Mett19基因对U251胶质瘤细胞增殖和凋亡的影响
发布时间：2018-10-21 14:44
【摘要】：胶质瘤是临床最常见的原发性中枢神经系统恶性肿瘤,它本质上是一种多基因异常疾病,通过一种或多种癌基因的过度表达、同时伴随抑癌基因的突变缺失从而使信号传导通路异常,肿瘤细胞逃逸了正常生长调控机制,结果出现细胞的异常增殖、自主侵袭、血管增生等恶性表型。Mettl9基因是新发现的一个p53下游基因,在肺癌、小鼠胎肺和SD大鼠中枢神经系统的发育过程中特异性表达,且该基因表达信号的强弱与凋亡细胞的强弱成正相关,其在胚胎发育过程中的作用很可能是通过参与细胞凋亡来实现的。因此,本研究中,我们观察不同表达水平的Mett19基因对胶质瘤U251细胞增殖和凋亡的影响,初步探讨及作用机制。 目的： 1.研究Mett19基因的不同表达水平对U251细胞生长的影响； 2.探讨Mett19基因对U251生长的作用机制。 方法： 应用RT-PCR方法,扩增出Mett19基因的cDNA,克隆至pGEM-T载体上并测序,再将该基因亚克隆至慢病毒载体pLenti6.3-MCS-IRES-EGFP,在293T细胞进行病毒包装,构建Mettl9基因的慢病毒过表达载体；根据RNA干扰序列设计原则,设计RNA干扰靶点序列,合成含干扰序列的双链DNA oligo,通过酶切连接构建Mett19基因的RNAi干扰载体。实验分为五组：正常培养细胞组,慢病毒空载体组(NC),慢病毒过表达载体组(Mett19组),干扰载体对照组(NC-RNAi组),Mett19干扰载体组(Mettl9-RNAi组)。将上述已经构建的各质粒分别瞬时感染或转染U251细胞,48h后,荧光显微镜观察GFP绿色荧光表达情况,qRT-PCR检测Mett19基因的表达水平,以此评价病毒感染或质粒转染后Mett19基因的表达效率；收集各组细胞,MTT方法检测细胞活力,流式细胞术检测细胞凋亡情况,qRT-PCR方法检测凋亡相关因子Bcl-2, Bax mRNA的表达水平。结果： 1.Mettl9cDNA的RT-PCR扩增产物连接到pGEM(?)-T载体,测序结果向GenBank递交获得收录号HQ898855； 2.成功构建了Mett19基因的慢病毒过表达载体和RNAi干扰载体； 3.荧光显微镜下可见GFP绿色荧光的表达阳性率约为50%,感染Mett19慢病毒过表达质粒,Mett19的表达水平明显高于NC对照组)；而转染Mettl9-RNAi组,Mett19的表达水平明显低于NC-RNAi组； 4.MTT结果显示,感染Mett19慢病毒过表达质粒组,细胞增殖能力明显降低；而转染Mettl9-RNAi组,细胞增殖能力明显增加； 5.流式细胞术结果显示,感染Mett19慢病毒过表达质粒组,细胞凋亡率明显增加；而转染Mettl9-RNAi组,细胞凋亡率明显降低)； 6.qRT-PCR结果显示,感染Mett19慢病毒过表达质粒组,凋亡相关因子Bcl-2mlNA的表达水平降低,Bax mRNA的表达水平明显升高,差异具有统计学意义；而转染Mettl9-RNAi组,凋亡相关因子Bcl-2mRNA的表达水平升高,Bax mRNA的表达水平明显降低,差异具有统计学意义。 结论：过表达Mett19基因抑制U251细胞增殖,促进其凋亡,可能与Bcl-2mRNA表达水平下降,Bax mRNA表达水平升高有关。
[Abstract]:Glioma is the most common primary malignant tumor of the central nervous system. It is essentially a polygenic disorder, which is overexpressed by one or more oncogenes. At the same time, with the mutation and deletion of tumor suppressor gene, the signal transduction pathway is abnormal, tumor cells escape the normal growth regulation mechanism, resulting in abnormal cell proliferation and spontaneous invasion. Angiogenesis and other malignant phenotypes. Mettl9 gene is a newly discovered p53 downstream gene that is specifically expressed in the central nervous system of lung cancer, mouse fetal lung and SD rats. Moreover, the intensity of the gene expression signal was positively correlated with that of apoptotic cells, and its role in embryonic development was probably realized by taking part in apoptosis. Therefore, in this study, we observed the effect of Mett19 gene at different levels on the proliferation and apoptosis of U251 glioma cells. Objective: 1. To study the effects of different expression levels of Mett19 gene on the growth of U251 cells. 2. To explore the mechanism of Mett19 gene acting on U251 growth. Methods: the cDNA, of Mett19 gene was amplified by RT-PCR and sequenced into the pGEM-T vector. Then it was subcloned into the lentivirus vector pLenti6.3-MCS-IRES-EGFP, for viral packaging in 293T cells to construct the lentivirus overexpression vector of Mettl9 gene. According to the principle of RNA interference sequence design, the RNA interference target sequence was designed, and the double-stranded DNA oligo, containing interference sequence was synthesized and ligated to construct the RNAi interference vector of Mett19 gene by restriction endonuclease ligation. The experiment was divided into five groups: normal culture cell group, lentivirus empty vector group, (NC), lentivirus overexpression vector group (Mett19 group), interference vector control group (NC-RNAi group), Mett19 interference vector group (Mettl9-RNAi group). The constructed plasmids were transiently infected or transfected into U251 cells respectively. After 48 hours, the green fluorescence expression of GFP was observed by fluorescence