Construction of ShRNA-Bmi1 Recombinant Vector and Its Effect on The Expression of hTERT in Transplantated Tumor of Nude Mice with Gallbladder Carcinoma

［Abstract］Objective　To construct and identify shRNA-Bmi1 recombinant vector in vitro， and to explore the effects of target silencing proto oncogene Bmi1 on the Bmi1mRNA and protein expression in human gallbladder carcinoma cell and the expression of hTERT on subcutaneously transplantated tumor of human gallbladder carcinoma in BALB/c Nude Mice. Methods　Four shRNABmi1 recombinant plasmids were successfully constructed according to different Bmi1 sites. Transfection efficiency was observed by inverted fluorescence microscope and RT-PCR and Western blot were used to measure mRNA and protein expression of Bmi-1 in gallbladder cancer cells. The effective interference group was selected for the vivo experiment. The subcutaneous transplantation tumor model of human GBC-SD cell line was successfully constructed in BALB/c nude Mice and randomly divided into shRNA-Bmi-4 group（experimental group），shRNA-Scramble（mismatch group），Lipofectamine（negative control group），GBC-SD（blank control group）. After established the subcutaneous transplantation tumor model， the nude mice were killed after the treatment for 6 weeks. The expression level of hTERT protein in nude mice transplanted tumor was detected by immunohistochemistry. Results shRNA-Bmi1 recombinant vector was successfully constructed. The most effective interferencing plasmids were transfected into GBC-SD cells for 48 hour. The highest green fluorescence intensity and transfection efficiency of shRNA-Bmi1-4 group GBC-SD were observed by inverted fluorescence microscope. RT-PCR and Western blot results showed that the expression levels of Bmi1mRNA and protein in shRNA-Bmi1-4 group were significantly lower than that in the control groups （P < 0.05）， and there was no significant difference between control groups（P > 0.05）.The shRNA-Bmi1-4 group was selected as the effective interference sequence for the following in vivo experiments. The expression level of hTERT protein on transplantation tumor in nude mice in shRNA-Bmi-4 group was significantly lower than that in control group（P < 0.05）， and there was no significant difference between the control groups（P > 0.05）. Conclusions　shRNA-Bmi1 recombinant vector has been successfully constructed in vitro， and the transformation and transfection experiments of the recombinant vector have been carried out successfully. Targeted silencing Bmi1 gene can effectively promote Bmi1mRNA degradation，inhibit the Bmil protein expression in the GBC-SD cell line and the expression of hTERT protein in subcutaneously transplantated tumor of GBC-SD in Nude Mice.