Research On Survivin Gene Silence Regulating The Radiosensitivity Of Laryngeal Squamous Cell Carcinoma

Background:Laryngeal squamous cell carcinoma (LSCC), accounting for 1.5% of all cancers and the vast majority (approximately 96%) of laryngeal carcinoma, is one of the most common malignancies of the head and neck. In the recent years, the incidence of Laryngeal squamous cell carcinoma is increasing overtime. Radiotherapy plays an important role in the treatment of laryngeal carcinomas, especially for the early stage, which is considered to be the primary treatment modality. Patients with early-staged LSCC are often cured by surgery or radiotherapy. However, patients with advanced inoperable LSCC are associated with poor prognosis. In addition, despite advances in diagnostic and therapeutic strategies including surgical management, radiotherapy, and chemotherapy, the clinical outcome has not been significantly improved in the last three decades, which might result from tumor recurrence, metastasis and radiosensitivity.Survivin, encoded by a single gene located on the human 17q25 chromosome (3%of the distance from the telomere), is the smallest member of the inhibitor of apoptosis protein (IAP) family with a molecular mass of 16.5 kD. Survivin functions to inhibit mitochondrial pathway of apoptosis through its single baculoviral IAP repeat (BIR) domain. On the other hand, survivin is expressed in the G2/M phase of the cell cycle to support the rapidly dividing cell machinery, leading to the suggestions by some investigators that the primary function of surviving lies in controlling cell division, rather than in inhibiting apoptosis. Except for the testis, thymus, and placenta, survivin is usually expressed at extremely low level in most normal adult tissues. In contrast, survivin is widely expressed during fetal development and is found in most human carcinomas, including LSCC. In recent years, large study shown that survivin is closely related to the apoptosis of tumor cells, but the related function of survivin in the radiationtherapy of LSCC was not reported. Mechanism research on the role of Survivin in LSCC, we can build a solid foundation for clinical study.Objective:Aim of this study was to analyze the influence of cell proliferation and apoptosis through the combine shRNA-Survivin with radiation dealing with the cells of Laryngeal squamous cell carcinoma, and to provide a strategy to therapy and prevention of Laryngeal squamous cell carcinoma.Methods:First, shRNA sequence used for slient gene were inserted into plasmid vector pGCsi-Hl containing GFP in this study. The resulting plasmids were named pGCsi-Hl-shRNA and-control, respectively. Then, MTS cell proliferation assay were used to analysis the proliferation of Hep-2 cells after the radiation dose (2Gy,4Gy, 8Gy,12Gy) treatment. We examined the effect of surviving knockdown in radiosensitivity and proliferation of Hep-2 cells under the radiation dose (2Gy,4Gy, 8Gy,12Gy) treatment, and each dose was cultured 24,48, and 72 h, respectively. Next, flow cytometry analysis was performed to explore the effect of survivin knockdown on cell cycle distribution and apotosis change after the cells subjected to 8 Gy radiation at 24 h. Finally, investigate the tumor growth of tumor-bearing mice which irradiated with 8 Gy of IR after Survivin slience. The tumor volume was compared.Result:Our results showed that Survivin mRNA and protein expression significant reduced in Hep-2 cells transfected with Survivin-shRNA by western blot and qRT-PCR analysis (P<0.01); Before transfection, MTS cell proliferation assay showed that the proliferation of Hep-2 cells was inhibited in dose-dependent-time manner after the radiation dose (2Gy,4Gy,8Gy,12Gy) treatment. After surviving knockdown, Hep-2 cells were treated with radiation (2Gy,4Gy,8Gy,12Gy), and each dose was cultured 24,48, and 72 h, respectively. The result was that cell viability was significantly reduced in group of surviving knockdown compared with contronl group. Next, flow cytometry analysis was performed after the cells subjected to 8 Gy radiation at 24 h.As shown in Figure 4,radiation treatment induced a significant cell arrest in G2/M phase at 24 hours compared with Hep-2 cells that did not received radiation(P<0.05). On the other hand surviving knockdown cause a marked cell cycle at Gl phase compared with parental Hep-2 cells(P>0.05).When radiation was given to the Hep-2 cells transfected with shRNA-survivin,the radiation-induced accumulation of cells at G2 phase was partially abrogated(P<0.01 VS. control).In contrast,Hep-2 cells transfected with scramble control shRNA did not cause change in Hep-2 cells before and after radiation treatment.As shown in Figure 5,transfection with shRNA-survivin significantly increased the population of apoptotic cells.In the tumor-bearing mice subjected to 8 Gy radiation at 24 h, shRNA surviving combined with irradiation caused a significant inhibition of tumor growth compared with shRNA alone in Hep-2 xenografts.Conclusion:1) The proliferation of Hep-2 cells of was inhibited by radiation treatment in a dose-dependent-time manner; 2) Survivin gene shRNA enhances the radiosensitivity of Hep-2 cells, and cell viability was significantly reduced in group of survivin-knockdown compared with contronl group after treated with radiation.3) When radiation was given to the Hep-2 cells transfected with shRNA-survivin,the radiation-induced accumulation of cells at G2 phase was partially abrogated(P<0.01 VS. control).Surviving knockdown group showed a significant cell arrest in Gl phase (P>0.05)and the percentage of apoptosis increased compared with control group of Hep-2 cells subjected to radiation (P<0.01); 4) shRNA surviving tumor-bearing mice combined with radiation showed a significant inhibition of tumor growth compared with shRNA alone in Hep-2 xenografts.