BTBD10在人脑胶质瘤中的表达及其对U251细胞株增殖和凋亡的影响
摘要
脑胶质瘤是成人中枢神经系统最常见的恶性肿瘤,统计资料表明其发生率约占颅内肿瘤的40-50%,其多呈浸润性生长,与正常脑组织间常无明显分界,易侵犯重要神经结构,难以做到手术完全切除,预后差,复发率、病死率高。而药物化疗和放射治疗不能高度特异性地杀伤肿瘤细胞,并且可能产生严重的不良反应。因此,目前脑胶质瘤仍为神经外科领域的难治性疾病,而阐明其发病机制、寻找新的治疗手段成为神经外科的研究热点。
和其他部位肿瘤一样,脑胶质瘤的发生、发展是一个非常复杂的过程,涉及多种肿瘤相关基因的表达异常或失活。胶质瘤本质上是一种多基因异常疾病,其发生是由于原癌基因的激活和抑癌基因的失活导致细胞信号传导通路的异常、细胞循环周期的改变、生命周期的延长以及细胞调亡缺陷等,从而出现细胞增殖失控及恶性转化。与脑胶质瘤的恶性进展密切相关的细胞通路主要有磷脂酰肌醇3-激醇(P13K)信号转导通路、Ras信号转导通路、Wnt信号转导通路和Notch信号转导通路等。寻找与胶质瘤发病相关的基因,在此基础上发现胶质瘤治疗的新靶点、新策略,是神经外科研究领域的主要内容之一。
BTBD10基因含BTB/POZ功能域,位于人染色体的11p15.2区域,全长2551bp,含245-16721bp开放阅读框架,编码475氨基酸的蛋白,蛋白分子量51.7Kd。前期研究在对18例脑胶质瘤进行表达谱芯片筛选及聚类分析中发现：芯片数据提示BTBD10表达量在各级别胶质瘤中均发生下调,平均Ratio值为0.218。基因表达谱Hierarchical聚类发现与BTBD10表达谱相近的基因有：血管性肠肽(VIP)、肿瘤蛋白p53结合蛋白(TP53BP1)、p淀粉样蛋白前体(APBB1)、8氧化鸟嘌呤糖基酶(OGG1)、促性腺素诱导阻抑蛋白(GIOT1)、MADS转录增强子2C(MEF2C)。MTC为模板RT-PCR结果显示BTBD10在检测的8种正常组织中,脑组织中高表达,而心、肺、肝、肾、胰腺组织中表达量较低。Northern杂交结果显示BTBD10在正常成人脑组织中表达量高,在7例不同级别胶质瘤中表达量都下降,BTBD10在胶质瘤／正常脑组织的表达量比率与芯片数据基本相符。在肝癌、卵巢癌和肺癌中BTBD10的表达没有显著性差异。提示BTBD10基因与脑胶质瘤高度相关,有可能成为新的脑胶质瘤治疗靶基因。为进一步研究BTBD10基因在胶质瘤发生发展中的作用,本实验通过检测BTBD10在大样本胶质瘤中的表达情况,培养过表达BTBD10基因的细胞株,初步探讨BTBD10基因调节胶质瘤细胞增殖水平的可能机制。
第一部分BTBD10在人脑胶质瘤组织中的表达及意义
目的：探讨BTBD1OmRNA和BTBD10蛋白在脑胶质瘤中的表达及意义。
方法：采用实时荧光定量PCR和Western blot杂交检测BTBD10mRNA和BTBD10蛋白在正常脑组织和各级别胶质瘤中的表达,分析其表达与胶质瘤临床病理特征的关系。
结果：在正常脑组织中BTBD10mRNA和蛋白的表达较高；各级别胶质瘤中的表达均低于正常脑组织(P<0.05)；在低级别胶质瘤(Ⅰ-Ⅱ级)中表达量较低,而在高级别胶质瘤(Ⅲ-Ⅳ)中表达水平更低,低级别和高级别之间的表达差异具有统计学意义(P<0.05)。BTBD1OmRNA和蛋白的表达水平与年龄、性别、病理类型等临床病理特征无明显相关(P>0.05)。
结论：胶质瘤中BTBD10表达明显低于正常脑组织中的表达,且随病理级别的增高而降低,提示BTBD10在胶质瘤的发生发展中起重要作用,并与胶质瘤的病理分级密切相关。
第二部分BTBD10过表达慢病毒载体的构建和鉴定
目的：构建BTBD10慢病毒表达载体,为后续实验研究提供依据。
方法：以BTBD10基因为模板PCR扩增目的基因,表达载体(pLV-UbC-GFP-3FLAG)酶切后进行胶回收载体片段,目的基因与载体片段同源重组后转化感受态细胞,用菌落PCR鉴定转化子,阳性克隆测序鉴定,质粒抽提,与无内毒素病毒包装质粒、膜蛋白表达质粒共转染293T细胞包装产生慢病毒颗粒,测定慢病毒滴度。
