| Glioma is the most frequently occurring tumor in the central nervous system, occupying 40% - 50% of the intracalvarium tumors. During the last twenty years, although there was great advance in the clinical diagnosis and treatment of glioma, the prognosis of these patients has not improved obviously and their mortality was still high. The median survival time was only 52 week in the malignant glioma patients. Therefore, human brain glioma was still the difficult dealt disease in the neurosurgical tumor, so most researchers focused on its etiology, pathogenesis, biological character and new and effective treatment techniques.Pituitary tumor transforming gene (PTTG) is a novel oncogene that is expressed at higher level in most of the tumors analyzed to date compared to normal tissues. PTTG could activate many kinds of tumor related genes, inhibited chromosome separation in the mitotic phase, induced tumor angiogenesis and it has close correlation with glioma generation, development and prognosis. At present there was not report about the treatment targeting PTTG gene on human glioma. Therefore, to study the mechanism of PTTG facilitating glioma development and explore the possibility RNAi treatment targeting PTTG gene, the present study observed the PTTG protein expression in human glioma tissues using immunohistochemical methods and analyzed the correlation between PTTG protein expression and clinical pathological characters. Using RNA interference technique silencing PTTG gene, the effects and its relative mechanism of PTTG shRNA on the human glioma cell line U87, U251 and SHG-44 cells was observed. The present study was divided into four parts.The follows are:1. Expression of PTTG protein in human glioma tissues and its correlation with micro-vessel density (MVD)Objective: To explore the expression of PTTG protein in human glioma tissues and its correlation with clinical pathogenic character and micro-vessel density (MVD). Meanwhile, PTTG protein expression in the human glioma cell line was observed and established the basis for the subsequent research work. Method: Seventy-two patients with glioma treated at the Institute of Neurosurgy in the present hospital, from January 2004 to September 2007 were studied respectively. There were 43 male and 29 female patients in the present study, aging between 5-71 years, and the average age is 42.9. The glioma tissue specimens were chosen according to the classification standard of central nervous system tumors of WHO in 1999. There were 12 cases I grade specimen and 20 cases II, III, IV grade respectively. Immonohistochemical method weas used to detect the PTTG protein expression and micro-vessel density in the human glioma tissues, and PTTG protein expression in the glioma cell line U251, U87 and SHG-44 cells was observed with immonohistochemical and Western blot method. Results: Among the 5 cases normal human brain tissues obtaining from brain decompression operation, there's nearly no positive PTTG protein expression in the 4 cases normal human brain tissue and there's only faint positive PTTG protein expression in one case normal human brain tissue. Different grade glioma tissues in the present study showed higher PTTG protein positive expression than that in the normal human brain tissues (P<0.01). The expression intensity of PTTG protein in the glioma tissues showed close correlation with the glioma pathogenic grade using Spearman rank correlation analysis (rs=0.540,P<0.01) . Non-parameter Kruskal-Wallis analysis was used to analyze the correlation of glioma pathogenic grade with MVD or the correlation of PTTG protein expression intentsity with MVD, and the results showed that MVD increased significantly with the glioma pathogenic grade (x2=98.755, P<0.01) and PTTG protein expression intensity increased (x2=92.798, P<0.01). There was high level PTTG protein expression in the U251, U87 and SHG-44 cells, especially in U251 cells and U87 cells showed relative higher level PTTG protein expression. Conclusions: There was high level PTTG protein expression in human brain glioma tissues and it has close correlation with glioma pathogenic grade and angiogenesis. That mean PTTG could be the molecular marker in clinic auxiliary diagnosis of glioma and it might be the potential target of glioma treatment. There was high level PTTG protein expression in human glioma cell lines and U251, U87, SHG-44 cells could be the target cells of PTTG gene silencing.2. Construction and its silencing efficiency assessment of shRNA targeting PTTG geneObjective: To construct and assess its silencing efficiency of shRNA targeting PTTG gene. Methods: Three pairs interfering sequences were designed and specific PTTG siRNAs were synthesized and inserted into the pGenesil2 vector. Using vector based RNA