| Purpose: Brain malignant gliomas have the poorest prognosis of intracranial primary tumors. Chemotherapy with temozolomide(TMZ), a kind of alkylating agent, is the most important component of glioma comprehensive therapy. However, the clinical result of brain malignant gliomas cannot satisfy human beings’ anticipation, which is closely related to the TMZ chemoresistance. The detailed process can be described as follows: TMZ may cause DNA damage while this adverse impact can activate DNA damage response(DDR) in return, resulting in the repair of DNA damage. For example, O6-methylguanine-DNA Methyltransferase(MGMT), a critical DNA repair protein, repairs the damaged DNA via transferring the methyl group from O6-methylguanine to a cysteine residue of itself, thus leading to TMZ chemoresistance, but the functions of MGMT depends on the initiation of DDR. Ataxia-telangiectasia mutated(ATM), as the key gene of DDR upstream plays important roles in cell cycle arrest, apoptosis, and DNA repair. But, the functions of ATM in glioma resistance to DNA alkylating agents are not fully elucidated yet. The previous study of our project found that the silence of ATM gene can induce glioma cells resistance to TMZ in half maximal inhibitory concentration(IC50), hence, we utilized the silence of ATM to verify the specific functions of ATM in TMZ chemotherapy in vitro and vivo. Furthermore, detail studies of a molecular pathway relevant to ATM silence induced TMZ chemoresistance of gliomas can be conducted, thus for further clinical translation research. All of these are aimed to discover the valid therapeutic targets for glioma chemoresistance or molecular markers for chemosensitive and prognostic prediction, and achieve the purpose of improving the prognosis of patients. Methods: 1) RNA interference(RNAi) technique was used to silence the ATM gene of U87 and U251 glioma cells, then, the U87 glioma cells with ATM silence were transplanted subcutaneously in nude mice to set up a xenograft tumor model; The cell growth inhibition rate of chemotherapy, cell cycle and cell apoptosis were detected by MTT assay and flow cytometry technique in vitro and the tumors volume and weight changes were recorded in vivo to analyze the influence of ATM silence on TMZ chemotherapy. 2) Human genome-wide oligonucleotide chips(Affymetrix Company, USA) were applied to detect the difference of gene expression pattern in ATM silent U251 cells. Additionally, Cellomics, a high content screening system, was used to screen the selected target genes relevant to ATM silence induced TMZ chemoresistance of glioma. 3) The proliferation changes were explored via a series of analysis methods, including MTT assay and flow cytometry technique, to comprehend the effect of target genes silence carried out by RNAi in U251 glioma cells on the TMZ chemotherapy. 4) Immunohistochemistry was utilized to evaluate the expression of phosphorylated ATM(p-ATM), target genes and MGMT in surgical specimens of glioblastoma, and the correlation of their expression and clinical prognosis were analyzed and studied. Results: 1)We obtained ATM silent U87 and U251 cell strains based on above methods. After 5 days TMZ(IC50) chemotherapy, the cell growth inhibition rate were 21.0±1.7% and 29.1±3.0 % of ATM silent U87 and U251 cells, lower than 27.5±3.8%(p=0.008) and 36.8±2.0%(p=0.001) of negative control groups, and the apoptosis rate were 2.23±0.05% and 15.31±1.71%, less than 3.73±0.15%(p=0.000) and 21.38±1.13%(p=0.001) of negative control groups, respectively; Meanwhile, we also obtained a xenograft tumor model of ATM silent U87 glioma cells in nude mice, and tumors of ATM silent group presented smaller tumor volume than that of negative control group. Compared with the volume at the start of TMZ chemotherapy, the fold changes of the tumor volume in ATM silent group were 4.00±3.68 and 5.72±4.92, higher than 1.18±0.18(p=0.027) and 1.22±0.19(p=0.012) in negative control group at 10 days and 13 days after TMZ withdrawal. Moreover, the average weight of xenograft tumors suffered ATM silence was 0.213±0.246 g, still lighter than 0.595±0.214g(p=0.002) of negative control group at the end of the experiment. 2) Differentially expressed genes in ATM silent U87 cells were 726 through the chip screening, 16 of them were picked out according to predetermined conditions to do the next detection. The gene of interferon-induced protein with tetratricopeptide repeat1(IFIT1) silence was found to get the lowest inhibition rate of 5 days’ TMZ(IC50) chemotherapy among the 16 screened genes, reaching to 8.77%, ever lower than 10.29% of ATM gene silent group( positive control). 3) The IFIT1 gene silent U251 strain was also generated based on above method. After 5 days TMZ(IC50) chemotherapy, the cell growth inhibition rate and cell apoptosis rate of IFIT1 silent U251 cells were 15.3±2.1 % and 6.61±0.28%, both lower than 29.3±4.9%(p=0.034) and 7.43±0.41%(p=0.000) of negative control group. 4) There were some corresponding relations of gene expression in the surgical specimens of glioblastoma, that is, the expression of p-ATM and IFIT1 gene was positive correlated(r=0.249,p=0.037), while the expression of IFIT1 and MGMT showed an opposite trend(r=-0.288,p=0.016). Univariate and multivariate analyses confirmed high IFIT1 expression as a favorable prognostic indicator for progression-free survival(PFS)(p=0.003 and0.017) and overall survival(OS)(p=0.001 and 0.001) compared with low IFIT1 expression group, respectively. Patients with 2 favorable factors(high IFIT1 and low MGMT) had an improved prognosis as compared with one favorable factor(high IFIT1 and MGMT or low IFIT1 and MGMT) and without any favorable factors(low IFIT1 and high MGMT). Conclusions: 1) U87 and U251 cell strains with stable ATM silent gene and a xenograft tumor model of ATM silent U87 glioma cells in nude mice were successfully set up in this study. Besides, the functions of restraining glioma cells apoptosis and promoting TMZ chemoresistance under the condition of ATM silence were verified in vivo and in vitro. 2) IFIT1, a gene with the property of inducing cell apoptosis and involving into the process of ATM silence caused TMZ chemoresistance, was screened out in U251 cells. 3) Another cell strain, U251 with stable silence of IFIT1 gene, was also generated in this study. Further, we still confirmed the functions of IFIT1 silence, suppressing the induction of apoptosis and leading to TMZ chemoresistance in U251 cells, and IFIT1 may be a predictive biomarker of chemosensitivity. 4) We demonstrated that the expression of IFIT1 significantly increased in glioblastoma tissue. High IFIT1 can be a predictive biomarker of favorable clinical outcome, and IFIT1 along with MGMT contributed to more accurate prognostic prediction of glioblastoma. |
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