The Mechanism Of Promoting Glioma Angiogenesis By Survivin

The inhibitor of apoptosis protein (IAP) family is a sort of important factors of inhibiting apoptosis, which is characterized by the presence of one or more baculoviral IAP repeat domains at the amino terminal, possessing or not a carboxyl terminal RING zinc-finger domain. Survivn is an important member of IAP family, which has a strong antiapoptosis activity. In addition, survivin also plays an important role in maintaining normal cell mitosis, promoting cell proliferation and angiogenesis. Aberrant angiogenesis is one of important characteristics of malignant tumors. Recent research results indicate that survivin may associate closely with the angiogenesis of most human cancers.Brain glioma is the most common malignancy of central nervous system, which occupies almost half of all intracranial tumors. The comprehensive treatment of brain glioma, including surgery, radiotherapy and chemotherapy, is progressing continuously. However, the outcome of this malignancy is still not improved drastically. The postoperative median survival is less than one year for patients with malignant brain glioma. The main reason for difficulties in treatment is associated closely with the malignant biological phenotype of brain glioma. As is demonstrated, abundant angiogenesis is one of important malignant phenotypes of brain glioma. It has important scientific significance and clinical application value for overcoming this malignancy to identify the key genes involved in brain glioma angiogenesis. At present, the role of survivin in malignant angiogenesis of brain glioma remains unclear completely. We presume that there is a positive feedback signaling pathway between survivin and angio-stimulating factors in glioma cells, which may partly explain the uncontrolled tumor angiogenesis. For this, the current study was performed from three aspects as follows.1. Angio-stimulating factors'effect on expression of survivin in glioma cellsObjective To investigate the influence of VEGF, bFGF and PDGF on expression of survivin in glioma cell lines SHG44 and U251. Methods In vitro, human glioma cell lines SHG44 and U251, which were divided into several groups, were cultured for 24 h in DMEM containing different concentrations of recombinant human VEGF (0, 25, 50, 100 ng/ml), bFGF (0, 1, 2.5, 5, 10, 20 ng/ml) and PDGF (0, 5, 10, 20, 40 ng/ml) respectively. After that, total protein was collected. Meanwhile, different groups of SHG44 and U251 cells were repectively cultured in 100 ng/ml VEGF, 10 ng/ml bFGF and 10 ng/ml PDGF for 0, 6, 24, 48, 72 h. After that, total protein was also collected. Expression levels of survivin protein of different groups were detected by Western blot. Furthermore, different groups of SHG44 and U251 cells were repectively cultured in 100 ng/ml VEGF, 10 ng/ml bFGF and 10 ng/ml PDGF for 0, 3, 6, 12, 24 h. After that, total RNA was collected. Expression levels of survivin mRNA of different groups were detected by Real-time RT-PCR. Results PDGF transcriptively upregulated survivin expression of SHG44 and U251 cells in a time and concentration dependent manner. In SHG44 cells, expression level of survivin mRNA and protein peaked at 3 h and 12 h respectively after exposure to 10 ng/ml PDGF, as low as 5 ng/ml PDGF can upregulate survivin protein expression after 24 h. In U251 cells, expression levels of survivin mRNA and protein peaked at 6~12 h and 24~48 h respectively after exposure to 10ng/ml PDGF, as low as 5 ng/ml PDGF can also upregulate survivin protein expression after 24 h exposure. BFGF transcriptively upregulated survivin expression in a time and concentration dependent manner only in U251 cells but not in SHG44 cells. In U251 cells, expression levels of survivin mRNA and protein peaked at 6 h and 12~24 h respectively after exposure to 10 ng/ml bFGF, less than 10 ng/ml bFGF can not upregulate survivin protein expression after 24 h exposure. Within designed concentration and time extents, VEGF did not alter expression of survivin. Conclusion The current study suggests that besides directly stimulate vascular endothelial cell proliferation and migration, bFGF and PDGF may upregulate survivin expression to exert an indirect angio-stimulating