DNA修复基因MGMT沉默对人脑胶质瘤化疗的影响
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摘要
胶质瘤细胞对烷化剂类化疗药物产生抗药性是化疗失败的一个主要原因。DNA修复酶—O6-甲基鸟嘌呤-DNA甲基转移酶(O6-methylguanine DNA methyltransferase ,MGMT)在脑胶质瘤组织中的表达与肿瘤细胞对双氯乙亚硝脲(Carmustine,BCNU)等亚硝脲类药物的耐药密切相关,并影响肿瘤的化疗效果和病人的预后。探讨通过特异性封闭MGMT基因,抑制MGMT mRNA的表达,达到逆转MGMT的目的,是MGMT研究的热点。目前MGMT基因治疗的策略主要有:使用MGMT酶活性抑制剂、核酶技术、反义RNA技术。RNAi(RNA interference)技术是诱导基因沉默的一种新技术。RNAi自诞生以来,不仅应用于基因功能分析,而且作为基因治疗的一种方法,在肿瘤学研究领域中获得了较大进展。
本课题从三个方面对MGMT进行了系统研究:①MGMT在胶质瘤组织、胶质瘤细胞系/株中的表达及其与MGMT基因启动子区甲基化之间的关系;②构建3条针对MGMT的siRNA质粒载体;③采用脂质体体外转染T98细胞系,观察转染后肿瘤细胞中MGMT的表达情况及其对BCNU敏感性的变化。
第一部分MGMT在胶质瘤组织、胶质瘤细胞系/株中的表达及意义
目的研究MGMT在胶质瘤组织、胶质瘤细胞系/株中的表达及其与MGMT基因启动子区甲基化之间的关系。
方法研究对象为13株人脑胶质瘤细胞系/株、35例临床胶质瘤患者的肿瘤组织。采用美国Gentra公司的试剂盒以盐析法提取基因组DNA,并采用美国Chemicon公司CpGenomeTM DNA修饰试剂盒对组织DNA进行亚硫酸氢盐修饰,采用甲基化特异性PCR法(Methylation-specific PCR, MSP)进行MGMT基因启动子区甲基化水平的检测;采用sulforhodamine B(SRB) Colorimetric抗癌药筛选法测定人脑胶质瘤细胞系/株对BCNU的敏感性;采用免疫组化的方法来检测胶质瘤患者肿瘤组织标本中MGMT蛋白的表达程度。
结果13株胶质瘤细胞系/株中有8株存在MGMT基因启动子区甲基化,其中有7株对BCNU敏感,1株耐药;其余5株为MGMT基因启动子区去甲基化,均对BCNU耐药。用Spearman相关系数分析:P<0.05;按性别分组,男性患者的肿瘤组织MGMT蛋白表达阳性率为59.1%(13/22),女性患者的肿瘤组织MGMT蛋白表达阳性率为69.2%(9/13),男女两组间差异无统计学意义,P>0.05;按年龄分组,≤53岁年龄组、>53岁年龄组患者组织的MGMT蛋白表达阳性率分别为66.7%(12/18)、58.8%(10/17)。两组间MGMT蛋白表达率差异无统计学意义,P>0.05;按病理分级分组,病理分级Ⅰ级、Ⅱ级、Ⅲ级、Ⅳ级患者组织的MGMT蛋白表达阳性率分别为100.0%(2/2)、53.8%(7/13)、61.1%(11/18)、100%(2/2),采用非参数多个独立样本秩和检验来进行统计分析,各组间MGMT蛋白表达阳性率差异无统计学意义,P>0.05;在35例胶质瘤患者的肿瘤组织标本中,22例MGMT蛋白表达呈阳性的胶质瘤组织中7例组织MGMT基因启动子区甲基化,阳性率为31.8%;13例MGMT蛋白表达呈阴性的胶质瘤组织中11例组织MGMT基因启动子区甲基化,阳性率为84.6%。用Spearman相关系数分析:P<0.05。
结论在人脑胶质瘤细胞系/株中,MGMT基因启动子区过甲基化与其对BCNU的敏感性相关。胶质瘤细胞MGMT基因启动子区过甲基化,胶质瘤细胞系/株对BCNU敏感;胶质瘤细胞MGMT基因启动子区去甲基化,胶质瘤细胞系/株对BCNU耐药。MGMT蛋白的表达与MGMT基因启动子区的甲基化状态相关,与胶质瘤患者的年龄、性别及病理分级无相关性。MGMT基因启动子区过甲基化,MGMT蛋白表达较低;MGMT基因启动子区去甲基化,MGMT蛋白表达较高。
第二部分MGMT基因RNAi质粒载体的构建及鉴定
目的构建MGMT基因的siRNA(small interfering RNA)质粒载体pRNAT-H1.1/Neo- MGMT siRNA,为进一步研究siRNA对MGMT基因的抑制作用,探讨MGMT在胶质瘤的化疗耐药及逆转胶质瘤细胞耐药表型中的作用奠定基础。
