blotting.Results: The transfect effection of pGFP -V-RS Notch2 shRNA plasmid in U251 was 90.4±2.6%. After the transfection the Notch2 was significantily down-regulate in mRNA and protein level. Compared with nontransfection and independence groups, the cell proliferation was slow down begin at the 5 th day, G0/G1(68.2 %±1.29%) state was significantly higer than the independence and nontransfection groups (49.1%±3.65% ,35.4%±2.42% , P<0.05), G2/S(20.4%±1.56%) state was significantly lower than the independence and nontransfection groups (49.1%±3.65%,35.4%±2.42%,P<0.05), apoptosis rate was significantly increased after transfected with Notch2 shRNA plasmid in U251 cell lines( P < 0. 05). Protein expression levels of Cyclin D1 and p-Rb was down-regulated and p21 was up–regulated after the cells transfected with Notch2 shRNA plasmid.Conclusion: RNA interference (RNAi) mediated by the Notch2 shRNA expression vector could significantly down-regulate the expression of Notch2, Inhibit cell proliferation, induce remarkable apoptosis and G0/G1 cell cycle arrest in glioma cell line U251. It could be a new target for therapy of glioma.Part three: Effects of shRNA- Notch2 stable transfection on the proliferation, cell cycle and apoptosis of U251 glioma cell lineObjective: The study was designed to investigate the long- term effect of stable expression shRNA-Notch2 on proliferation, cell cycle and apoptosis of U251 cells.Methods: The shRNA expressed vector that expresses the specific small hairpin RNA targeting Notch2 mRNA and independence sequence was transfected into U251 cells, the stably expressing shRNA cells were selected and continuously cultured. Then, the Notch2 mRNA,Notch2 signaling 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 nonsence sequence group and the nontransfection group, the mRNA and protein expression of Notch2 in interference group was significantly suppressed. In interference group the cell proliferation was slow down begin at the 5th day, the apoptosis rate in interference group(15.14±4.26% )was significantly higher than the nonsence sequence group and nontransfection group (2.7±1.45%, 2.1±1.72% P<0.05). The cells in S phase (23.1±3.4% ) was significantly lower than the nonsence sequence group and nontransfection group (41.2±6.9% , 53.2±7.8 P<0.01), The cells in G1 phase (51.8±3.9%) was significantly higher than the other two groups (38.9±9.7% , 25.2±7.7 P<0.05), Protein expression levels of Cyclin D1 and MCM2 were down-regulated, p21 and p27 were up–regulated after the cells stably expressed Notch2-shRNA.Conclusion: Our study demonstrated that shRNA- Notch2 has long- term effect to decrease the Notch2 mRNA transcription, the cell cycle was arrested, apoptotic rate increased and cellular proliferation inhibited subsequently, which implies that regulating Notch2 genes by RNAi may be a potential approach in glioma gene therapy.Part four: Vivo studies of Notch2shRNA stably transfected U251 cell lineObjective: To observe the impact of stable transfection of specific short hairpin RNA (shRNA) eukaryotic expression vector targeting Notch gene on tumorigenesis and angiogenesis of human brain glioblastoma U251 cells in nude mice in vivo.Methods: U251 cells transfected stably with shRNA eukaryotic expression vector targeting Notch2 gene, and U251 cells 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 contents 10 mice. The tumor growth status was observed and tumor volume was measured termly, and the tumor growth curve was drawn. The 4 animals in each group were killed and the tumor weight was investigated 40 days after inoculation and tumor inhibition rate was calculated. Protein expression of Notch2 was investigated by immunohistochemistry method.Results: Comparing with those in U251 and U251 blank vector transfection groups, in U251 pGFP-V-RS Notch2 shRNA stable transfection group, the tumorigenesis time delayed, tumor grew slow, both tumor volume and tumor weight decreased significantly. tumor volume of nude mice in pGFP-V-RS Notch2 shRNA stable transfection group was significantly lower than other groups different from 35 days after transplantation (F = 6.336, P = 0.003). 40 days after transplantation the tumor weight inU251, and pGFP-V-RS vector stably transfected group (1.375±0.236) g, (1.231±0.558) g were significantly greater than pGFP-V-RS Notch2 shRNA stable transfected group (0.372±0.079) g, (F = 7.862, P<0.01). In pGFP-V-RS Notch2 shRNA stable transfected group the tumor inhibition rate was 72.9%. nude mice inU251 and pGFP-V-RS vector stably transfected groups were all dead within 88 days after transplantation, the average survival time was 73±4 and 72±3 days, 3 nude mice in pGFP-V-RS Notch2 shRNA stable transfection group to were still alive at the 130 days and the average survival time was 112±8 days, significantly longer than other goups. Notch2 protein expression was downregulated markedly in Notch2 shRNA stable transfected group than other groups (Z=9.87, P=0.00).Conclusion: The specific shRNA targeting Notch gene can inhibit significantly tumorigenesis and angiogenesis of U251 cells in vivo. For this, Notch gene may act as a valuable target for gene therapy of glioma, meanwhile, RNA interference (RNAi) technology may provide a novel and important means for gene therapy of glioma.