interference techniques, three interfering (pGenesil2-PTTG siRNA1, pGenesil2-PTTG siRNA2 and pGenesil2-PTTG siRNA3) vectors were constructed to transcribe functional siRNA specially targeting PTTG. The interfering plasmids were used to transfect U251, U87 and SHG-44 cells with lipofectmine2000 transfection reagent. Transfection efficiency was measured with pGensil1 plasmid with EGFP fluorescence protein. PTTG mRNA and protein expression levels in the cells transfected with PTTG shRNA were analyzed with RT-PCR and flow cytometry methods. Results: With restriction endonuclease and DNA sequencing analyzing, eukyaryotic expression plasmids of PTTG shRNA were successfully constructed. Transfection efficiency of U251 and U87 cells was 60% and 40% respectively, while the transfection efficiency of SHG-44 cell was about 22%. PTTG mRNA and its protein level in transfected U251 and U87 cells with PTTG RNAi plasmid decreased significantly compared with the normal control group (P<0.01). There was no statistic difference between PTTG shRNA transfectd SHG-44 cells and normal SHG-44 cells. Conclusions: The PTTG siRNA expression vectors were successfully constructed, and they silenced the PTTG gene level in U251 and U87 cells with high efficiency.3. Effects of PTTG gene silencing on human glioma cells behaviorsObjective: To explore the effects of PTTG shRNA on the U251 and U87 cell proliferation, apoptosis, migration and invasion abilities. Methods: pGenesil-2 PTTG shRNA plasmids were transfected into the U251 and U87 cells with lipo2000. Cell proliferation ability was measured with MTT method in U251 and U87 cells after they were transfected with PTTG shRNA plasmids 24 h, 48 h and 72 h. The change of U251 and U87 apoptosis percentage was detected with Annexin V-PI kits after they were transfected with PTTG shRNA plasmids for 48 h. Scorching method and Transwell chamber were used to observe the transfected U251 and U87 cells migration ability and Transwell chamber combining matrigel was used to detect the transfected U251 and U87 cells invasion ability. Results: In this part, stable PTTG shRNA expression cell line was established with G418 screening method previously. However, the stable PTTG shRNA expression cell line grew slowly in poor conditions. Transient transfection method was used in the following experiment. U251 and U87 cells transfected with PTTG shRNAs showed slower proliferation, increased apoptosis percentage, slower migration ability and attenuating invasion ability compared with normal U251 and U87 cells. U251 and U87 cells transfected with PTTG shRNA showed dilating endoplasmic reticulum, increasing vacuoles and diminishing villus with transmitting electron microscope. Conclusions: PTTG gene is a kind of proto-oncogene which is related with brain glioma development closely. PTTG shRNA in this study inhibited transfected U251 and U87 cells proliferation and migration, invasion ability and facilitate cell apoptosis. RNAi targeting PTTG gene might offer the new direction for the treatment of glioma. 4. Possible mechanism of PTTG shRNA on the behavior of glioma cellsObjective: To explore the possible mechanism of PTTG shRNA on the behavior of glioma cells. Methods: Propidium iodide (PI) was used to detect the change of cell cycle in the transfected U251 and U87 cells. P53, bcl-2 and bax protein percentage were measured with flow cytometry method. C-myc protein expression was observed with Western blot method. Caspase3 and caspase9 activities were measured with spectrophotometry. Ca2+ content in the cell with Fluo-3/AM reagent. Results: Compared with the negative HK group, s phase percentage of transfected U251 cells decreased significantly (P<0.01), G0/G1 phase cell percentage increased significantly (P<0.01), caspase3 and caspase9 activities and Ca2+ content increased significantly (P<0.01), p53å’Œbcl-2 protein level increased significantly (P<0.01), while c-myc and bax protein level decreased significantly (P<0.01). Conclusions: Bcl-2, bax, p53 and c-myc proteins changed obviouslt in the U251 and U87 cells with PTTG shRNA plasmids, combing caspases activities and Ca2+ content change, which showed that transfected U251 or U87 cells apoptosis might be caused by the mitochondria regulation pathway.In summary, PTTG gene expression products might be the auxiliary diagnosis way in assessing glioma malignance and invasion ability, which might direct the glioma operation and further treatment. The present study showed that PTTG shRNA plasmids could silence PTTG gene effectively and RNAi gene therapy targeting PTTG might offer the new direction for treatment glioma. There were still no reports about the application of siRNA targeting PTTG gene in the human glioma treatment.
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