结果：PCR扩增和测序结果均证实,插入序列完全正确,包装病毒产生病毒悬液的滴度为3.42×108TU／ml。
结论：成功构建了携带BTBD10基因的慢病毒载体,为研究其在胶质瘤细胞中的生物学功能和以其为靶点的基因治疗奠定基础。
第三部分BTBD10慢病毒载体转染人胶质瘤细胞株U251及其作用机制研究
目的：了解BTBD10重组慢病毒载体转染人胶质瘤细胞株U251后,对U251细胞增殖、凋亡的影响,探讨其发挥肿瘤调控作用的机制。
方法：应用BTBD10慢病毒载体、空载体分别转染体外培养的U251人胶质瘤细胞株,正常培养U251细胞作为空白对照。荧光定量PCR检测转染前后BTBD10和CyclinDl mRNA的表达变化,Western blot杂交检测BTBD 10、磷酸化Akt(pAkt)和CyclinD1蛋白水平变化,流式细胞仪检测细胞周期和凋亡水平,MTT法分析细胞增殖情况并绘制生长曲线。
结果：荧光定量PCR和Western blot杂交结果显示,与未转染的U251细胞和转染空白载体的U251细胞相比,转染重组BTBD10病毒载体的U251细胞中BTBD10 mRNA和蛋白水平明显提高,差异具有统计学意义(P<0.05)；Akt的磷酸化水平下调,CyclinDl mRNA和蛋白水平下调,差异具有统计学意义(P<0.05)。流式细胞仪分析各组细胞的凋亡水平,检测结果表明,与未转染的U251细胞和转染空白载体的U251细胞相比,转染BTBD10的U251细胞中凋亡细胞明显增多,早期凋亡细胞达14.64%,统计学上有差异(P<0.05)：细胞周期检测结果显示,未转染的U251细胞和转染空载体的U251细胞的细胞周期和细胞状态基本相同,部分细胞处于G2／M期,转染BTBD10病毒载体的U251细胞则被明显的抑制在G0／G1期,提示增殖活性降低。MTT法检测细胞增值率,发现外源性BTBD10基因转染U251细胞后,细胞生长速度明显下降。
结论：慢病毒表达载体对人胶质瘤细胞系U251具有很高的转染效率,感染后可显著上调靶基因BTBD10的表达和活化。BTBD10过表达后将肿瘤细胞阻滞在G0／G1期,抑制肿瘤细胞增殖,促进凋亡,可能与下调pAkt和CyclinD1水平,负调控PI3K/Akt信号通路有关。
Gliomas are the most common primary tumor of the central nervous system in adults, and accounts for approximately 40-50% of total intracranial tumors. These tumors are highly invasive, and have no obvious dividing line with normal brain tissue, often infiltrate critical neurological areas within the brain. It is difficult to achieve totally surgical resec-tion, and the recurrence and fatality rate is high. In addition, chemotherapy and radioatio-therapy can not be a high degree of specificity in killing tumor cells, and may cause serious adverse reactions. Therefore, glioma is still a refractory disease in the neurosurgical field. To elucidate its pathogenesis and to find new treatments is a research hotspot in neurosurgery.
