Study On The Clinical Prognosis Of Glioma And The Molecular Mechanism Of Inhibition Of Glioblastoma By Nifulmic Acid

The doctoral thesis consists of two parts.The first part is the clinical prognosis of glioma.The second part is the molecular mechanism of inhibition of glioblastoma by niflumic acid.Part I Study on clinical prognosis of gliomaObjective To analyze the related factors affecting the prognosis of glioma in order to provide theoretical basis for the diagnosis,treatment and prognosis evaluation of glioma.Methods The prognosis of 246 patients with glioma in Ningxia Medical University General Hospital was analyzed.Includes demographic information;Clinical data;Degree of surgical resection;Radiotherapy,radiation dose,chemotherapy,chemotherapy dose,Molecular markers such as EMA,Vimentin,GFAP,IDH1,S100,MGMT,Neun,CD34,1p19 q co-deletion,Oligo 2,Ki67,and P53.The expression of IDH1 and Ki67 was detected by immunohistochemical staining.SPSS22.0 software was used for statistical analysis.Log-rank method was used for univariate analysis and Cox proportional regression model was used for multivariate analysis.The independent prognostic factors affecting the prognosis of patients with glioma were determined.The corresponding survival curves were drawn by Kaplan-Meier method.Spearman method was used to analyze the correlation.Results 1.The 1-year,3-year and 5-year survival rates of high-grade and low grade glioma patients were 67.3%,43.3%,15.6%;100%,95.7%,75.0% respectively.The mortality rate of high grade glioma patients was significantly higher than that of low grade glioma group(37.0% vs 5.0%,P<0.001).2.Univariate analysis showed that marriage,KPS score,blood type and CD34 were factors affecting progression free survival in patients with low grade glioma(P<0.05),while KPS score,S100 and CD34 were factors affecting overall survival in patients with low grade glioma(P<0.05).Smoking,drinking,KPS score,tumor boundary,the occurrence of preoperative epilepsy,surgical resection degree,chemotherapy,radiotherapy,GFAP,IDH1 and Ki67 were factors affecting progression free survival in patients with high grade glioma(P<0.05).While age,marriage,KPS score,surgical resection degree,chemotherapy,radiotherapy,radiotherapy dose,IDH1,Ki67,MGMT were factors affecting the overall survival of high grade glioma patients(P<0.05).3.In order to further clarify the independent prognostic factors of the two groups,Cox multivariate analysis was performed.The results showed that in low grade glioma group,negative expression of CD34(HR=0.019,P=0.012)and(HR=0.142,P=0.003)were independent protective factors affecting overall survival and progression free survival,while KPS score ? 70(HR=0.055,P=0.040)and(HR=0.106,P=0.001)were independent protective factors affecting overall survival and progression free survival.In high grade glioma group,High expression of Ki67(HR=2.170,P=0.032)and age?60 years(HR=1.889,P=0.039)were independent risk factors affecting the overall survival of patients.Postoperative chemotherapy(HR=0.359,P=0.043)and IDH1 mutation(HR=0.227,P=0.018)were independent protective factors affecting overall survival of patients.High expression of Ki67(HR=1.685,P=0.017)is an independent risk factor for progression free survival in patients.IDH1 mutant(HR=0.541,P=0.021)and postoperative chemotherapy(HR=0.588,P=0.007)are independent protective factors affecting progression free survival.By Spearman correlation test,there was a negative correlation between the expression of IDH1 mutant and the high expression of Ki67 in high grade glioma patients(r=0.319,P < 0.001).Conclusion 1.The prognosis and survival rate of high grade glioma patients with gliomas in Ningxia is lower than that of low grade glioma patients.(the 1-,3-and 5-year survival rates are 67.3% vs 100%,43.3% vs 95.7%,15.6% vs 75.0%,respectively).2.Negative expression of CD34 and high KPS score are protective factors for the prognosis of patients with low grade glioma.3.IDH wild type and high expression of Ki67 are poor prognostic factors in patients with high grade glioma.Postoperative chemotherapy is a protective factor for the prognosis of patients with high grade glioma.Part II Study on the molecular mechanism of inhibition of glioblastoma by niflumic acidObjective The aim of this study was to investigate the molecular mechanism of apoptosis and autophagy induced by niflumic acid(NFA)in glioblastoma multiforme(GBM)cells in vitro,and to explore the molecular mechanism of NFA induced changes in the resistance of GBM cells to temozolomide(TMZ).Methods In this study,the effects of different concentrations of NFA on the viability of U87 cells and U251 cells were detected by CCK-8 method;the effects of NFA on the apoptosis degree of U87 cells were detected by TUNEL method;the effects