Study Of The Effects Of RNAi Silencing PTTG And Survivin Gene On The Human Brain Glioma Cells U251 Cytobiological Behaviors And Its Relative Mechanism

Glioma is the most frequently occurring tumor in the central nervous system,occupying 40%-60% of the intracalvarium tumors. Although there was great advance in the clinical diagnosis and treatment of glioma,the prognosis of these patients has not improved obviously and their mortalities are still high. Therefore,human brain glioma was still the difficult dealt disease in the neurosurgical tumors,so most researchers focused on its etiology, pathogenesis, biological character,new and effective treatment techniques.PTTG and Survivin are expressed at higher level in most of the malignant tumors,there is a close correlation between the two genes and the glioma's genesis and developments.At present there was not report about the treatment targeting PTTG and Survivin at the same time on human glioma.Therefore,to study the mechanism of PTTG and Survivin in facilitating glioma development and explore the superiority of RNAi treatment targeting PTTG and Survivin gene comparing treatment targeting PTTG or Survivin.The present study observed the PTTG and Survivin protein expression in human glioma tissues using immunohistochemical methods and analyzed the correlation between the two gene's expression and clinical pathological characters.Using RNA interference technique silencing PTTG and Survivin gene at the same time,the effects and its relative mechanism on the human glioma cell line U251 cells was observed.The present study was divided into four parts. The follows are:1.Expression of PTTG and Survivin protein in human glioma tissues and their correlation with micro-vessel density(MVD).Objective:To explore the expression of PTTG and Survivin protein in human glioma tissues and its correlation with clinical pathogenic character and micro-vessel density(MVD). Meanwhile, PTTG and Survivin protein expression in the human glioma cell line was observed and established the basis for the subsequent research.Method: 81 patients with glioma treated at the Institute of Neurosurgy in the present hospital,from January 2005 to September 2008 were studied respectively.There were 43 male and 38 female patients in the present study, aging between 5-73 years,and the average age is 43.7.The glioma tissue specimens were chosen according to the classification standard of central nervous system tumors of WHO in 1999.There were 14 cases I grade specimen, 22 cases II grade specimen, 24 cases III grade specimen,21 cases IV grade specimen grade respectively. Immonohistochemical method was used to detect the PTTG and Survivin protein expression and micro-vessel density in the human glioma tissues. PTTG and Survivin protein expression in the glioma cell line U251 cells was observed with Western blot method.Results:Among the 9 cases normal human brain tissues obtaining from brain decompression operation,there's nearly no positive PTTG protein expression in the 8 cases normal human brain tissue and there's only faint positive PTTG protein expression in 1 case normal human brain tissue,as for Survivin there is no positive protein expression.Different grade glioma tissues in the present study showed higher PTTG and Survivin protein positive expression than that in the normal human brain tissues(P<0.01). The expression intensity of the two kinds of protein in the glioma tissues showed close correlation with the glioma pathogenic grade using Spearman rank correlation analysis(rs=0.508,P<0.01 for PTTG; rs=0.520,P<0.01 for Survivin). Non-parameter Kruskal-Wallis analysis was used to analyze the correlation of glioma pathogenic grade with MVD or the correlation of the two kinds of protein(PTTG and Survivin) expression intentsity with MVD,and the results showed that MVD increased significantly with the glioma pathogenic grade(χ2=49.337,P<0.01),the PTTG(χ2=51.567, P<0.01)and Survivin(χ2=49.613, P<0.01) protein expression intensity increased.There was high level PTTG and Survivin protein expression in the U251 cells.Conclusions:There was high level PTTG and Survivin protein expression in human brain glioma tissues and it has close correlation with glioma pathogenic grade and angiogenesis.That mean PTTG and Survivin could be the molecular marker in clinic auxiliary diagnosis of glioma and the two genes might be the potential target of glioma treatment.There was high level PTTG and Survivin protein expression in human glioma cell lines U251,so the cell line could be the target cells of PTTG and Survivin gene silencing.2.Construction and its silencing efficiency assessment of siRNA targeting PTTG and Survivin gene.Objective:To construct and assess its silencing efficiency of siRNA targeting PTTG and Survivin gene.Methods: Acording to our preliminary experiment we designed the interfering sequence targeting PTTG and Survivin,then specific PTTG siRNA and Survivin siRNA were synthesized and inserted into the pGenesil2.1 vector.By subcloning we obtain the pGenesil2.1- PTTG-Survivin-siRNA