It has been showed that the devolpment of glioma, just the same as the tumors in other sites of the body, is a very complex process, involving expression disorder or inactivation of a variety of tumor-related genes. Glioma is essentially a multi-gene disorders. Its occurrence is due to the activation of proto-oncogenes and inactivation of tumor suppressor genes, which lead to excessive cell proliferation and malignant transformation. The malignant progression of glioma cells is closely related with several pathways, such as PI3K (phosphatidylinositol 3-kinase) signaling pathway, Ras signaling pathway, Wnt signaling pathway and Notch signaling pathway. However, up to now, the molecular mechanism of glioma is not fully elucidated. Seeking glioma pathogenesis-related genes, finding new target and new strategy for glioma therapy, plays a important role in the field of neurosurgery.
BTBD10 contained a BTB/POZ domain, located in human chromosome 11p15.2 region. It was 2489 bp in length, containing open reading frame from 245 to 1672 bp, encoding 475 amino acid protein with a molecular weight of 51.7 Kd. Preliminary study of 18 glioma samples of cDNA microarray screening and cluster analysis found that the expression level of BTBD10 descend in all samples, with an average Ratio value of 0.218. The result of RT-PCR showed that a ubiquitous expression pattern of BTBD10 in the 8 tissues examined with high expression in adult brain, testis and small intestine and weak expression in the heart, lung, liver, kidney, pancreas, spleen, thymus. Northern blot showed that BTBD10 was highly expressed in mixed normal brain while it was sharply down-regulated in all grades of glioma tissues. RT-PCR showed that there was no significant difference in hepatocellular carcinoma, ovary cancer and lung cancer tissues. That means the BTBD10 gene is highly correlated with glioma, and it may become a new target gene in gene therapy of gliomas. For further studying the role of BTBD10 gene in glioma, we began with examining the expression of BTBD10 in a large number of glioma specimens in the present study, then increased the expression of BTBD10 in glioma cell lines, evaluated the effects of BTBD10 on glioma proliferation and apoptosis.
Part I:Study on the expression of BTBD10 in human gliomas and its significance
Objective:To investigate the expression of BTBD10 mRNA and protein in brain gliomas and its significance.
Methods:The mRNA expression of BTBD10 in 52 cases of human glioma tissues and 8 cases of normal brain tissues was detected with quantitative real-time PCR. The protein expression in these samples were detected using western blot.
Results:The expression of BTBD10 mRNA and protein is higher in normal brain tissue than in all grades of glioma tissue (P<0.05) and significantly decreased ascending the degree of glioma. The difference between the low-grade and high-grade tumors is statisticly significant(P<0.05). A statistically significant association was found between expression of BTBD10 mRNA and protein.
Conclusion:BTBD10 mRNA and protein showed markedly lower expression in glioma than normal brain tissues, and the expression decreased with the malignancy of glioma. These results suggested that BTBD10 was significantly associated with the proliferation activity and may play an improtant role in human glioma.
Part II:Construction and identification of lentiviral vector plasmid containing BTBD10
Objective:To construct lentiviral vector plasmid containing BTBD10 and provide the basis for further experiments.
Methods:The vector was obtained from pLV-UbC-GFP plasmid digested with EcoR I and Nhe I restriction enzymes. Then BTBD10 gene was inserted into the vector to construct pLV-BTBD10, which was subsequently confirmed by PCR and DNA sequencing analysis. After these identification, the resultant pLV-BTBD10 plasmid, pCD/NL-BH*DDD and pLTR-G were transfected into 293T cell to produce a replication incompetent lentivirus. Finally the titer of lentivirus was determined.
Results:PCR analysis and DNA sequencing confirmed that the BTBD10 sequences were successfully inserted into the lentiviral vectors. The titer of concentrated virus was 3.42 x 108TU/ml.
Conclusion:The lentiviral vector plasmid containing BTBD10 has been successfully constructed, which will provide a foundation for the further study on function of BTBD10 gene in glioma and gene therapy.