方法针对MGMT的靶序列设计合成三条shRNA(small hair RNA)的DNA模板单链,同时模板链两端分别设计BamHⅠ和HindⅢ限制酶切位点。退火形成siRNA载体插入片断。用限制性内切酶将pRNAT-H1.1/Neo线性化,T4连接酶将插入片断插入pRNAT-H1.1/Neo中。重组质粒转化大肠杆菌DH5α后培养出阳性克隆,抽提质粒,经酶切、电泳和测序的方法鉴定质粒构建是否成功。
结果设计构建三条针对MGMT基因的siRNA质粒载体,经双酶切,在2%琼脂糖凝胶电泳结果显示:PCR产物大小76bp,产物大小与设计的完全相同。测序结果证明序列正确。
结论成功设计构建了三条针对MGMT基因的siRNA质粒载体,为研究RNA干扰逆转胶质瘤细胞的耐药表型提供了良好的实验材料。
第三部分siRNA干扰MGMT基因表达的初步研究
目的探讨三条针对MGMT基因的siRNA质粒载体逆转T98细胞MGMT mRNA、MGMT蛋白表达及对BCNU敏感性的效果。
方法通过LipofectimineTM 2000将三条MGMT的siRNA质粒载体及空载体分别转入T98细胞中,以绿色荧光蛋白(GFP)基因为报告基因,用流式细胞仪和荧光显微镜观察转染效率和G418筛选4周后质粒表达效率。采用实时定量PCR(Real-time Quantitative Polymerase Chain Reaction,RQ-PCR)法测定MGMT mRNA的表达,Western Blot测定MGMT蛋白的表达。MTT法测定siRNA质粒载体转染前后T98细胞系对烷化剂BCNU的敏感性变化。
结果在荧光显微镜下观察:经LipofectimineTM 2000转染的T98细胞在转染后48h,在显微镜下可见细胞呈现明显的绿色荧光,说明转染成功。用流式细胞仪检测转染效率分别为15.9%(siRNA1)、15.5%(siRNA2)、16.7%(siRNA3)、11.1%(阴性对照),4周后再次检测质粒表达率分别为81.1%(siRNA1)、79.6%(siRNA2)、88.1%(siRNA3)、86.1%(阴性对照);由实时定量RT-PCR可知:三种MGMT siRNA质粒载体转染T98细胞后,其MGMT mRNA的表达受到抑制。相对阴性对照而言,siRNA2抑制作用最为明显,达到87%,siRNA1次之,为74%,siRNA3为65%;采用Western blot法对MGMT蛋白表达测定的结果显示:干扰组(T98/MGMT siRNA1;T98/MGMT siRNA2;T98/MGMT siRNA3)MGMT蛋白表达量较空白对照组及阴性对照组明显减少,MGMT蛋白的表达在干扰组间无显著差异;采用MTT法检测转染后T98细胞对BCNU的敏感性:转染siRNA质粒载体后,BCNU对T98细胞作用的剂量-反应曲线,相对于未转染及转染空载体T98细胞的曲线左移。未转染的T98细胞对BCNU耐药,其IC50为140.56μg/ml,而转染后的细胞其IC50分别为126.18μg/ml(阴性对照)、44.50μg/ml(siRNA1)、23.12μg/ml(siRNA2)、56.87μg/ml(siRNA3)。结果表明转染siRNA质粒载体的T98细胞对BCNU的敏感性增加。
结论LipofectimineTM 2000在转染中表现出稳定、高效和低毒的特点。针对MGMT的三条靶序列的shRNA均能有效下调其mRNA表达。转染后的T98细胞MGMT蛋白表达下降,转染后的T98细胞对BCNU的敏感性升高。
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 lines
objective 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 vector
Objective 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 expression
Objective 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.