of NFA on the apoptosis degree of U87 cells were detected by flow cytometry;after the treatment of U87 cells with NFA,the expression of apoptosis related proteins(Bcl-2,Bax and caspase-3)and apoptosis pathway related proteins(p53 and p21)in the control group and the experimental group were detected by Western blot;the expression of tumor cell proliferation related antigen Ki67 and apoptosis related protease caspase-3 in the tumor tissue of mice were analyzed by immunohistochemistry;the effect of NFA on autophagy of GBM cells was observed by fluorescence microscope;The expression of autophagy related proteins LC3 II/I and beclin-1in GBM cells after NFA intervention was detected by Western blot;the TMZ resistant cell line of U87 cells was constructed;the viability of U87 / TMZ-R cells was detected by CCK-8method;the apoptosis of U87 / TMZ-R cells was detected by flow cytometry;the apoptosis of U87 / TMZ-R cells was detected by TUNEL method.Results In the study of the effect of NFA on the apoptosis and autophagy of U87 cells,CCK-8 assay showed that compared with the control group,the survival rates of U87 and U251 cells were decreased when NFA concentration was 200,300,400 and 500 ?M(P < 0.05 or P < 0.01),and NFA inhibited the growth of cells in a dose-dependent manner,with IC50 of350 ?M;Compared with the control group,TUNEL staining showed significant apoptosis in U87 cells after NFA intervention(P < 0.05);The results of flow cytometry showed that compared with the control group,the apoptosis of U87 cells after NFA intervention was significantly increased(P < 0.05);Western blot analysis showed that the apoptosis of U87 cells after NFA intervention was significantly increased(P < 0.05)Compared with the control group,the expression of Bcl-2 was significantly decreased in NFA treated cells,while the expression of Bax and Caspase3 was significantly increased in NFA treated cells(P < 0.05).Compared with the control group,the expression of p53 and p21 were significantly increased(P < 0.05);NFA could not only inhibit the growth of tumor cells,but also induce the apoptosis of tumor cells;immunohistochemical results showed that the expression of Ki67 protein in tumor tissues of NFA group was significantly decreased(P < 0.05),the results of immunohistochemistry showed that the expression of caspase-3 protein in tumor tissue of NFA group was significantly higher than that of the control group(P < 0.05);The results of electron microscopy showed that GBM cells in NFA treatment group had irregular cell boundary,disordered organelle and nuclear structure,and autophagosome could be seen in cytoplasm;Fluorescence microscopy results showed that LC3 red fluorescence was significantly expressed in GBM cells treated with NFA,indicating that NFA could promote autophagy in GBM cells.The expression of LC3 II/I and beclin-1 in NFA treated GBM cells was significantly higher than that in the control group(P < 0.05),indicating that NFA can promote the occurrence of autophagy in GBM cells;Western blot analysis showed that the protein expression levels of LC3 II/I and beclin-1 in NFA treated GBM cells were significantly higher than those in the control group(P < 0.05).In the study of the effect of TMZ-R cell apoptosis and autophagy,CCK-8 detection results showed that compared with the control group of TMZ sensitive cells,U87 / TMZ-R cell line showed resistance to TMZ treatment,and NFA(350 ?M)was detected in U87/TMZ-R cells/ TMZ-R cells showed mild killing effect,but the combination of NFA and TMZ significantly inhibited cell growth(P <0.05);Flow cytometry results showed that compared with TMZ sensitive cells in the control group,NFA(350 ?M)group and TMZ(500 ?M)cells had mild apoptosis,and TMZ(500 ?M)+ NFA(350 ?M)group cells had significant apoptosis(P < 0.05);TUNEL assay showed that NFA(350 ?M)group and TMZ(500 ?M)+ NFA(350 ?M)group cells had significant apoptosis(P < 0.05)The results showed that there were almost no positive cells in the control group,and apoptotic cells were occasionally seen in NFA(350 ?M)group and TMZ(500 ?M)group.The number of positive staining cells in TMZ(500 ?M)+ NFA(350 ?M)group was significantly higher than that in the control group(P < 0.05).Conclusion 1.NFA can inhibit the proliferation of U87 cells;2.The expression of Bcl-2in U87 cells after NFA treatment was significantly reduced,Bax and caspase 3 were significantly increased in U87 cells after NFA treatment;3.The expression of p53 and p21 in U87 cells after NFA treatment was significantly increased;4.The expression levels of LC3II/I and Beclin-1 in U87 cells after NFA intervention were significantly increased;5.NFA can increase the sensitivity of U87/TMZ-R resistant cells.NFA and TMZ could inhibit the proliferation of U87 / TMZ-R cells and induce apoptosis.