microscope and the expression level of Mett19 gene was detected by qRT-PCR to evaluate the expression efficiency of Mett19 gene after virus infection or plasmid transfection. The cell viability was detected by MTT, apoptosis was detected by flow cytometry, and the expression of apoptosis-related factor (Bcl-2, Bax mRNA) was detected by qRT-PCR. Results: the RT-PCR amplification product of 1.Mettl9cDNA was ligated to pGEM (?) -T vector, and the result of sequencing was submitted to GenBank to obtain HQ898855; 2. The lentivirus overexpression vector and RNAi interference vector of Mett19 gene were successfully constructed. Under fluorescence microscope, the positive rate of GFP green fluorescence was about 50%, the expression level of Mett19 in infected Mett19 lentivirus overexpression plasmid was significantly higher than that in NC control group, but the expression level of Mett19 in Mettl9-RNAi group was significantly lower than that in NC-RNAi group. The results of 4.MTT showed that the ability of cell proliferation was significantly decreased in the group infected with Mett19 lentivirus overexpression plasmid, while in the group transfected with Mettl9-RNAi, the ability of cell proliferation was significantly increased. The results of flow cytometry showed that the rate of apoptosis was significantly increased in the group infected with Mett19 lentivirus overexpression plasmid, while the rate of apoptosis was significantly decreased in the group transfected with Mettl9-RNAi. The results of 6.qRT-PCR showed that the group infected with Mett19 lentivirus overexpression plasmid group. The expression level of apoptosis-related factor (Bcl-2mlNA) decreased significantly, and the expression level of, Bax mRNA increased significantly (P < 0.05), while in Mettl9-RNAi transfected group, the expression level of Bcl-2mRNA increased and the expression of, Bax mRNA decreased significantly. The difference is statistically significant. Conclusion: overexpression of Mett19 gene inhibits the proliferation of U251 cells and promotes its apoptosis, which may be related to the decrease of Bcl-2mRNA expression level and the increase of, Bax mRNA expression level.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R739.41
本文编号：2285416
[Abstract]:Glioma is the most common primary malignant tumor of the central nervous system. It is essentially a polygenic disorder, which is overexpressed by one or more oncogenes. At the same time, with the mutation and deletion of tumor suppressor gene, the signal transduction pathway is abnormal, tumor cells escape the normal growth regulation mechanism, resulting in abnormal cell proliferation and spontaneous invasion. Angiogenesis and other malignant phenotypes. Mettl9 gene is a newly discovered p53 downstream gene that is specifically expressed in the central nervous system of lung cancer, mouse fetal lung and SD rats. Moreover, the intensity of the gene expression signal was positively correlated with that of apoptotic cells, and its role in embryonic development was probably realized by taking part in apoptosis. Therefore, in this study, we observed the effect of Mett19 gene at different levels on the proliferation and apoptosis of U251 glioma cells. Objective: 1. To study the effects of different expression levels of Mett19 gene on the growth of U251 cells. 