本课题从三个方面对MGMT进行了系统研究:①MGMT在胶质瘤组织、胶质瘤细胞系/株中的表达及其与MGMT基因启动子区甲基化之间的关系;②构建3条针对MGMT的siRNA质粒载体;③采用脂质体体外转染T98细胞系,观察转染后肿瘤细胞中MGMT的表达情况及其对BCNU敏感性的变化。
第一部分MGMT在胶质瘤组织、胶质瘤细胞系/株中的表达及意义
目的研究MGMT在胶质瘤组织、胶质瘤细胞系/株中的表达及其与MGMT基因启动子区甲基化之间的关系。
方法研究对象为13株人脑胶质瘤细胞系/株、35例临床胶质瘤患者的肿瘤组织。采用美国Gentra公司的试剂盒以盐析法提取基因组DNA,并采用美国Chemicon公司CpGenomeTM DNA修饰试剂盒对组织DNA进行亚硫酸氢盐修饰,采用甲基化特异性PCR法(Methylation-specific PCR, MSP)进行MGMT基因启动子区甲基化水平的检测;采用sulforhodamine B(SRB) Colorimetric抗癌药筛选法测定人脑胶质瘤细胞系/株对BCNU的敏感性;采用免疫组化的方法来检测胶质瘤患者肿瘤组织标本中MGMT蛋白的表达程度。
结果13株胶质瘤细胞系/株中有8株存在MGMT基因启动子区甲基化,其中有7株对BCNU敏感,1株耐药;其余5株为MGMT基因启动子区去甲基化,均对BCNU耐药。用Spearman相关系数分析:P<0.05;按性别分组,男性患者的肿瘤组织MGMT蛋白表达阳性率为59.1%(13/22),女性患者的肿瘤组织MGMT蛋白表达阳性率为69.2%(9/13),男女两组间差异无统计学意义,P>0.05;按年龄分组,≤53岁年龄组、>53岁年龄组患者组织的MGMT蛋白表达阳性率分别为66.7%(12/18)、58.8%(10/17)。两组间MGMT蛋白表达率差异无统计学意义,P>0.05;按病理分级分组,病理分级Ⅰ级、Ⅱ级、Ⅲ级、Ⅳ级患者组织的MGMT蛋白表达阳性率分别为100.0%(2/2)、53.8%(7/13)、61.1%(11/18)、100%(2/2),采用非参数多个独立样本秩和检验来进行统计分析,各组间MGMT蛋白表达阳性率差异无统计学意义,P>0.05;在35例胶质瘤患者的肿瘤组织标本中,22例MGMT蛋白表达呈阳性的胶质瘤组织中7例组织MGMT基因启动子区甲基化,阳性率为31.8%;13例MGMT蛋白表达呈阴性的胶质瘤组织中11例组织MGMT基因启动子区甲基化,阳性率为84.6%。用Spearman相关系数分析:P<0.05。
结论在人脑胶质瘤细胞系/株中,MGMT基因启动子区过甲基化与其对BCNU的敏感性相关。胶质瘤细胞MGMT基因启动子区过甲基化,胶质瘤细胞系/株对BCNU敏感;胶质瘤细胞MGMT基因启动子区去甲基化,胶质瘤细胞系/株对BCNU耐药。MGMT蛋白的表达与MGMT基因启动子区的甲基化状态相关,与胶质瘤患者的年龄、性别及病理分级无相关性。MGMT基因启动子区过甲基化,MGMT蛋白表达较低;MGMT基因启动子区去甲基化,MGMT蛋白表达较高。
第二部分MGMT基因RNAi质粒载体的构建及鉴定
目的构建MGMT基因的siRNA(small interfering RNA)质粒载体pRNAT-H1.1/Neo- MGMT siRNA,为进一步研究siRNA对MGMT基因的抑制作用,探讨MGMT在胶质瘤的化疗耐药及逆转胶质瘤细胞耐药表型中的作用奠定基础。
方法针对MGMT的靶序列设计合成三条shRNA(small hair RNA)的DNA模板单链,同时模板链两端分别设计BamHⅠ和HindⅢ限制酶切位点。退火形成siRNA载体插入片断。用限制性内切酶将pRNAT-H1.1/Neo线性化,T4连接酶将插入片断插入pRNAT-H1.1/Neo中。重组质粒转化大肠杆菌DH5α后培养出阳性克隆,抽提质粒,经酶切、电泳和测序的方法鉴定质粒构建是否成功。
