Research On The Effect Of SAA1 On Human Glioblastoma Cell Apoptosis And Its Mechanism

Background and Objective:Glioma is the most common primary malignant tumor of central nervous system,in which the incidence of glioblastoma(GBM)is the highest.GBM is highly malignant with invasive growth.It is difficult to completely remove the tumor tissue by surgery.Thus,patients with GBM are prone to relapse,which contributes to poor prognosis.It is of great importance to find effective therapeutic targets and prognostic markers for the treatment of GBM.The purpose of this study was to find the expression features of serum amyloid A1(SAA1)in GBM,and explore the association between down-regulation of SAA1 and apoptosis of GBM.In view of the association between apoptosis and chemotherapy resistance of Temozolomide(TMZ),we further studied the effects of down-regulation of SAA1 expression on regulating the sensitivity of TMZ.Meanwhile,we discussed the application value of SAA1 in the treatment of glioma.Methods:1.Find the expression features of SAA1 in glioma by bioinformatics methods.The GSE52009 dataset was downloaded from The Gene Expression Omnibus Dataset(GEO).And data of the RNA Expression of SAA1 in different grade gliomas were downloaded from The Cancer Genome Atlas and Chinese Glioma Genome Atlas databases.Statistical analyses were conducted:(1)the expression of SAA1 in different grade gliomas;(2)the expression of SAA1 in different subtype GBMs;(3)the association between SAA1 expression level and survival prognosis of patients with different grade gliomas.2.Immunohistochemical staining(IHC)was performed on human glioma tissue chips.Tumor tissues and peripheral blood samples of glioma patients were collected for Western blot and ELISA to find the expression differences of SAA1 in different grades of glioma.Following-up data was collected to analyze the association between SAA1 expression level and progression-free survival in patients with different grades of glioma.3.The expression level of SAA1 in human GBM cells was down-regulated by si RNA interference of human GBM cell lines U87 and U251.Western blot assay,JC-1 mitochondrial membrane potential staining,and PE / 7-AAD flow cytometry analysis was performed to observe the effect of down-regulation of SAA1 protein expression on apoptosis of GBM cells.Western blot assay was performed to detect the expression changes of apoptosis-related proteins(such as Bcl2,Bax,and Cleaved caspase3)in U87 and U251 cells after down-regulation of SAA1 protein expression level.4.CCK-8 assay,PE / 7-AAD flow cytometry assay,and TUNEL fluorescence staining assay was used to investigate whether down-regulation of SAA1 expression level could enhance the sensitivity of human glioma cells to Temozolomide(TMZ).Bioinformatics was used to analyze the effect of SAA1 expression levels on the overall survival of glioma patients who received chemotherapy or radiotherapy alone.At the same time,we analyzed the relationship between SAA1 expression level and molecular typing of glioma(such as MGMT methylation status,and 1P / 19 q gene combined deletion status).5.Western blot assay was used to detect the expression changes of AKT related proteins after the down-regulation of SAA1 expression.Furthermore,SC79 which is an AKT signaling pathway modulator was used to reverse the AKT signal of human GBM cells after the down-regulation of SAA1 expression.And the changes of AKT,P-Akt,and downstream apoptotic protein expression were detected.The changes of apoptosis ratio and sensitivity of human GBM cells to TMZ before and after the treatment with SC79 were detected by JC-1 mitochondrial membrane potential staining and flow cytometry.Results:1.Bioinformatics analysis showed that SAA1 expression was different in different grades of glioma.The expression level of SAA1 in high-grade gliomas was significantly higher than that in low-grade gliomas.And in GBM,the expression level of SAA1 in mesenchymal tumors was significantly higher than other types.At the same time,immunohistochemical staining and Western blot on human glioma tissue samples verified the difference of SAA1 protein expression in different grades of glioma.ELISA showed that SAA1 protein content in peripheral blood was associated with intracranial expression level,and was different in patients with different grades of glioma(p<0.01).2.Bioinformatics analysis showed that the overall survival of glioma patients with high expression of SAA1 was significantly lower than that of glioma patients with low expression of SAA1.Survival analysis showed that the progress-free survival of glioma patients with different grades was related with SAA1 expression level(p<0.01).3.JC-1 mitochondrial membrane potential staining and flow cytometry analysis showed that down-regulated SAA1 protein expression could significantly induce apoptosis of U87 and U251 cells.At the same time,the level of SAA1 protein in glioma was positively correlated with a series of anti-apoptotic genes(BFL1,MCL1,Fas,etc.),and negatively correlated with the expression levels of pro-apoptotic genes(Bim,Caspase 9,APAF1).Western blot analysis showed that the expression levels of apoptotic genes Bax and Cleaved caspase3 were increased in glioblastoma cells after down-regulation of SAA1 expression,while the expression levels of anti-apoptotic genes Bcl-2 were decreased.4.CCK-8 assay showed that down-regulation of SAA1 protein expression in GBM cells enhanced the efficacy of TMZ.The results of flow cytometry and TUNEL fluorescence staining showed that GBM cells with down-regulated SAA1 expression significantly increased apoptosis compared with normal cells after the same dose of TMZ treatment(p<0.01).Kaplan-Meier analysis showed that the overall survival of glioma patients with low SAA1 expression level was significantly longer than that of glioma patients with high SAA1 expression level under TMZ treatment alone.However,there was no significant correlation between SAA1 expression level and survival time of glioma patients who received radiotherapy only.In addition,glioma patients with MGMT promoter methylation and 1P / 19 q gene combined deletion had significant higher expression level of SAA1 and better overall survival,compared with glioma patients without such molecular pathological type.In glioma patients with MGMT methylation promoter,SAA1 expression level was negatively correlated with prognosis.5.Western blot results showed that down-regulation of SAA1 expression level in U87 and U251 cell lines could effectively reduce the content of intracellular AKT phosphorylated protein,and the total amount of AKT protein remained basically unchanged.After si RNA transfected GBM cells were treated with AKT phosphorylated agonist SC79,the content of p-AKT protein in GBM cells was significantly increased,and the apoptosis of GBM cells was significantly improved.In addition,GBM cells treated with SC79 were less sensitive to TMZ than before,and the apoptosis level of cells treated with TMZ at the same concentration was significantly lower than before(p<0.01).Conclusion:Results of this study show the differences of the SAA1 protein expression in different grades of glioma.High expression of SAA1 indicates poor prognosis.And the content of SAA1 in peripheral blood of glioma patients is related with that in tumor tissues.Down-regulation of SAA1 protein expression could induce GBM cell apoptosis and enhance its sensitivity to TMZ.Down-regulation of SAA1 affects GBM cell apoptosis by regulating the phosphorylation level of AKT.SC79 activation of AKT/Bcl2/Bax signaling pathway to a certain extent reduces the sensitivity of GBM cells to TMZ.It suggests that SAA1 may be involved in the mechanism of TMZ resistance.This study finds that SAA1 expression level has certain significance in evaluating TMZ efficacy in glioma patients,which could be a potential therapeutic target and prognostic indicator of glioma.