| Glioma is the mostcommon type of primary intraeranial tumors,which derived from glial cell deterioration. It account for the vast majority of adult malignant brain tumors that are graded according to the WHO classification system, which has implications for prognosis and management. The current standard therapy, includes maximal safe resection followed by radiotherapy in combination with temozolomide. Great progress have been made in surgery, radiotherapy and chemotherapy in recent years,and many researches have confirmed that those methods could prolonged the survival time of some patients with high grade glioma, however, overall prognosis of patients with high grade glioma have no improved. Moreover, because glioma is a kind of high malignant tumor and shows invasion during its growth, a majority of patients succumb to the disease within 2 years of diagnosis. Furthermore, the malignant degree of glioma usually increase after recurrence. 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. Currently, the role of PTTG1 in glioma,and the exact mechanisms which mediate its expression and function in glioma are not well understood. To know the exact function of PTTG1 in glioma,and analyze the the application of siRNA targeting PTTG1 gene in the human glioma treatment. In our study,we evaluated the PTTG1 expression in human patient samples by Immunohistochemistry,then analyzed the relationship between the expression of PTTG1 and pathological grade and prognosis. Subsequently, to explore its associated molecular mechanisms in glioma cells, we examined the effect of targeted silencing of PTTG1 gene on cell proliferation, invasion and chemotherapeutic sensitivity using siRNA in U373 cell.The present study was divided into three parts.1.Expression of PTTG1 in human glioma tissues and the correlation with clinical pathogenic characterObjective:To explore the expression of PTTG1 protein in human glioma tissues and glioma cell line,meanwhile, to analyze the correlation with clinical pathogenic character.Methods:Eighty patients with glioma were obtained from the neurosurgery in Inner Mongolia medical university affiliated hospital and Nanfang Hospital of Southern Medical University,from September 2012 to August 2014. There were 48 male and 32 female patients in the present study, aging between 35-68 years, and the average age is 47. The glioma tissue specimens were chosen according to the classification standard of central nervous system tumors of WHO in 2007. There were 10 cases I grade specimen,20 cases II and 25 cases III, IV grade respectively. Immonohistochemical method was used to detect the PTTG1 expression in the human glioma tissues, and PTTG1 expression in the glioma cell line U373 and LN-229 cells was observed with real-time PCR and western blot method.Results:PTTG1 protein expression was investigated by Immunohistochemistry and found to be upregjxlated in 80 cases of glioma compared with 10 NB tissues.Furthermore,PTTG1 positive expression mainly located in the cytoplasm of tumor cells, diffuse distribution in cells, a small amount of localization in the nucleus. PTTGl protein was highly expressed in 67.5%(54/80) of glioma samples, while only in 10.0%(1/10) of NB samples (P<0.01),and the expression intensity of PTTGl protein in the glioma tissues showed close correlation with the glioma pathogenic grade.Among the 10 cases normal human brain tissues obtaining from brain decompression operation, there’s nearly no positive PTTG1 expression in the 9 cases normal human brain tissue and only faint positive PTTGl protein expression in one case normal human brain tissue.Compared with NB tissues,glioma tissues exhibited higher expression levels of PTTG1 mRNA (4.37±0.58 vs 1.00±0.22,P=0.003).Furthermore,PTTGl protein expression was investigated by Westernblot and found to be upregulated in cases of glioma tissues.Conclusions:Significant up-regulation of PTTG1 expression was observed in glioma,and there is a positive correlation between the expression and the pathological grade,while there is a negative correlation between the expression and the prognosis. PTTGl expression may have significant value as an unfavorable progression indicator for glioma patients.2.Construction siRNA targeting PTTGl gene and the effects of its on glioma cell proliferation and invasion in vitroObjective:To construct and assess its silencing efficiency of siRNA targeting PTTG1 gene and explore the effects of PTTGl siRNA on the U373 cell proliferation, migration and invasion abilities.Methods:Three pairs interfering sequences were designed and specific PTTGl siRNAs were synthesized and inserted into the pGenesil2 vector. Using vector based RNA interference techniques, three interfering vectors were constructed to transcribe functional siRNA specially targeting PTTG1. The interfering plasmids were used to transfect U373 cells with lipofectmine2000 transfection reagent. Silencing efficiency,the levels of MMP2 and VEGF expression in the cells transfected with PTTG1 siRNA were analyzed by RT-PCR and Western blot. The migration and invasion ability of U373 cells were detected by scorching method and Transwell chamber method.Results:The plasmids of PTTG1 siRNA were successfully constructed by restriction endonuclease and DNA sequencing analyzing. The level of PTTG1 was assessed by RT-PCR,with the most efficient knockdowns from PTTG1 siRNA in U373 cell line compared to the controls vector (P<0.01). Consistent results for protein levels were observed by western blot.U373 cells transfected with PTTG1 siRNA showed slower proliferation, migration ability, attenuating invasion ability and lower levels of MMP2 and VEGF expression compared with normal control cells(0.34±0.02 vs 1.00±0.35 p<0.05;0.51±0.03 vs 1.00±0.09 p<0.05).Conclusions:The PTTG1 siRNA expression vectors were successfully constructed, and they silenced the PTTG gene level in U373 cells with high efficiency. PTTG1 siRNA inhibited proliferation and migration,invasion ability in U373 cell.3.Possible mechanism of PTTG siRNA on the apoptosis and chemothcrapeutic sensitivity of glioma cellsObjective:To explore the possible mechanism of PTTG shRNA on the apoptosis and chemotherapeutic sensitivity of glioma cells.Methods:The interfering plasmids were used to transfect U373 cells with Iipofectmine2000 transfection reagent.The proliferation and apoptosis of U373 cells combined with TMZ(in 24h,48h and 72h)were measured with MTT and flow cytometry method.Furthermore,the expression of PI3K、P-PI3K、Akt and p-Akt were detected by RT-PCR and western blot.Results:PTTG1 siRNA group could inhibit the proliferation and increase apoptosis ability of U373 cells.The outcome of MTT showed that the growth and survive were influenced in PTTG1 siRNA group and TMZ group (400umol/L).Especially, there was a statistically significance in PTTG1 siRNA combined with TMZ group compare to control.The apoptosis rate was detected by the flow cytometry analysis, there was a statistically significance between PTTG1 siRNA group and control group.We examined the effect of PTTG1 on PI3K/AKT pathway and found that reduced PTTG1 significantly decreased the expression of phosphorylated PI3K and AKT, but not their total protein levels.Conclusions:PTTG1 siRNA could inhibit the proliferation and increase apoptosis ability of glioma cell,furthermore,combined with TMZ could upregulate chemotherapeutic sensitivity. In addition, we provided that PTTG1 protein leads to suppressed cell growth,migration and invasion progression by activating the PI3K/Akt pathway in glioma cells.In conclusion, the expression of PTTG1 protein were related to pathological grade and the prognosis of glioma, and can be considered as the indicator of the maligant degree and the prognosis. Further mechanism research indicated that PTTG1 protein leads to suppressed cell growth,migration and invasion progression by promoting the expression of MMP2 and VEGF and activating the PI3K/Akt pathway in glioma cells.PTTG RNA interference combined with TMZ may upregulate chemotherapeutic sensitivity and Provide new strategies for the prognostic evaluation and treatment of glioma. |
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