Part III Growth inhibition of human glioma cell line U251 by transfer of lentiviral vector containing BTBD10 and its mechanism
Objective:To investigate the effects of recombinant lentiviral vector containing BTBD10 on tumor proliferation and apoptosis of human glioma cells U251, and to explore the mechanism of regulating tumors.
Methods:BTBD10 lentiviral vector and empty vector were transfected into cultured U251 human glioma cell lines separately. Parental U251 cells was cultured as a negative control. Real-time quantitative PCR was chosen for detection the mRNA expression of BTBD10 and CyclinDl. The protein expression of BTBD10, phosphorylated Akt(pAkt), and CyclinDl were detected using western blot. Cell apoptosis and cell cycle were analyzed by flow cytometry. MTT assay was used to detected cell viability.
Results:The real-time quantitative PCR and western blot hybridization results showed that compared with non-transfected U251 cells and U251 cells(transfected with empty vector), the expression levels of BTBD10 mRNA and protein were significantly increased in U251 cells(transfected with BTBD10 lentiviral vector), and the difference was statistically significance (P<0.05). The expression levels of pAkt protein was decreased, and the difference was statistically significant (P<0.05). The expression levels of CyclinDl mRNA and protein were down regulated, and the difference was statistically significant (P<0.05). The quantitative analysis of apoptotic cells by FCM showed that compared with non-transfected U251 cells and U251 cells(transfected with empty vector), the apoptotic cells increased significantly in U251 cells(transfected with BTBD10 lentiviral vector) with the apoptotic rate of 14.64%. The difference was statistically significant (P<0.05). The results on cell cycle showed that in non-transfected U251 cells and U251 cells (transfected with empty vector), cell cycle and cell statement were basically the same, some cells were in the G2/M phase. U251 cells(transfected with BTBD10 lentiviral vector), were markedly inhibited in the G0/G1 phase. MTT assay showed that the group transfected with BTBD10 lentiviral vector had a much lower cell viability than other group.
Conclusion:The lentiviral vector plasmid containing BTBD10 showed high efficiency on infecting glioma U251 cells line and significantly increased the expression and activation of BTBD10 in cells. The over-expression of BTBD10 expression was able to inhibit the glioma cell proliferation acivity and to induce cell apoptosis. The mechanism may be related to negative regulation of PI3K/Akt signaling pathways by reducing the expresssion of pAkt and CyclinDl.
和其他部位肿瘤一样,脑胶质瘤的发生、发展是一个非常复杂的过程,涉及多种肿瘤相关基因的表达异常或失活。胶质瘤本质上是一种多基因异常疾病,其发生是由于原癌基因的激活和抑癌基因的失活导致细胞信号传导通路的异常、细胞循环周期的改变、生命周期的延长以及细胞调亡缺陷等,从而出现细胞增殖失控及恶性转化。与脑胶质瘤的恶性进展密切相关的细胞通路主要有磷脂酰肌醇3-激醇(P13K)信号转导通路、Ras信号转导通路、Wnt信号转导通路和Notch信号转导通路等。寻找与胶质瘤发病相关的基因,在此基础上发现胶质瘤治疗的新靶点、新策略,是神经外科研究领域的主要内容之一。
