Expression Of BTBD10 In Gliomas And Its Effect On Proliferation And Apoptosis In U251 Cell Line

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 significanceObjective: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 BTBD10Objective: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 mechanismObjective: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.