| Deregulation of cell proliferation and cell apoptosis underlies neoplastic initiation and development, which involves multiple gene alterations, and is regulated by complicated signal transduction pathways. 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 (BIR) domains at the amino terminal, possessing or not a carboxyl terminal RING zinc-finger domain. Survivn is a novel member of IAP family, which has potential antiapoptosis activity. In addition, survivin also plays an important role in maintaining normal cell mitosis, promoting cell proliferation and angiogenesis. Survivin is generally expressed in embryonic tissues, but is not expressed in most normal adult tissues, whereas is overexpressed in the majority of human cancers, indicating that survivin may associate with the tumorigenesis and progress of most human cancers.Brain glioma is the most common malignancy of central nervous system, which occupies almost half of all intracranial tumors. Although the comprehensive treatment level 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 forpatients with malignant brain glioma. The main reason for difficulties on treatment is associated closely with the malignant biology phenotype of brain glioma, which including excessive proliferation, antiapoptosis and abundant angiogenesis. So, it has important science significance and clinical application value to identify the key genes involved in the malignant biology behavior of this malignancy for overcoming it. At present, research on the relationship between survivin and brain glioma is little, and the role of survivin in malignant proliferation, antiapoptosis and angiogenesis of brain glioma is not completely clear. For this, the current study was performed from four aspects as follows.1 Expression of mRNA of survivin and its splice variants survivin-AEx3 and survivin-2B in brain glioma and its significance: Four human brain glioma cell lines (U251, U87, BT325 and SHG-44), 62 cases of human brain glioma tissue and 10 cases of normal human brain tissue were investigated by reverse transcription polymerase chain reaction (RT-PCR) for expression of mRNA of survivin and its splice variants survivin-AEx3 and survivin-2B. The results showed that, all mRNA of survivin and its splice variants survivin-AEx3 and survivin-2B were expressed in U251 and U87 cells, mRNA of survivin and survivin-2B were expressed in BT325 cells, whereas no mRNA of survivin, survivin-AEx3 and survivin-2B were expressed in SHG-44 cells. It was 67.7%, 30.6% and 17.7% respectively that the positive expression rate of mRNA of survivin, survivin-AEx3 and survivin-2B in human brain glioma tissues, while none was expressed in normal human brain tissues. There were specially significant difference (P<0.01) and significant difference (P<0.05) respectively between the positive expression rate of mRNA of survivin and survivin-AEx3 in brain glioma tissues and those in normal brain tissues, whereas no significant difference (P>0.05) for positive expression rate of survivin-2B mRNA. There were specially significant difference (P<0.01 for both) respectively between the positive expression rate of survivin mRNA in benign brain glioma and that in malignant brain glioma and among those in different pathological grades of glioma, whereas no significant difference (P>0.05 for all) for positive expression rate of mRNA of survivin-AEx3 and survivin-2B as above comparison. Therewere no significant difference (P>0.05 for all) respectively for positive expression rate of mRNA of survivin, survivin-AEx3 and survivin-2B among different sex of patient, age of patient, size of tumor and site of tumor. The results suggested that, survivin mRNA be highly expressed in brain glioma, and which may associate closely with initiation and progress of brain glioma; although mRNA of survivin-AEx3 and survivin-2B be certainly expressed in brain glioma, however their role is limited in tumorigenesis of brain glioma.2 Survivin protein expression and its relationship with clinicopathological features, proliferation, apoptosis and angiogenesis in brain glioma: Four human brain glioma cell lines (U251, U87, BT325 and SHG-44), 83 cases of human brain glioma tissue and 12 cases of normal human brain tissue were investigated by immunohistochemistry SABC method for protein expression of survivin. The results showed that, survivin protein was expressed in U251, U87 and BT325 cells, while no expression of survivin protein in SHG-44 cells. It was 57.8%, 3.75±3.89 and 8.3%, 0.17±0.58 respectively that the positive expression percent and immunoreactivity score (IRS) of survivin in brain glioma tissues and normal brain tissues, there were specially significant difference (PO.01 for both) respectively in the positive expression percent and IRS of survivin between them. There were specially significant difference (PO.01 for all) respectively in the positive expression percent and IRS of survivin between benign and malignant brain glioma and among different pathological grades of glioma, whereas no significant difference (P>0.05 for all) respectively among different sex of patient, age of patient, size of tumor and site of tumor. There was no significant difference between survivin