BTBD10基因含BTB/POZ功能域,位于人染色体的11p15.2区域,全长2551bp,含245-16721bp开放阅读框架,编码475氨基酸的蛋白,蛋白分子量51.7Kd。前期研究在对18例脑胶质瘤进行表达谱芯片筛选及聚类分析中发现：芯片数据提示BTBD10表达量在各级别胶质瘤中均发生下调,平均Ratio值为0.218。基因表达谱Hierarchical聚类发现与BTBD10表达谱相近的基因有：血管性肠肽(VIP)、肿瘤蛋白p53结合蛋白(TP53BP1)、p淀粉样蛋白前体(APBB1)、8氧化鸟嘌呤糖基酶(OGG1)、促性腺素诱导阻抑蛋白(GIOT1)、MADS转录增强子2C(MEF2C)。MTC为模板RT-PCR结果显示BTBD10在检测的8种正常组织中,脑组织中高表达,而心、肺、肝、肾、胰腺组织中表达量较低。Northern杂交结果显示BTBD10在正常成人脑组织中表达量高,在7例不同级别胶质瘤中表达量都下降,BTBD10在胶质瘤／正常脑组织的表达量比率与芯片数据基本相符。在肝癌、卵巢癌和肺癌中BTBD10的表达没有显著性差异。提示BTBD10基因与脑胶质瘤高度相关,有可能成为新的脑胶质瘤治疗靶基因。为进一步研究BTBD10基因在胶质瘤发生发展中的作用,本实验通过检测BTBD10在大样本胶质瘤中的表达情况,培养过表达BTBD10基因的细胞株,初步探讨BTBD10基因调节胶质瘤细胞增殖水平的可能机制。
第一部分BTBD10在人脑胶质瘤组织中的表达及意义
目的：探讨BTBD1OmRNA和BTBD10蛋白在脑胶质瘤中的表达及意义。
方法：采用实时荧光定量PCR和Western blot杂交检测BTBD10mRNA和BTBD10蛋白在正常脑组织和各级别胶质瘤中的表达,分析其表达与胶质瘤临床病理特征的关系。
结果：在正常脑组织中BTBD10mRNA和蛋白的表达较高；各级别胶质瘤中的表达均低于正常脑组织(P<0.05)；在低级别胶质瘤(Ⅰ-Ⅱ级)中表达量较低,而在高级别胶质瘤(Ⅲ-Ⅳ)中表达水平更低,低级别和高级别之间的表达差异具有统计学意义(P<0.05)。BTBD1OmRNA和蛋白的表达水平与年龄、性别、病理类型等临床病理特征无明显相关(P>0.05)。
结论：胶质瘤中BTBD10表达明显低于正常脑组织中的表达,且随病理级别的增高而降低,提示BTBD10在胶质瘤的发生发展中起重要作用,并与胶质瘤的病理分级密切相关。
第二部分BTBD10过表达慢病毒载体的构建和鉴定
目的：构建BTBD10慢病毒表达载体,为后续实验研究提供依据。
方法：以BTBD10基因为模板PCR扩增目的基因,表达载体(pLV-UbC-GFP-3FLAG)酶切后进行胶回收载体片段,目的基因与载体片段同源重组后转化感受态细胞,用菌落PCR鉴定转化子,阳性克隆测序鉴定,质粒抽提,与无内毒素病毒包装质粒、膜蛋白表达质粒共转染293T细胞包装产生慢病毒颗粒,测定慢病毒滴度。
结果：PCR扩增和测序结果均证实,插入序列完全正确,包装病毒产生病毒悬液的滴度为3.42×108TU／ml。
结论：成功构建了携带BTBD10基因的慢病毒载体,为研究其在胶质瘤细胞中的生物学功能和以其为靶点的基因治疗奠定基础。
第三部分BTBD10慢病毒载体转染人胶质瘤细胞株U251及其作用机制研究
目的：了解BTBD10重组慢病毒载体转染人胶质瘤细胞株U251后,对U251细胞增殖、凋亡的影响,探讨其发挥肿瘤调控作用的机制。
方法：应用BTBD10慢病毒载体、空载体分别转染体外培养的U251人胶质瘤细胞株,正常培养U251细胞作为空白对照。荧光定量PCR检测转染前后BTBD10和CyclinDl mRNA的表达变化,Western blot杂交检测BTBD 10、磷酸化Akt(pAkt)和CyclinD1蛋白水平变化,流式细胞仪检测细胞周期和凋亡水平,MTT法分析细胞增殖情况并绘制生长曲线。
结果：荧光定量PCR和Western blot杂交结果显示,与未转染的U251细胞和转染空白载体的U251细胞相比,转染重组BTBD10病毒载体的U251细胞中BTBD10 mRNA和蛋白水平明显提高,差异具有统计学意义(P<0.05)；Akt的磷酸化水平下调,CyclinDl mRNA和蛋白水平下调,差异具有统计学意义(P<0.05)。流式细胞仪分析各组细胞的凋亡水平,检测结果表明,与未转染的U251细胞和转染空白载体的U251细胞相比,转染BTBD10的U251细胞中凋亡细胞明显增多,早期凋亡细胞达14.64%,统计学上有差异(P<0.05)：细胞周期检测结果显示,未转染的U251细胞和转染空载体的U251细胞的细胞周期和细胞状态基本相同,部分细胞处于G2／M期,转染BTBD10病毒载体的U251细胞则被明显的抑制在G0／G1期,提示增殖活性降低。MTT法检测细胞增值率,发现外源性BTBD10基因转染U251细胞后,细胞生长速度明显下降。
结论：慢病毒表达载体对人胶质瘤细胞系U251具有很高的转染效率,感染后可显著上调靶基因BTBD10的表达和活化。BTBD10过表达后将肿瘤细胞阻滞在G0／G1期,抑制肿瘤细胞增殖,促进凋亡,可能与下调pAkt和CyclinD1水平,负调控PI3K/Akt信号通路有关。
Gliomas are the most common primary tumor of the central nervous system in adults, and accounts for approximately 40-50% of total intracranial tumors. These tumors are highly invasive, and have no obvious dividing line with normal brain tissue, often infiltrate critical neurological areas within the brain. It is difficult to achieve totally surgical resec-tion, and the recurrence and fatality rate is high. In addition, chemotherapy and radioatio-therapy can not be a high degree of specificity in killing tumor cells, and may cause serious adverse reactions. Therefore, glioma is still a refractory disease in the neurosurgical field. To elucidate its pathogenesis and to find new treatments is a research hotspot in neurosurgery.
