The Expression And Cloning Of SEPT7 In Human Gliomas And The Experimental Study On Gene Therapy Of Malignant Gliomas With SEPT7

Gliomas are the most common primary brain tumors. The pathogenesis of gliomas remains largely undefined. As the knowledge of tumor biology and molecular genetics increased, it has been shown that the development of gliomas, just the same as the tumors in other sites of the body, results from the activation of proto-oncogenes and inactivation of tumor suppressor genes. However, up to now, the molecular mechanism of gliomagenesis is not fully elucidated. To optimize treatment strategies and to develop novel therapeutic approaches for gliomas, a more precise understanding of the cellular and molecular basis of gliomas is necessary, so it is important to continue seeking more genes which may play a key role in the gliomagenesis.Our previous study on the gene expression profiles of gliomas by cDNA microarray analysis demonstrated that the expression of CDC10 gene, a member of septin family, is lowered in a variety of gliomas. Septins belong to a family of cytoskeleton proteins and are identified in yeast at first. Former nomenclature for septin proteins is very confused. A unified nomenclature is proposed later as SEPT and CDC10 is named as SEPT7. For further studying the aberration and the role of SEPT7 in gliomas, we began with examining the expression of SEPT7 in a large number of glioma specimens in the present study. A recombinant vector carrying SEPT7 cDNA was then constructed and transfected into glioma cells with lowered expression of SEPT7 or without SEPT7 expression for observing its biological effect on gliomas in vitro. Moreover, the established subcutaneous gliomas in nude mice were treated with SEPT7 cDNA for further defining the role of SEPT7 in the progression of gliomas.In the first part of this study, SEPT7 expression was examined in 47 freshly resected glioma samples and 4 malignant glioma cell lines at mRNA and protein level by RT-PCR and immunohistochemical staining. Meanwhile, SEPT7 expression was also detected with immunohischemical staining in a tissue array consisting of 63 glioma samples. It was found that the normal brain tissues hadconstitutional expression of SEPT7 while the expression of SEPT7 mRNA and protein was reduced in most of the gliomas and its expression was negatively correlated with the tumor grade. The expression level of SEPT7 in high grade gliomas was significantly decreased than that of low grade gliomas. TJ905 and TJ899 human glioblastoma cell lines did not express SEPT7, but there was a lowered expression of SEPT7 in human glioblastoma U251 and TJ862 cell lines. This result further confirmed that SEPT7 expression was reduced or deleted in majority of gliomas especially in high grade gliomas. Based on this finding, an eukaryotic expression vector of SEPT7 was constructed for studying its biological effect on glioma cells. Recombinant vector was identified by DNA sequencing. As compared to the cDNA sequence of SEPT7 registered in Genebank, a nonsense mutation at 404 site (g replaced a) was found. This finding was submitted to and accepted by Genebank. The accession number is DQ232879.In the second part of this study, SEPT7 constructs was transfected to human glioblastoma TJ905 cell line without SEPT7 expression and U251 cell line with reduced expression of SEPT7. The positive clones were selected by G418 and identified by RT-PCR, immunofluorence and Western blot analysis. For observing the phenotypic changes of the cells transfected with SEPT7, The cell proliferation was determined by MTT assay and flowcytometry, cell apoptosis was detecteded with Annexin V staining and cell invasion was evaluated by motility in sandwich-matrigel. Moreover, the molecules regulating the cell cycle progression were examined by immunofluorence staining and Western blot analysis. It was found that after transfection with SEPT7 the cell proliferation activity was inhibited, the S phase fraction (SPF) was lowered and cell cycle was arrested in G0/G1 phase. Apoptotic cells were increased and cell invasive ability was attenuated. The positive regulatory molecules for cell cycle progression including cyclin Dl, CDK4, cyclin E and CDK2 were downregulated while the negative modulators including pl6 and p21 were upregulated. These results revealed that SEPT7 exerted the suppressive effect on the glioma cell growth and invasion, and induced apoptosis.In the third part of the present study, in vivo experiment was carried out. Nude mice with well-established subcutaneous explanted TJ905 and U251 gliomas were divided into three groups: 1) control group: gliomas treated with PBS;2) empty vecter group;gliomas treated with empty vectors;3) SEPT7 group: gliomas treated with SEPT7-lipofectamine complex. Each group consists often nude mice for both TJ905 and U251 gliomas. During the observationperiod of 4 weeks, the growth rate of TJ905 or U251 gliomas in nude mice of SEPT7 group was significantly slow down and the tumor volumes were much smaller than those in control and empty vector group. The expression of SEPT7 and proteins involved in the cell cycle progression, apoptosis and invasion were examined and compared in the gliomas taken from the mice of three groups. The SEPT7 expression was significantly upregulated in the U251 and TJ905 gliomas treated with SEPT7. It was also demonstrated that the apoptotic cells were increased and the expression of cyclin Dl, CDK4, cyclin E and CDK2 was markedly inhibited while the expression of pi6 and p21 was significantly increased in gliomas treated wit SEPT7 cDNA, similar to the results found in vitro study. The expression of prosurvival protein BCL-2 was dowregulated. In contrast, the expression of proapoptotic protein caspase 3 was upregulated. Meanwhile, the expression of the most important proteases for glioma invasion-MMP2 and MMP9 was reduced. All these findings proposed that SEPT7 inhibited the tumor growth by the molecular mechanisms which arrest the cell cycle progression and induction of apoptosis. Additionally, we had tried to knockdown SEPT7 by RNAi technology via injection of siRNA targeting SEPT7 into the subcutaneous explanted U251 gliomas with lowered expression of SEPT7 in nude mice and found that the tumor growth rate in the siRNA treated group tended to be higher than those in the control group and scrambled siRNA treated group. This result further suggested that SEPT7 was intimately related to the inhibition of tumor growth.In conclusion, the results of the current study demonstrate that normal brain tissues have constitutive expression of SEPT7. However, SEPT7 expression is significantly reduced in the majority of the gliomas and its expression is negatively correlated with the tumor grade. Transfection of SEPT7 construct into the glioma cells with lowered expression of SEPT7 or without SEPT7 expression is able to inhibit the glioma cell proliferation acivity and invasive ability and to induce cell apoptosis. SEPT7 also can inhibit tumor growth and invasion, induced cell apoptosis in vivo. Treated with siRNA targeting SEPT7, tumor of nude mice grew with accelerative rate than that of control group. Consequently, SEPT7 may be considered as a tumor suppressor gene and a promising candidate for gene therapy of gliomas. To our knowledge, this is the only comprehensive study on the biological effect of human SEPT7 on gliomas and provide the first molecular evidence that SEPT7 is a tumor suppressor gene.