The Experiment Study On Survivin-siRNA Inhibiting The Growth Of Retinoblastoma

Retinoblastoma is the most common primary ocular malignancy of childhood. Today over 90% of these children can be saved by modern therapeutic strategies. The best aim of the management of this disease is not only keeping globe salvage, but also maintaining useful visual function. The therapeutic methods including chemotherapy, focal treatment methods (cryotherapy and photocoagulation, chemothermotherapy, thermotherapy, brachytherapy or plaque radiotherapy, stereotactic conformal radiotherapy, accelerated proton beam irradiation), enucleation and external beam radiotherapy. With the development of tumor molecular biology, gene therapy becomes a new biotreatment method. As a gene therapy target, eyeball has its own virtues. The anatomical structure of ocular is clear; the refractive medium is transparent; and the immune of eye is relatively priviledge. It is easy to transfer a target gene into an eyeball accurately and also to observe the curative effect. The research of this field includes suicide gene therapy, tumor suppressor gene therapy, anti-angiogenesis gene therapy and inducing apoptosis gene therapy.Survivin, a member of the inhibitor of apoptosis (IAP) gene family, has been shown a bifunctional role in cell division and apoptosis control by the experimental evidence. It is highly expressed in most cancers. In tumor cells, elevated Survivin is commonly associated with enhanced proliferative index, reduced levels of apoptosis, resistance to chemotherapy, and increased rate of tumor recurrence. Comparing with the limited expression in normal differentiated tissues, Survivin has a different distribution in cancers. Moreover, Survivin is a critical anti-apoptosis factor in the oncogenesis and tumor development. According to these characters, researchers put their heads together to find methods to treat cancer and other deseases by controlling the apoptosis with Survivin. The inhibition of the expression of Survivin in the tumor cells has been demonstrated dual function, inhibiting tumor growth through an increase in spontaneous apoptosis, and enhancing tumor cellular response to apoptosis-inducing agents. Different approaches of gene therapies against Survivin have shown that a spontaneous apoptosis can be promoted by the inhibition of Survivin, whereas almost no effect for the normal tissues.RNA interference (RNAi) is an important regulating mechanism of gene expression. Specific short interfering double-stranded RNAs (siRNAs) serve as the effector molecules for sequence-specific gene silencing. Because of the character of highly sequence-specific, siRNAs can recognise the target gene mRNA quickly and accurately, and downregulate the expression of target gene. Using RNAi technology can inhibit the target gene expression specificly, chronically and completely. RNAi-mediated gene therapy may against tumor development by reversing malignant phenotype, inducing apoptosis and reducing the drug tolerance of tomor cells.Objective: To evaluate the effects of Survivin-siRNA eukaryotic expression vectors on growth and apoptosis of retinoblastoma.Methods: 1.The expression of Survivin in retinoblastoma was detected by immunohistochemical staining. 2. Accroding to the design principles of shRNA, pGCsilencer-U6-siRNA-Survivin eukaryotic expression vector ( Survivin-siRNA ) and pGCsilencer-U6-siRNA-Scramble eukaryotic expression vector(Scramble-siRNA)were synthesized. Restriction enzyme digestion (Hindâ…¢) and sequence analysis were used to confirm the sequences of vectors. 3. In vitro study: (1) Y79 retinoblastoma cells were cultured. The cells were divided into three groups including mock, Scramble-siRNA and Survivin-siRNA. Vectors were trasfected into Y79 cells with lipofectamine. (2) Semi-quantitative RT-PCR was performed to analyze Survivin mRNA in tumor cells. (3) Western Blotting was used to analysis the expression of Survivin protein. (4) The inhibition of cell proliferation was detected by MTT. (5) AO/EB staining was used to observe the morphological changes of typical apoptosis. (6) The apoptosis was assessed by FACS. 4. In vivo study: (1) Animal models were generated by injection Y79 retinoblastoma cells into the vitreous cavity of the nude mice. (2) Mice were divided into three groups: mock, Scramble-siRNA and Survivin-siRNA group. (3) Semi-quantitive RT-PCR was used to detect Survivin mRNA in tumor tissues. (4) Western Blotting was used to analyse the expression of Survivin protein. (5) The tumor tissues were observed by HE staining. The expression of Survivin and Ki67 were observed by immunohistochemical staining. (6) TUNEL method was used to analysis the apoptosis of retinoblastoma cells.Results: 1. High expression of Survivin was found in retinoblastoma cases by immunohistochemical staining and no expression was found in the normal retina. Significant difference was found between two groups (P<0.01). 2. siRNA sequence and pGCsilencer-U6/Neo/GFP vector were connected correctly, and it was confirmed by restriction enzyme digestion and sequence analysis. 3. In vitro study: (1) The vectors were trasfected into Y79 cells. Morphological changes were observed by phase contrast microscope 48h later. The growth of cells were normal in control groups whereas the quantities of cells decreased and the fragments of cells increased in experimental group.The green fluorescence was found in Survivin-siRNA group and Scramble-siRNA group while none was found in mock group by fluorescence microscope. (2) The expression of Survivin was significantly inhibited at mRNA level comparing with the control group(sP<0.01), and no significant difference was found between two control groups(P>0.05)by semi-quantitive RT-PCR. (3) Comparing with the control groups, the expression of Survivin was significantly inhibited at protein level by Western Blotting(P<0.01), and no significant difference was found between two control groups(P>0.05). (4) The inhibition of cell proliferation was present significantly in treated group comparing with the control groups(P<0.01)by MTT. (5) Apoptosis cells were observed in experimental group by AO/EB staining. (6) After 72h treatment, significant cell apoptosis (P<0.01)and blocking in G1 (P<0.01)stage were demonstrated between treated group and control groups by FACS analysis. 4. In vivo study: (1) The Y79 retinoblastoma vitreous cavity model was generated successfully. (2) Survivin-siRNA significantly inhibited the transcription of survivin gene at mRNA level as it did in vitro study. (3) Survivin-siRNA inhibited the expression of Survivin protein significantly as it did in vitro study. (4) More dead cells were detected in experimental group by HE staining. The expression of Survivin and Ki67 was inhibited significantly(P<0.01)in experimental group. (5) More apoptosis cells were observed in experimental group, and apoptosis index was significant higher in experimental group than in control groups(P<0.01).Conclusion: Survivin-siRNA can be used for interrupting the gene transcription and downregulating the protein expression for Y79 retinoblastoma cell line and tumor tissues. Survivin-siRNA induces the apoptosis of retinoblastoma cells. RNAi may be a useful method to treat retinoblastoma as a new gene therapy in future.