The Experimental Study Of Growth Inhibition Induced By ShRNA To Silence Survivin Gene In Human Nasopharyngeal Carcinoma CNE2 Cell

AIM: Survivin shRNA recombinant plasmid vector was constructed and transfected into human nasopharyngeal carcinoma CNE2 cells to observe the influence of survivin gene/protein expression, cell apoptosis, cell proliferation and radiation sensibility of CNE2 cells in vivo and in vitro, which provides an experimental foundation for the further clinical nasopharyngeal carcinoma gene therapy.METHODS:①According to the cDNA sequences of survivin, shRNA survivin recombinant plasmid pGenesil-1-survivin and pGenesil-1-HK were constructed, after sequencing and enzyme digestion identification, recombinant plasmids were extract ed and transfected into CNE2 cells, respectively;②The following experiments in vivo and in vitro were divided into three groups: blank control group (CNE for abbreviation), pGenesil-1-HK group (HK for abbreviation) and pGenesil-1-survivin group (SURVIVIN for abbreviation);③At 48h, cell apoptosis and relative amount of survivin mRNA were detected by FCM and RT-PCR respectively to observe the inhibition effect of survivin gene in CNE2 cells transiently transfected with interfering recombinant plasmid;④Stable CNE2 cells transfected with respective recombinant plasmid were obtained by G418 selection. Expression of survivin mRNA and protein was estimated by using RT-PCR, FCM and Western-blotting. Cell proliferation ability was evaluated by MTT experiment and radiation sensibility was observed by Cell Clone experiment;⑤Models of nude mice bearing cancer were established by using BALB/c nude mice inoculated with respective stable transfection CNE2 cells. To study the growth inhibition of transplanted human nasopharyngeal carcinoma induced by survivin shRNA, tumorigenic time, tumor size and tumor weight were observed, and survivin protein expression was detected by using immunohistochemistry .RESULTS:①The results of sequencing and restrictive enzyme digestion confirmed that recombinant plasmid expression vectors of pGenesil-1-survivin and pGenesil-1-HK were successfully constructed.②The apoptotic index after 48 h post-transfection was (3.57±0.34)%, (3.77±0.33)%, (6.33±0.37)% respectively in blank control,pGenesil-1-HK and pGenesil-1-survivin groups, and the apoptotic index was 1.78 times in pGenesil-1-survivin group as much as that in blank control group; The inhibition rate of survivin mRNA in pGenesil-1-survivin group was 63.53%, statistically significant difference was observed when compared with the blank control group (P<0.05). However, no statistical difference was observed between pGenesil-1-HK and blank control groups (P>0.05);③Stable CNE2 cells transfected with various recombinant plasmids were selected with G418. FCM results showed that the percentage of stable transfection cells was above 85% in each group. The inhibition rate of survivin mRNA was 64.55% in pGenesil-1-survivin group, when compared with the blank control group and pGenesil-1-HK group, statistically significant differences were observed (P<0.05). The inhibition rate of survivin protein detected by FCM and Western-blotting was 50.29% and 51.24% respectively and two results were almost consistent. When compared with the blank control group and pGenesil-1-HK group, statistically significant differences were seen (P<0.05);④The comparison results of cell proliferation rate between each group indicated that the proliferation ability of stably transfected cells was inhibited obviously;⑤The results of colony forming experiment certified that the radiosensitivity of CNE2 cells could be increased after survivin shRNA making survivin gene silence;⑥Compared with the blank control group and pGenesil-1-HK group, the average tumorigenic time was significantly delayed, the average tumor sizes and weights both decreased significantly in pGenesil-1-survivin group(P<0.05); The immunohistochemical results further confirmed the survivin protein expression was significantly reduced in the pGenesil-1-survivin group.CONCLUSION: Survivin shRNA recombinant plasmid vectors named pGenesil-1-survivin was successfully constructed; Recombinant plasmid pGenesil-1-survivin could interfere the survivn gene expression of human nasopharyngeal carcinoma CNE2 cells and inhibit the proliferation of CNE2 cells in vitro and in vivo, at the same time, it could increase radiosensitivity of CNE2 cells. 5'-GGACCACCGCATCTCTACA-3'could be used as the RNAi targets specific to survivin gene in human nasopharyngeal carcinoma CNE2 cells.