It has been showed that the devolpment of glioma, just the same as the tumors in other sites of the body, is a very complex process, involving expression disorder or inactivation of a variety of tumor-related genes. Glioma is essentially a multi-gene disorders. Its occurrence is due to the activation of proto-oncogenes and inactivation of tumor suppressor genes, which lead to excessive cell proliferation and malignant transformation. The malignant progression of glioma cells is closely related with several pathways, such as PI3K (phosphatidylinositol 3-kinase) signaling pathway, Ras signaling pathway, Wnt signaling pathway and Notch signaling pathway. However, up to now, the molecular mechanism of glioma is not fully elucidated. Seeking glioma pathogenesis-related genes, finding new target and new strategy for glioma therapy, plays a important role in the field of neurosurgery.
BTBD10 contained a BTB/POZ domain, located in human chromosome 11p15.2 region. It was 2489 bp in length, containing open reading frame from 245 to 1672 bp, encoding 475 amino acid protein with a molecular weight of 51.7 Kd. Preliminary study of 18 glioma samples of cDNA microarray screening and cluster analysis found that the expression level of BTBD10 descend in all samples, with an average Ratio value of 0.218. The result of RT-PCR showed that a ubiquitous expression pattern of BTBD10 in the 8 tissues examined with high expression in adult brain, testis and small intestine and weak expression in the heart, lung, liver, kidney, pancreas, spleen, thymus. Northern blot showed that BTBD10 was highly expressed in mixed normal brain while it was sharply down-regulated in all grades of glioma tissues. RT-PCR showed that there was no significant difference in hepatocellular carcinoma, ovary cancer and lung cancer tissues. That means the BTBD10 gene is highly correlated with glioma, and it may become a new target gene in gene therapy of gliomas. For further studying the role of BTBD10 gene in glioma, we began with examining the expression of BTBD10 in a large number of glioma specimens in the present study, then increased the expression of BTBD10 in glioma cell lines, evaluated the effects of BTBD10 on glioma proliferation and apoptosis.
Part I:Study on the expression of BTBD10 in human gliomas and its significance
Objective:To investigate the expression of BTBD10 mRNA and protein in brain gliomas and its significance.
Methods:The mRNA expression of BTBD10 in 52 cases of human glioma tissues and 8 cases of normal brain tissues was detected with quantitative real-time PCR. The protein expression in these samples were detected using western blot.
