Studies On Survivin Targeting Therapy Of Human Osteosarcoma And Its Relative Mechanism

Background and objective: Osteosarcoma is most frequent primary of all primary bone tumors of the skeleton and occurring mainly in children and adolescent. Survivin is a member of inhibitor of apoptosis family which is recently described. The most notable feature of Survivin expression is its absence in most terminally differentiated normal tissues, but it is highly expressed in malignancies and embryonic tissues. But whether it is highly expressed in osteosarcoma tissue and cells and whether it is relative to the degree of malignancy and the capable of invasion? In this experiment, we study the expression of Survivin, Cox-2 in osteosarcoma tissues and research the effect on carcinogenesis and development of osteosarcoma. Researchers have discovered that selective inhibitors of Cox-2 can induce the apoptosis of tumor cells. But the mechanism and whether it can kill osteosarcoma cells or not is still unknown. We investigate the effects of NS-398, a Cyclooxygenase-2 selected inhibitor, induced growth inhibitory and apoptosis in osteosarcoma cells line MG-63 and the influences of NS-398 on the expression of Survivin in MG-63 cells and then make a pilot study for interrelated mechanism.Can we use antisense technology block the expression of Survivin to induce the apoptosis of osteosarcoma cells because Survivin is highly expression in osteosarcoma? We investigate the effect of antisense oligonucleotide (ASODN) targeting Survivin on proliferation and apoptosis of osteosarcoma cells line MG-63. And then observe the influence of expression of Survivin and Fas in osteosarcoma cells line MG-63. To further research the effect of Survivin in osteosarcoma, we construct and identify the antisense eukaryotic expression vectors for human Survivin gene. Observe the influence on the expression of Survivin in osteosarcoma cells line MG-63 by inhibiting Survivin expression specifically by Survivin antisense RNA. And then observe the inhibitory effects of an antisense Survivin vector on proliferation and invasion of osteosarcoma cells line MG-63, and then make a pilot study for interrelated mechanism.Methods: Immunohistochemical assay (SP method) was used to detect the expression of Survivin and Cox-2 in osteosarcoma tissues.Osteosarcoma cells line MG-63 was cultured in vitro. Methyl thiazolyl tetrazolium (MTT) and flow cytometry (FCM) were used to study the effects of NS-398 with different concentration induced apoptosis in osteosarcoma cells line MG-63. The expression of Survivin of osteosarcoma cells line MG-63 was analyzed by RT-PCR.Survivin ASODN was designed and transfected into MG-63 cells mediated by liposome reagent. Proliferation of osteosarcoma cells was detected by MTT; the morphological alterations were confirmed by Hochest 33258 staining. Apoptosis was determined by flow cytometry and DNA ladder. Survivin and Fas protein and mRNA expression was detected by Western bolt and RT-PCR.Gene recombination method was used to construction and identification of antisense eukaryotic expression vectors for Survivin gene. The recombinant plasmid was confirmed by double digestion with restriction endonucleases BamH I and EcoRI, and then certified by DNA sequencing. The recombinant plasmid was transfected into human osteosarcoma cells line MG-63 by lipofectamine. The expression levels of Survivin before and after transfection were detected by RT-PCR. MTT assay was used to detect the capability of proliferation, Boyden chamber in vitro invasion assay was applied to observe the changes of invasion capability. Then, the effect on the expression of Ang-2 was evaluated by RT-PCR method. Furthermore, the protein expression levels of Ang-2 before and after transfrction were detected by Western blot method. Results: 1. The expression rate of Survivin and Cox-2 were significantly highly in osteosarcoma than in osteochondroma. The positive signals were marked in cytoplasm. And the expression of Survivin and Cox-2 were strongly correlated with tumor stage and poor differentiation. Survivin positive cases were strongly correlated with Cox-2 expression.2. The apoptosis of osteosarcoma cells line MG-63 could be induced by NS-398, and the apoptosis rates were different with different concentration. The apoptosis rate rose significantly when the concentration of NS-398 was over 200μmol/L. The expression of Survivin of osteosarcoma cells line MG-63 declined obviously than before medication.3. The proliferation of MG-63 cells was inhibited notably after ASODN transfection and the apoptosis rate of ASODN groups was significantly higher than that of other groups. DNA ladder-shaped was observed. Apoptosis and inhibition of proliferation were observed by morphological detect After ASODN being transfected. Survivin protein and mRNA expression in ASODN groups was significantly decreased compared with that in the control groups. In contrast, Fas protein and mRNA expression in ASODN groups was significantly increased compared with that in the control groups.4. The antisense recombinant eukaryotic expression vector for Survivin was successfully constructed and identified. Expression level of Survivin mRNA decreased sharply in antisense Survivin transfected cells. The recombinant plasmid was successfully transfected into MG-63 cells. The cells antisense Survivin transfected grew slowly than other groups. The invasion ability was remarkably decreased after antisense transfection compared with the control groups. Moreover, the protein and the mRNA expression of Ang-2 in antisense Survivin transfected cells were much lower than another compared groups.Conclusions:1. The abnormal expression of Survivin and Cox-2 in osteosarcoma suggested they might be involved in the pathogenesis of osteosarcoma. It illustrate that Survivin is relate to the occurrence and development of neoplasm. It can be a target of oncotherapy.2. NS-398 has lethal effect on osteosarcoma cells line MG-63. It may induce apoptosis of osteosarcoma cells line MG-63 by inhibiting the expression of Survivin.3. ASODN targeting Survivin can inhibit cell proliferation; induce cell apoptosis. ASODN has lethal effect on osteosarcoma cells line MG-63. It may induce apoptosis of osteosarcoma cells line MG-63 by inhibiting the expression of Survivin and increase the expression of Fas.4. The eukaryotic expression vector for Survivin antisense RNA may play a specific inhibitory role in highly malignant proliferation of human osteosarcoma cells line MG-63, and may provide a good experimental model for further studying molecular characteristic of Survivin.5. The antisense Survivin vector could inhibit markedly the proliferation and invasion of osteosarcoma cells line MG-63, partially reversed their malignant phenotype. Those seemed to have relations with down-regulating the expression of Ang-2, which indicated that Survivin might be a new target for antisense gene therapy of osteosarcoma.