The Study Of The Effects On The Radiosensitivity Of Sw1990 After XIAP And Survivin Expression Being Stably Inhibited By RNAi

Objective:Pancreatic cancer is one of the most aggressive human tumors,with surgical resection is the only curative treatment, but less than 15% of patients are candidates for potentially curative resection. Even among those patients with curative resection(R0), survival rates after surgical resection remain poor, with less than 20% survive 5 years, the high incidence of tumor recurrence both locally and at distant sites,and the low resection rate make the adjuvant therapy a rational treatment.Over the years, radiotherapy receives more attention, thus in conjunction to surgery or chemotherapy, radiotherapy may achieve better clinical results. However, pancreatic cancer is striking resistance to radiotherapy and chemotherapy. So,it has become a hotspot in the field of pancreatic cancer therapy to seek a scientific and applicable way to enhance the curative effect and improve the prognosis of pancreatic cancer. XIAP and Survivin,both as the most potent one of inhibitor of apoptosis (IAP)families,up-regulated in many cacer tissues and cells, including pancreatic cancer,is thought as an important factor comtributed to carcinoma proliferation transformation and radioresistance.The main object of our study include①To evaluate the expression of XIAP and S(?)(?)vivin in pancreatic carcinoma cell lines and further investigate the relationship between their expression and the radioresistance.②To explore the possibility of XIAP or Survivin (and combined inhibition of XIAP and survivin) inhibited by Lentiviral vector-mediad RNA interference technique, and to investigate the sensitivity to radiation changed of pancreatic carcinoma cells Sw1990 after XIAP and/or survivin being inhibited.Methods:①Expression of XIAP and Survivin in 4 pancreatic carcinoma cell lines (Panc-1,Sw1990,Bxpc-3 and Miapaca-2) before or after exposure to sublethal dose (1 Gy) of X-irradiation was evaluated by quantitative real-time RT-PCR and Western-blot;Cell survival was determined using a clonogenic assay to evaluate radiosensitivity of cells; After being exposured to a lethal dose of X-irradiation,Enzymatic activity of Caspase-3/7 and percent cytotoxicity determined by trypan blue staining were measured in 3 pancreatic carcinoma cell lines(Panc-1,sw1990 and Miapaca-2) which pretreated with sublethal dose of X-irradiation or not.②The Lentiviral vector of SiRNA targeted against XIAP(LV-shRNA-XIAP-1, LV-shRNA-XIAP-2,LV-shRNA-XIAP-3) and Survivin (Lv-shRNA-Survivin-1,Lv-shRNA-Survivin-2,Lv-shRNA-Survivin-3) were constructed, then virus was packaged for transfecting Sw1990 cells and the inhibition rate of XIAP and Survivin was measured respectively; stable transfective clones was selected by puromycin (for XIAP-shRNA) or G418(for Survivin-shRNA),after then, in Sw1990 cell lines in which XIAP or survivin expression was stably inhibited,the influence on the cell proliferation, apoptosis and radiosensitivity was measured. Inoculated Sw1990,Sw1990-Xnc,Sw1990-X1 and Sw1990-Snc,Sw1990-S1 respectively in flank subcutaneous tissue of nude mice to establish xenograft models of human pancreatic carcinoma,observed the tumor growth status and compared the tumorigenesis ability of these cells.③Sw1990 was transfected by XIAP-and Survivin-shRNA Lentivirus simultaneously, after selected by puromycin and G418, the inhibition rate of XIAP and Survivin was measured by quantitative real-time RT-PCR and Western-blot. After then, in Sw1990 cell lines in which XIAP and Survivin expression was stably combined inhibited,the influence on the cell proliferation, apoptosis and radiosensitivity was evaluated and compared with the result of single gene silence strategy used in Sw1990 targeted against XIAP or survivin.Results:①XIAP and Survivin were simultaneously existed in 4 pancreatic carcinoma cell lines (Panc-1,Sw1990,Bxpc-3 and Miapaca-2), and we found an inverse relationship between XIAP and Survivin mRNA expression and radiosensitivity. PANC-1 cells, which had the highest XIAP and Survivin mRNA levels, was most resistant to X-irradiation; Sw1990 cells, which had the moderate XIAP and Survivin mRNA levels,had the modest sencitivity to X-irradiation;MIAPaCa-2 cells, which showed the least XIAP and Survivin mRNA expression, showed the most sensitivity to X-irradiation. The mRNA and protein expression of XIAP and Survivin increased significantly in all four cell lines (Panc-1,Sw1990,Bxpc-3 and Miapaca-2) by treatment with a sublethal dose of X-irradiation. Three cells (Panc-1,Sw 1990 and Miapaca-2),were subjected to sublethal doses of X-irradiation followed by a lethal dose, result showed that the total cytotoxicity was decreased significantly and the caspase-3/7 activivty was significantly suppressed.②Three lentiviral vector-Xiap-shRNA (Lv-X1,Lv-X2,Lv-X3)and three lentiviral vector-Survivin-shRNA(Lv-S1,Lv-S2,Lv-S3) were constructed success-fully, and relative high-titer lentivirus were gain. The results of transfection showed that lentivirus can transfect Sw1990 cells efficiently and stablely. The results of instantaneous transfection show that Lv-X1,Lv-X2,Lv-X-3 all could decrease the XIAP mRNA expression level more than 70%, while the protein expression level was decreased more than 95%(P<0.05);Lv-S1 could effectively decrease the Survivin mRNA and protein expression level to (73.5±1.85)% and (87.64±5.57) % respectively(P<0.05), while Lv-S2 and Lv-S3 couldn't inhibit Survivin in Sw1990 cells significantly(P>0.05).