Results:The expression of BTBD10 mRNA and protein is higher in normal brain tissue than in all grades of glioma tissue (P<0.05) and significantly decreased ascending the degree of glioma. The difference between the low-grade and high-grade tumors is statisticly significant(P<0.05). A statistically significant association was found between expression of BTBD10 mRNA and protein.
Conclusion:BTBD10 mRNA and protein showed markedly lower expression in glioma than normal brain tissues, and the expression decreased with the malignancy of glioma. These results suggested that BTBD10 was significantly associated with the proliferation activity and may play an improtant role in human glioma.
Part II:Construction and identification of lentiviral vector plasmid containing BTBD10
Objective:To construct lentiviral vector plasmid containing BTBD10 and provide the basis for further experiments.
Methods:The vector was obtained from pLV-UbC-GFP plasmid digested with EcoR I and Nhe I restriction enzymes. Then BTBD10 gene was inserted into the vector to construct pLV-BTBD10, which was subsequently confirmed by PCR and DNA sequencing analysis. After these identification, the resultant pLV-BTBD10 plasmid, pCD/NL-BH*DDD and pLTR-G were transfected into 293T cell to produce a replication incompetent lentivirus. Finally the titer of lentivirus was determined.
Results:PCR analysis and DNA sequencing confirmed that the BTBD10 sequences were successfully inserted into the lentiviral vectors. The titer of concentrated virus was 3.42 x 108TU/ml.
Conclusion:The lentiviral vector plasmid containing BTBD10 has been successfully constructed, which will provide a foundation for the further study on function of BTBD10 gene in glioma and gene therapy.
Part III Growth inhibition of human glioma cell line U251 by transfer of lentiviral vector containing BTBD10 and its mechanism
Objective:To investigate the effects of recombinant lentiviral vector containing BTBD10 on tumor proliferation and apoptosis of human glioma cells U251, and to explore the mechanism of regulating tumors.
Methods:BTBD10 lentiviral vector and empty vector were transfected into cultured U251 human glioma cell lines separately. Parental U251 cells was cultured as a negative control. Real-time quantitative PCR was chosen for detection the mRNA expression of BTBD10 and CyclinDl. The protein expression of BTBD10, phosphorylated Akt(pAkt), and CyclinDl were detected using western blot. Cell apoptosis and cell cycle were analyzed by flow cytometry. MTT assay was used to detected cell viability.
Results:The real-time quantitative PCR and western blot hybridization results showed that compared with non-transfected U251 cells and U251 cells(transfected with empty vector), the expression levels of BTBD10 mRNA and protein were significantly increased in U251 cells(transfected with BTBD10 lentiviral vector), and the difference was statistically significance (P<0.05). The expression levels of pAkt protein was decreased, and the difference was statistically significant (P<0.05). The expression levels of CyclinDl mRNA and protein were down regulated, and the difference was statistically significant (P<0.05). The quantitative analysis of apoptotic cells by FCM showed that compared with non-transfected U251 cells and U251 cells(transfected with empty vector), the apoptotic cells increased significantly in U251 cells(transfected with BTBD10 lentiviral vector) with the apoptotic rate of 14.64%. The difference was statistically significant (P<0.05). The results on cell cycle showed that in non-transfected U251 cells and U251 cells (transfected with empty vector), cell cycle and cell statement were basically the same, some cells were in the G2/M phase. U251 cells(transfected with BTBD10 lentiviral vector), were markedly inhibited in the G0/G1 phase. MTT assay showed that the group transfected with BTBD10 lentiviral vector had a much lower cell viability than other group.
Conclusion:The lentiviral vector plasmid containing BTBD10 showed high efficiency on infecting glioma U251 cells line and significantly increased the expression and activation of BTBD10 in cells. The over-expression of BTBD10 expression was able to inhibit the glioma cell proliferation acivity and to induce cell apoptosis. The mechanism may be related to negative regulation of PI3K/Akt signaling pathways by reducing the expresssion of pAkt and CyclinDl.
引文
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