The Experimental Study Of Growth Inhibition Induced By RNAi To Silence Survivin Gene In Human Pancreatic Cancer Patu 8988 Cell

AIM: Survivin is one of the newly discovered members of inhibition apoptosis protein family,and is the strongest discovered inhibitor of apoptosis so far. It highly express in pancreatic carcinoma and not express in normal tissue, and its expression are closely related to pancreatic cancer cell proliferation, apoptosis and angiogenesis. Reducing the expression of Survivin gene will help to inhibit normal cell proliferation and reverse tumor formation. RNA interference (RNAi) can effectively and specifically down-regulate targeted gene expression and has been widely applied to the study of tumor therapy. In this study, Survivin shRNA recombinant plasmid vectors were constructed and transfected human pancreatic cancer Patu 8988 cells. Influence of Survivin gene expression and growth inhibition of Patu 8988 cells induced by Survivin expression vectors were observed in vivo and in vitro to explore the role of Survivin gene in pancreatic cancer, which will provide an experimental foundation for the further investigation of pancreatic cancer gene therapy.METHODS:①Structure of Survivin mRNA was analysed by software to select targeted interference sites. Survivin shRNA recombinant plasmids named pGenesil-1-Survivin-1, pGenesil-1-Survivin-2, pGenesil-1-Survivin-1+2 and negative recombinant plasmid named pGenesil-1-NC were constructed.②The following experiments were divided into five groups: blank control group, pGenesil-1-Survivin-1 group, pGenesil-1-Survivin-2 group, pGenesil-1-Survivin-1+2 group and pGenesil-1-NC group.③At 48h, cell apoptosis was detected and relative amount of Survivin mRNA and Survivin protein were estimated by using RT-PCR and FCM to observe Survivin gene inhibition effect of Patu 8988 cells transiently transfected with respective recombinant plasmid.④Cell proliferation capacity was established by MTT, and expression of Survivin protein was estimated by Western-blotting.⑤Stable Patu 8988 cells transfected with respective recombinant plasmid were obtained by G418 selection, models of nude mice bearing pancreatic cancer were established by injecting BALB/c mice with respective stable transfection Patu 8988 cells. To study the growth inhibition of transplanted pancreatic cancer tumor induced by Survivin shRNA, tumorigenic time, tumor size and tumor weight were observed, Survivin protein expression was detected by using immunohistochemistry and cell ultra structure was observed under transmission electron microscope.RESULTS:①The results of restrictive enzyme digestion and sequencing confirmed that recombinant plasmids of pGenesil-1-Survivin-1, pGenesil-1-Survivin-2, pGenesil-1-Survivin-1+2 and pGenesil-1-NC were successfully constructed.②FCM results showed that the percentage of transfection fluorescence cells was above 60% in each group. The transient experimental results at 48h indicated that the inhibition rate of Survivin mRNA on Patu 8988 cells was 65.62%,54.89%,64.96% respectively in pGenesil-1-Survivin-1 group, pGenesil-1-Survivin-2 group and pGenesil-1-Survivin-1+2 group, compared with the control group, statistically significant differences were observed (vs control, p<0.05). FCM showed that the apoptotic index was very low and no difference in each group.③The comparison results of cell proliferation ability between each groups indicated that the transfected cell proliferation ability of pGenesil-1-Survivin-1 group and pGenesil-1-Survivin-1+2 group was decreased obviously, however, transfected cell proliferation ability of pGenesil-1-Survivin-2 group was decreased slowly which weaker than the first two groups. The inhibition rate of Survivin protein detected by FCM was 64.61%,59.49% and 32.68% respectively(vs control, p<0.05) , and the result of Western-blotting corresponded to that of FCM.④The average tumorigenic time was slightly delayed respectively in pGenesil-1-Survivin-1 group and pGenesil-1-Survivin-1+2 group, statistically no differences were observed (vs control, p>0.05), and the average tumor sizes and weights significantly decreased respectively in pGenesil-1-Survivin-1 group, pGenesil-1-Survivin-2 group and pGenesil-1-Survivin-1+2 group. The immunohistochemistry results indicated that large flake necrosis in tumor tissue which effected the expression of Survivin protein. A little proportion of cell apoptosis was found under transmission electron microscope in all the interfering groups, which corresponded to cell apoptotic results detected by FCM.⑤The results also showed that the interfering effect of pGenesil-1-Survivin-1 against Survivin gene was the best and the inhibition effect of tumor cells'proliferation in pGenesil-1-Survivin-1 group was also the most obvious, which superior to pGenesil-1-Survivin-1+2, the interfering effect of pGenesil-1-Survivin-2 group was the worst. No obvious effect of interfering Survivin gene was observed between pGenesil-1-NC group and control group, and also no significant effect of Patu 8988 cell proliferation was found in these groups.CONCLUSION: Survivin shRNA recombinant plasmid vectors named Genesil-1- Survivin-1, Genesil-1-Survivin-2, Genesil-1-Survivin-1+2 and pGenesil-1-NC were successfully constructed. The three interfering recombinant plasmids could interfere the Survivin gene expression of Patu 8988 cells and inhibit the proliferation of pancreatic cancer Patu 8988 cells in vitro and in vivo. 5'–GCATTCGTCCGGTTGCGCT-3'and 5'-GACTTCATAAGGCGCATGC-3'could be used as the RNAi targets specific to Survivin gene in pancreatic cancer Patu 8988 cells, and the former obviously superior to the latter.