function.2. Construction, identification and cell transfection of survivin eukaryotic expression vector and survivin specific targeting RNA interference vectorObjective To construct human survivin eukaryotic expression vector and the specific RNA interference vector targeting human survivin gene, further to transfect them to human glioma cell lines SHG44 and U251 respectively to establish stably transfected cell lines. Methods By RT-PCR, the specific coding sequence fragment of survivin cDNA was amplified, which was cloned into eukaryotic expression vector pcDNA3.1, and the eukaryotic expression vector pcDNA3.1-SVV was constructed. The specific RNA interference fragments targeting survivin and firefly luciferase gene were designed and synthesized, which were cloned into pSilencer3.1-H1neo plasmid vector respectively, thus the specific RNA interference vector pSilencer3.1-SVV targeting survivin gene and the human non-specfic interference sequence vector pSilencer3.1-FLF were constructed. By lipofectamine 2000, the vectors pcDNA3.1, pcDNA3.1-SVV were transfected respectively into SHG44 cells, while the vectors pSilencer3.1-SVV, pSilencer3.1-FLF and pSilencer3.1-H1neo were transfected respectively into U251 cells, and the transfected cells were selected by G418. Expression levels of mRNA and protein of survivin were investigated by Real-time RT-PCR, Western blot and immunocytochemistry methods. Results The survivin gene eukaryotic expression vector pcDNA3.1-SVV and the specific RNA interference vector pSilencer3.1-SVV targeting survivin gene were constructed successfully, which were identified by restriction endonuclease and DNA sequence analysis. The stable transfectants SHG44-P and SHG44-S cells transfectanted respectively with pcDNA3.1 and pcDNA3.1-SVV, U251-S, U251-F and U251-P cells transfectanted respectively with pSilencer3.1-SVV, pSilencer 3.1-FLF and pSilencer3.1-H1neo, were obtained. By identification, no expression of survivin gene in SHG44-P and expression of survivin gene in SHG44-S were demonstrated, and expression levels of mRNA and protein of survivin were inhibited significantly in U251-S cells with the inhibitory rate of 70%, whereas survivin gene expression levels were hardly changed in U251-F and U251-P cells. Conclusion Survivin gene eukaryotic expression vector pcDNA3.1-SVV is expressed in SHG44 successfully, and survivin gene expression is suppressed markedly in U251 cells by specific RNA interference vector pSilencer3.1-SVV. The current results establish the experimental foundation for further studying the angio-stimulating function and its mechanisms of survivin in glioma cell lines. 3. Expression of angio-stimulating factors VEGF, bFGF and PDGF in stable transfected glioma cell linesObjective To contrast the expression differences of VEGF, bFGF and PDGF between pre-transfection and post-transfection in SHG44 and U251 cells. Investigate whether or not survivin can promote vascular formation mediated by angio-stimulating factors. Methods Extracted total protein and RNA from SHG-44, SHG44-P, SHG44-S, U251, U251-P, U251-F and U251-S cell lines. Expression levels of mRNA and protein of three angio-stimulating factors in different cell lines were investigated by real-time RT-PCR and Western blot. Collected supernatant of different cell lines, secretion levels of three angio-stimulating factors were detected by ELISA. Results At protein level in cytoplasm or supernatant, presence of bFGF, absence of VEGF and PDGF, were observed in SHG44-S, whereas VEGF, bFGF and PDGF were expressed neither in SHG44-P or in SHG44, expression levels of VEGF and bFGF in U251-S were suppressed significantly to 50% and 30% respectively, whereas VEGF and bFGF expression levels were hardly changed in U251-F and U251-P, PDGF was absent in U251, U251-P, U251-F and U251-S. At mRNA level, the results corresponded well with the protein results. Conclusion Upregulation of survivin expression switchs on bFGF expression in SHG44, while downregulation of survivin expression results in suppression of VEGF and bFGF in U251. The present results indicate that survivin can promote glioma angiogenesis mediated by VEGF and bFGF, which provides evidences for further studying the detailed signaling pathway involved in this process.