2. To explore the mechanism of Mett19 gene acting on U251 growth. Methods: the cDNA, of Mett19 gene was amplified by RT-PCR and sequenced into the pGEM-T vector. Then it was subcloned into the lentivirus vector pLenti6.3-MCS-IRES-EGFP, for viral packaging in 293T cells to construct the lentivirus overexpression vector of Mettl9 gene. According to the principle of RNA interference sequence design, the RNA interference target sequence was designed, and the double-stranded DNA oligo, containing interference sequence was synthesized and ligated to construct the RNAi interference vector of Mett19 gene by restriction endonuclease ligation. The experiment was divided into five groups: normal culture cell group, lentivirus empty vector group, (NC), lentivirus overexpression vector group (Mett19 group), interference vector control group (NC-RNAi group), Mett19 interference vector group (Mettl9-RNAi group). The constructed plasmids were transiently infected or transfected into U251 cells respectively. After 48 hours, the green fluorescence expression of GFP was observed by fluorescence microscope and the expression level of Mett19 gene was detected by qRT-PCR to evaluate the expression efficiency of Mett19 gene after virus infection or plasmid transfection. The cell viability was detected by MTT, apoptosis was detected by flow cytometry, and the expression of apoptosis-related factor (Bcl-2, Bax mRNA) was detected by qRT-PCR. Results: the RT-PCR amplification product of 1.Mettl9cDNA was ligated to pGEM (?) -T vector, and the result of sequencing was submitted to GenBank to obtain HQ898855; 2. The lentivirus overexpression vector and RNAi interference vector of Mett19 gene were successfully constructed. Under fluorescence microscope, the positive rate of GFP green fluorescence was about 50%, the expression level of Mett19 in infected Mett19 lentivirus overexpression plasmid was significantly higher than that in NC control group, but the expression level of Mett19 in Mettl9-RNAi group was significantly lower than that in NC-RNAi group. The results of 4.MTT showed that the ability of cell proliferation was significantly decreased in the group infected with Mett19 lentivirus overexpression plasmid, while in the group transfected with Mettl9-RNAi, the ability of cell proliferation was significantly increased. The results of flow cytometry showed that the rate of apoptosis was significantly increased in the group infected with Mett19 lentivirus overexpression plasmid, while the rate of apoptosis was significantly decreased in the group transfected with Mettl9-RNAi. The results of 6.qRT-PCR showed that the group infected with Mett19 lentivirus overexpression plasmid group. The expression level of apoptosis-related factor (Bcl-2mlNA) decreased significantly, and the expression level of, Bax mRNA increased significantly (P < 0.05), while in Mettl9-RNAi transfected group, the expression level of Bcl-2mRNA increased and the expression of, Bax mRNA decreased significantly. The difference is statistically significant. Conclusion: overexpression of Mett19 gene inhibits the proliferation of U251 cells and promotes its apoptosis, which may be related to the decrease of Bcl-2mRNA expression level and the increase of, Bax mRNA expression level.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R739.41
【参考文献】
相关期刊论文 前6条
1 邬力祥,刘发益,曹莉,赵彦,马志成;低(无)糖、缺氧和缺氧/复氧对培养鼠脑胶质细胞活力及致损的影响[J];湖南医科大学学报;2003年05期
2 孙迎娟,时飞,孙坚萍,黄葆华,罗兵;胃癌组织中p53基因突变及p53和mdm2蛋白表达的研究[J];肿瘤防治杂志;2005年13期
3 李明;闫军;王春芳;李鹏飞;;胃癌标本和正常胃组织中P53基因及其下游基因METTL9表达研究[J];中国医疗前沿;2010年23期
4 王洪滨;张学新;苏君;张锐;金华;常亮;;P53、Ki-67在不同级别胶质瘤中的表达及其意义[J];中国误诊学杂志;2011年34期
5 邬力祥,王绮如,刘发益,曹莉,赵彦;缺氧处理后鼠脑星形胶质细胞基因表达谱的初步分析[J];中国现代医学杂志;2003年18期
6 舒坤贤;夏燕;冉启煤;;PAP1/METTL9基因在肺癌细胞中表达谱的生物信息学分析[J];中国现代医学杂志;2011年33期
，本文编号：2285416
本文链接：https://www.wllwen.com/yixuelunwen/shenjingyixue/2285416.html
最近更新
教材专著