expression vector.The interfering plasmids were used to transfect U251 cells with lipofectmine2000 transfection reagent.Transfection efficiency was measured with pGensil1.1 plasmid with EGFP fluorescence protein.The two genes'(PTTG and Survivin) mRNA and protein expression levels in the cells transfected with three siRNA expression vectors(pGenesil2.1- PTTG–siRNA,pGenesil2.1-Survivin-siRNA,pGenesil2.1- PTTG-Survivin- siRNA) were analyzed with RT-PCR and flow cytometry methods.Results:With restriction endonuclease and DNA sequencing analyzing, three siRNA expression vectors ( pGenesil2.1-PTTG–siRNA , pGenesil2.1- Survivin-siRNA ,pGenesil2.1-PTTG-Survivin-siRNA)were successfully constructed.Transfection efficiency of U251 was 67.2%.The mRNA and protein level of the two genes(PTTG and Survivin) in U251 cells transfected with three kinds of RNAi expression plasmid decreased significantly compared with the normal control group(P<0.01),especially in the cells transfected with pGenesil2.1-PTTG-Survivin-siRNA where there is the lowest mRNA and protein level of the two genes.Conclusions:The pGenesil2.1-PTTG-Survivin-siRNA expression vectors were successfully constructed,and it has a most strongest silencing effect on the two genes(PTTG and Survivin)in U251 compared with pGenesil2.1-PTTG -siRNA,pGenesil2.1- Survivin-siRNA.3.Effects of PTTG and Survivin gene silencing on human glioma cells behaviors Objective:To explore the effects of pGenesil2.1-PTTG-Survivin-siRNA on the U251 cell proliferation,apoptosis,migration and invasion abilities.Methods: pGenesil2.1-PTTG–siRNA,pGenesil2.1- Survivin-siRNA,pGenesil2.1-PTTG- Survivin-siRNA plasmids were transfected into the U251 cells with lipo2000.Cell proliferation ability was measured with MTT method in U251 cells after they were transfected with plasmids 24 h,48 h and 72 h.The change of U251 apoptosis percentage was detected with Annexin V-PI kits after they were transfected with the three kinds of plasmids for 48 h. Transwell chamber were used to observe the transfected U251 cells migration ability and Transwell chamber combining matrigel was used to detect the transfected U251 cells invasion ability.Results:In this part,stable vector expression cell line transfected with three kinds of siRNA vectors was established with G418 screening method previously.However,the three stable expression cell lines grew slowly in poor conditions.Transient transfection method was used in the following experiment.U251 cells transfected with the three siRNA vector plasmids showed slower proliferation,increased apoptosis percentage,slower migration ability and attenuating invasion ability compared with normal control cells. Especially in the cells transfected with pGenesil2.1- PTTG -Survivin-siRNA where all the changes above was the most obvious.Conclusions:Compare with the pGenesil2.1-PTTG–siRNA and pGenesil2.1- Survivin-siRNA,the pGenesil2.1-PTTG-Survivin-siRNA can silence PTTG and Survivin at the same time, furthermore it can achieve a better inhibit effect on transfected U251 cell's proliferation and migration,invasion ability and facilitate cell apoptosis.RNAi targeting PTTG and Survivin gene at the same time might offer the new direction for the treatment of glioma. 4.Possible mechanism of pGenesil2.1- PTTG -Survivin-siRNA on the behavior of glioma cellsObjective:To explore the possible mechanism of pGenesil2.1- PTTG -Survivin-siRNA on the behavior of glioma cells.Methods:Propidium iodide(PI)was used to detect the change of cell cycle in the transfected U251 cells with the three different siRNA plasmid vectors. VEGF,P53,HDM2,P14ARFand Rb protein percentage were measured with flow cytometry method.Caspase3 and caspase9 activities were measured with spectrophotometry.Results:Compared with the negative HK group,s phase percentage of three groups U251 cells transfected with different siRNA expression vector decreased significantly (P<0.01),G0/G1 phase cell percentage increased significantly (P<0.01) ,caspase3 and caspase9 activities increased significantly (P<0.01) ,p53和P14ARF protein level increased significantly (P<0.01),while VEGF and HDM2 protein level decreased significantly(P<0.01),there was no change in every group for the level of Rb.Conclusions:whether the pGenesil2.1-PTTG–siRNA or pGenesil2.1- Survivin -siRNA can produce an obvious impact on U251 cell's apoptosis,generation cycle and the expression of some proteins(such as VEGF,P53,HDM2,P14ARF,Rb) which have a close relation to cell cycle.But the pGenesil2.1- PTTG– Survivin -siRNA which can silence the two genes at the same time can obtain a more obvious effect.In summary,PTTG and Survivin 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 the pGenesil2.1- PTTG– Survivin -siRNA plasmids which can silence the two genes at the same time can obtain a better suppress effect on glioma than the pGenesil2.1-PTTG–siRNA or the pGenesil2.1- Survivin -siRNA.There were still no reports about the application of siRNA targeting PTTG and Survivin gene at the same time in the human glioma treatment.