结果设计构建三条针对MGMT基因的siRNA质粒载体,经双酶切,在2%琼脂糖凝胶电泳结果显示:PCR产物大小76bp,产物大小与设计的完全相同。测序结果证明序列正确。
结论成功设计构建了三条针对MGMT基因的siRNA质粒载体,为研究RNA干扰逆转胶质瘤细胞的耐药表型提供了良好的实验材料。
第三部分siRNA干扰MGMT基因表达的初步研究
目的探讨三条针对MGMT基因的siRNA质粒载体逆转T98细胞MGMT mRNA、MGMT蛋白表达及对BCNU敏感性的效果。
方法通过LipofectimineTM 2000将三条MGMT的siRNA质粒载体及空载体分别转入T98细胞中,以绿色荧光蛋白(GFP)基因为报告基因,用流式细胞仪和荧光显微镜观察转染效率和G418筛选4周后质粒表达效率。采用实时定量PCR(Real-time Quantitative Polymerase Chain Reaction,RQ-PCR)法测定MGMT mRNA的表达,Western Blot测定MGMT蛋白的表达。MTT法测定siRNA质粒载体转染前后T98细胞系对烷化剂BCNU的敏感性变化。
结果在荧光显微镜下观察:经LipofectimineTM 2000转染的T98细胞在转染后48h,在显微镜下可见细胞呈现明显的绿色荧光,说明转染成功。用流式细胞仪检测转染效率分别为15.9%(siRNA1)、15.5%(siRNA2)、16.7%(siRNA3)、11.1%(阴性对照),4周后再次检测质粒表达率分别为81.1%(siRNA1)、79.6%(siRNA2)、88.1%(siRNA3)、86.1%(阴性对照);由实时定量RT-PCR可知:三种MGMT siRNA质粒载体转染T98细胞后,其MGMT mRNA的表达受到抑制。相对阴性对照而言,siRNA2抑制作用最为明显,达到87%,siRNA1次之,为74%,siRNA3为65%;采用Western blot法对MGMT蛋白表达测定的结果显示:干扰组(T98/MGMT siRNA1;T98/MGMT siRNA2;T98/MGMT siRNA3)MGMT蛋白表达量较空白对照组及阴性对照组明显减少,MGMT蛋白的表达在干扰组间无显著差异;采用MTT法检测转染后T98细胞对BCNU的敏感性:转染siRNA质粒载体后,BCNU对T98细胞作用的剂量-反应曲线,相对于未转染及转染空载体T98细胞的曲线左移。未转染的T98细胞对BCNU耐药,其IC50为140.56μg/ml,而转染后的细胞其IC50分别为126.18μg/ml(阴性对照)、44.50μg/ml(siRNA1)、23.12μg/ml(siRNA2)、56.87μg/ml(siRNA3)。结果表明转染siRNA质粒载体的T98细胞对BCNU的敏感性增加。
结论LipofectimineTM 2000在转染中表现出稳定、高效和低毒的特点。针对MGMT的三条靶序列的shRNA均能有效下调其mRNA表达。转染后的T98细胞MGMT蛋白表达下降,转染后的T98细胞对BCNU的敏感性升高。
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 lines
objective 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 vector
Objective 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 expression
Objective 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.
引文
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