Effect Of Silencing MGMT Gene On The Chemotherapy For Glioma

Clinically, O6-methylguanine-DNA-methyltransferase (MGMT) is one of the major causes for chemotherapeutic treatment failure in glioma patients. Drug-resistance genes are some of the most important elements of tumors themselves in determining drug-resistance and MGMT is drug-resistance gene for nitrosoureas. Directed blocking of MGMT gene, inhibiting the MGMT mRNA and MGMT expression, reversing MGMT and restoring drug sensitivity to drug-resistant glioma has been in great demand. The strategies of gene therapies for reversing MGMT mainly included the substrate analogue inhibitor,ribozymes and antisense RNA. Since the advent of RNAi approach, RNAi has been widely used for gene function analyzing and gene therapy. This study contains three parts of work: The first, study the expression of MGMT in glioma tissues and glioma lines, and relationship between the hypermethylation of CpG islands in the promoter regions of MGMT genes and the expression of MGMT. The second, construct MGMT siRNA recombinant vector in order to lay the foundation for further investigation on the function of MGMT. The third, transfect the recombinant vector into T98 cell by lipid Lipofectimine 2000 in order to assess the inhibiting effect on MGMT and assess the sensitivity of transfected T98 to BCNU. PARTⅠ: Study on the expression of MGMT in glioma tissues and glioma linesobjective to study the expression of MGMT in glioma tissues and glioma lines and to investigate the relationship between the hypermethylation of CpG islands in the promoter regions of MGMT genes of glioma cells and the expression of MGMT.Methods In this research, 13 human glioma cell lines, 35 primary glioma tissues were studied. Genomic DNA was extracted from tissues using protocols provided by Gentra Puregene DNA purification kit. The DNA was modified by bisulfite reaction using the CpGenomeTM DNA modification kit. Modified DNA was amplified by methylation-specic PCR (MSP) procedure to analyze the status of methylation. The cell resistance to BCNU in human glioma cell lines was determined by SRB assay; MGMT expression was examined by immunohistochemistry method.Result In the 8 cell lines which were hypermethylated, sensitivity to BCNU was noted in 7 cell lines; in the 5 cell lines which were unmethylated,resistance to BCNU was noted in all of the 5 cell lines. Spearman correlation coefficient was used to analysis the result. The relation was significant, P<0.05; The rate of the expression of MGMT in glioma tissues in male and female were 59.1%(13/22) and 69.2%(9/13), respectively. However, the difference was not significant, P>0.05; The rate of the expression of MGMT in glioma tissues was 66.7%(12/18) in patients aged no more than 53 years, the rate of the expression of MGMT in glioma tissues was 58.8%(10/17) in patient aged more than 53 years. The difference between the both groups was not significant, P>0.05; Of the 35 glioma tissues, the rate of the expression of MGMT in GradeⅠ,GradeⅡ,GradeⅢand GradeⅣgroups were 100.0%(2/2),53.8%(7/13),61.1%(11/18)and 100%(2/2), respectively. There were no significant differences among these groups, P>0.05; Among the 35 glioma samples, MGMT expression was noted in 22 samples, in which hypermethylation was detected in 7(31.8%) cases,while MGMT expression was not noted in 13 samples, in which hypermethylation was detected in 11(84.6%) cases. Spearman correlation coefficient was used to analysis the result. The relation was significant, P<0.05.Conclusion In human glioma cell lines, the relation between the aberrant methylation of MGMT gene promoter and the cell resistant to BCNU is significant. If the MGMT gene promoter is hypermethylated, the cell lines are sensitive to BCNU, while if the MGMT gene promoter is not hypermethylated, the cell lines are resistant to BCNU. In human glioma tissues, if the MGMT gene promoter is hypermethylated, the glioma tissues don't express MGMT, while if the MGMT gene promoter is not hypermethylated, the glioma tissues express MGMT. The relationship between the aberrant methylation of MGMT gene promoter and the expression of MGMT in glioma is significant. But the expression of MGMT does not show any significant relationship with clinical condition such as age, gender and pathological grading.PartⅡstudy on designing siRNA of blocking MGMT expression, construction expression vectorObjective to construct MGMT combinative siRNA vector—pRNAT -H1.1/Neo-MGMT, which may lay the foundation for further investigation on the chemotherapeutic treatment failure in glioma patients , and may conduce the development of strategy of gene therapy targeting MGMT in gliomas.Methods Three shRNAs for blocking MGMT gene was designed. And two single strand DNA templates were designed according to the sequence of each shRNA. When the DNA templates synthesized, two different restriction sites were superimposed, respectively, to the two end of it. The insertion element formed after the DNA templates annealed. Make the blank plasmid linearization by use of restriction enzyme, and then the insertion element was inserted into the blank plasmid by T4 ligase. Enzyme cutting and sequencing was performed to check whether MGMT siRNA plasmid be constructed successfully or not.Result Three siRNA eukaryotic expression vectors—pRNATin-H1.1 /Neo MGMT siRNA were constructed. We gained positive clone after recombinant vector transforming, extracted the vector and gained products by enzyme cutting. And the 2% gel electrophoresis result showed: The product is near 76bp and accord with the design, and the results of DNA sequence proved its preciseness.Conclusion Successfully constructed the siRNA eukaryotic expression vector pRNATin-H1.1/Neo MGMT siRNA. These vectors may provide experimental bases for study of reversing the drug-resistance of gliomas with siRNA and provide more information about the mechanisms of siRNA.PartⅢEffect of RNA interference on MGMT expressionObjective to investigate the effect of three combinative siRNA vectors—pRNAT-H1.1/Neo-MGMT on MGMT expression and its effect on the sensitivity of transfected T98 to BCNU.Methods Transfect T98 cells with MGMT siRNA plasmid by Lipofectamine 2000. As a reporter gene, Green Fluorescence Protein(GFP) was evaluated by flow cytometry and fluorescent microscopy to estimate the transfection efficiencies and expression efficiencies when the positive cell clones were selected with G418. Use RQ-PCR(Real-time Quantitative Polymerase Chain Reaction)to detect the expression of MGMT mRNA; Expression of MGMT protein was detected by Western blot. And MTT was used to assesse the sensitivity of T98 cell to alkylating agent before and after siRNA eukaryotic expression vector being transfected into it. Result GFP expression in transfected T98 cell : 48h after LipofectimineTM 2000 transfecting, the T98 cell showed significant green fluorescence, which implicated that transfection was successful, and the transfected T98 cell also showed significant green fluorescence after it was screened by G418 for four weeks; The real time RT-PCR result showed:Of the 3 kinds of interfere sequence transfected into T98 cell, the inhibiting effect for MGMT gene expression is about 74%, 87%, 65%, respectively. The Western blot result showed: Expression of MGMT protein after siRNA transfected intoT98 cells is less than the expression of MGMT protein of the T98 cells without siRNA. The MTT test result showed: The dose-effect curve for reaction of transfected T98 cell to BCNU shifted left when compared to that of non-transfected T98 cell. The sensitivity of T98 cell to BCNU has increased after transfected with MGMT-siRNA. And the IC50 of transfected T98 cell was 44.50μg/ml(siRNA1),23.12μg/ml(siRNA2),56.87μg/ml(siRNA3),respectively.Conclusion Lipofectamine 2000 appears stability,high efficiency and low toxicity. All MGMT-targeted siRNA could downregulate MGMT mRNA markedly. The expression of MGMT of T98 cell decreased after siRNA eukaryotic expression vector being transfected into it, and the sensitivity of T98 cell to alkylating agent increased after being transfected.