③Three pancreatic cancer cell line in which XIAP expression is stably suppressed was successfully set up(Sw1990-X1,X2,X3), the inhibition rate of XIAP mRNA was (81.79±6.59)%, (71.89±3.14)% and (73.67±0.36)% respectively, while the inhibition rate of XIAP protein was (98.54±0.82)%,(97.33±0.49)% and (97.62±0.40)% respectively(P<0.05)。 MTT showed that the cell number of X1 and X3 decreased significantly, and the radiation-induced Caspase-3/7 activity, total cytotoxicity and apoptotic index measurd by flow cytometry were increased significantly. With the rise of radiation dose, cell clone formation rate declined in all groups of cells and at the same dose of radiation, clone formation rate of X1 declined notably; the cell survival curve also showed a significant decrease of X1-Do and X1-SF2,were 1.32 and 21.4% respectively, and the radiation enhancement ratio of X1 was 1.65(a ratio of D0)(Multi-target click model).These data imply that the radiosensitivity of X1 was increased significantly.3 groups of xenograft models of human pancreatic carcinoma were established(Sw1990, Sw1990-Xnc, Sw1990-X1). compared with Xnc and Sw1990 group,the tumorigenesis time of X1 group delayed,tumor grew slowly,the tumor volume of X1 group was obviously less than that of Xnc and Sw1990 group at every checkpoint except the first one.At the terminal of observation,the tumor weight of X1 group was lower than that of Xnc group and Sw1990 group.The tumor growth inhibitive rate of X1 group is 44.14%.④A pancreatic cancer cell line in which survivin expression is stably suppressed was successfully set up(Sw1990-S1), the inhibition rate of Survivin mRNA and protein was (76.51±0.99)% and(49.32±2.20)% respectively, and the radiation-induced Caspase-3/7 activity, total cytotoxicity and apoptotic index measurd by flow cytometry were increased significantly(P<0.05). With the rise of radiation dose, cell clone formation rate declined in all groups of cells and at the same dose of radiation, clone formation rate of X1 declined notably; the cell survivalcurve also showed asignificant decrease of S1-D0 and S1-SF2,were 1.11 and 16.35% respectively, and the radiation enhancement ratios of S1 was 1.82(a ratio of D0)(Multi-target click model).These data imply that the radiosensitivity of S1 was increased significantl.3 groups of xenograft models of human pancreatic carcinoma Sw1990,Sw1990-Snc,Sw1990-S1) were established successfully. Compared with Snc and Sw1990 group,the tumorigenesis time of S1 group delayed dramaticly, tumor grew slowly, the tumor volume of S1 group was obviously less than that of Snc and Sw1990 group at every checkpoint.At the terminal of observation,the tumor weight of S1 group was significantly lower than that of Snc group and Sw1990 group.The tumor growth inhibitive rate of S1 group is 78.95%.⑤Sw1990 was transfected by XIAP-and Survivin-shRNA Lentivirus simultaneously, after selected by puromycin and G418, a pancreatic cancer cell line in which XIAP and Survivin expression is stably suppressed was successfully set up(Sw1990-X1S1), the inhibition rate of XIAP-and survivin mRNA was (56.53±3.38)% and (56.19±3.31)% respectively, while the data was(56.26±5.18)% and(30.72±8.16)% in protein level. AS expected, the radiation-induced Caspase-3/7 activity and total cytotoxicity were both increased significantly in X1S1 cells. When compared with X1 cells and S1 cells, the radiation-induced Caspase-3/7 activity increased significantly in X1S1 cells, but there is no significant difference on total cytotoxicity after radiation among the three cell linces. With the rise of radiation dose, cell clone formation rate declined in all groups of cells and at the same dose of radiation, clone formation rate of X1S1 declined notably, besides, when exposure to low-dose X-irradiation (2 and 3 Gy), the clonogenic survival of X1S1 cells was significantly less than S1 cells and X1 cells; the cell survival curve also showed a significant decrease of X1S1-Do and X1S1-SF2,were 0.90 and 10.64% respectively, and the radiation enhancement ratios of X1 was 2.26 (a ratio of D0)(Multi-target click model).These data imply that the radiosensitivity of X1S1 was increased significantly, and the radiosensitivity of X1S1 is better than that of X1 and S1.Conclusion:①XIAP and Survivin were simultaneously existed in 4 pancreatic carcinoma cell lines (Panc-1,Sw1990,Bxpc-3 and Miapaca-2),and there was an inverse relationship between XIAP and Survivin mRNA expression and radiosensitivity. XIAP and Survivin both act as a constitutive and inducible radioresistance factor in pancreatic cancer cells.②Stablely inhibit XIAP or Survivin expression in Sw1990 cell lines could significant inhibit its cell proliferation and long-term survival, weaken the ability of turmorigenesis and enhance the rediosensitivity significantly.③Combined Stable-inhibition of XIAP and Survivin expression could enhance rediosensitivity of Sw1990 more effective than single stable-inhibition of XIAP or survivin expression in pancreatic carcinoma cell lines Sw1990.