protein expression level and survivin mRNA expression level (P>0.05). Further, expression of proliferating cell nuclear antigen (PCNA) and factor VI related antigen (FWRAg) was investigated by immunohistochemistry SABC method, apoptotic cells were screened by TUNEL method, proliferative index (PI), apoptotic index (AI), overall daily growth (ODG) and microvessel density (MVD) in brain glioma tissues were measured respectively. The results showed that, it was (28.39±19.49)%, (1.00±0.80)%, (12.19±10.2I)% and 62.75±31.50 respectivelythat the PI, AI, ODG and MVD of brain glioma, and all of them markedly ascended with the increasing of pathological grade of brain glioma (/*<0.01 for all). PI, ODG and MVD in survivin protein positive group were significantly higher than those in survivin protein negative group (PO.01 for all), in addition, PI, ODG and MVD were positively correlated with survivin IRS (P<0.01 for all). Although there was no significant difference between AI in survivin protein positive group and that in survivin protein negative group (P>0.05), however, AI was inversely correlated with survivin IRS (PO.01). The results suggested that, survivin protein be highly expressed in brain glioma, and which may associate closely with initiation, progress, malignant proliferation, antiapoptosis and angiogenesis of brain glioma.3 Impact on proliferation and apoptosis of human brain glioma cells in vitro by RNAi targeting survivin gene: According to survivin cDNA sequence, the specific RNAi fragments targeting survivin were designed and synthesized, which were cloned into pWHl vector, and the eukaryotic expression vector pWHl-SR of survivin shRNA was constructed. The pWHl-SR vector and blank pWHl vector were transfected respectively into human brain glioblastoma U251 cells by lipofectin medium, and the permanent transfectants U251-SR and U251-P cells were established through selecting with G418. RT-PCR results showed that mRNA expression of survivin was inhibited markedly with the inhibitory rate of 87%, while the results of immunohistochemistry and Western blot indicated that protein expression of survivin was also suppressed significantly with the inhibitory rate of 91% in U251-SR cells. The cell growth curve drawn by cell counting showed that U251-SR cells grew significantly slow than U251 and U251-P cells (PO.01). The results of plate colony formation showed that the colony number of U251-SR cells markedly decreased than those of U251 and U251-P cells (P<0.01). The cell cycle analysis by flow cytometry (FCM) showed that the Gl phase cells significantly increased, whereas S phase cells markedly decreased in U251-SR cells, as compared with those in U251 and U251-P cells. The results of observation by phase contrast microscopy, HE staining, Hoechst staining, TUNEL staining and observation by transmission electro microscopyshowed that the apoptotic cells markedly increased in U251-SR cells compared with those in U251 and U251-P cells. The quantitative analysis of apoptotic cells by FCM showed that the apoptotic cells increased about 6-fold with the apoptotic rate of 14.4% compared with U251 (2.1%) and U251-P (2.7%) cells. The above results suggested that the survivin shRNA can suppress significantly expression of mRNA and protein of survivin, thereby markedly inhibit growth and induce apoptosis of human brain glioma cells in vitro.4 Impact on tumorigenesis, proliferation, apoptosis and angiogenesis of human brain glioma cells in nude mice by RNAi targeting survivin gene: U251, U251-P and U251-SR cells were inoculated respectively in flank subcutaneous tissue of nude mice to establish xenograft models of human brain glionia. The tumor growth status was observed and tumor volume was measured termly, and the tumor growth curve was drawn. The animals were killed and the tumor weight was investigated 45 days after inoculation. The results showed that the tumorigenesis time delayed, tumor grew slow, both tumor volume and tumor weight decreased significantly (PO.01) in U251-SR group as compared with those in U251 and U251-P groups. HE staining for each group of tumor specimen indicated that apoptotic cells increased in U251-SR group in comparison with those in U251 and U251-P groups. Further, expression of survivin, PCNA and FWRAg was investigated by immunohistochemistry SABC method, apoptotic cells were screened by TUNEL method, PI, AI and MVD were measured respectively in each group of tumor specimen. The results showed that survivin protein expression was downregulated markedly in U251-SR group in comparison with that in U251 and U251-P groups. In U251, U251-P and U251-SR groups, PI was (84.85±7.63)%, (82.44±6.87)% and (33.77±9.24)% respectively, and PI decreased significantly in U251-SR group (PO.01); AI was (2.32±0.90)%, (3.13±1.15)% and (10.93±1.99)% respectively, and AI increased significantly in U251-SR group (PO.01); MVD was 53.60±6.73, 50.20±5.80 and 25.40±5.18 respectively, and MVD decreased significantly in U251-SR group (PO.01). The above results suggested that RNAi targeting survivin can suppress significantly tumorigenesis and growth of human brain glioma in nude mice, which may be...
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