The Experimental Study Of Silencing Bcl-2 Gene And Inhibiting B Cell Lymphoma Growth By ShRNA Expression Vector

AIM: There is a close relationship between B cell lymphoma and high expression of Bcl-2 gene. Reducing the expression of Bcl-2 gene will help to restore normal cell apoptosis and reverse tumor formation. RNA interference (RNAi) can effectively and specifically down-regulate targeted gene expression and has been applied to the study of tumor therapy. In this study, Bcl-2 shRNA recombinant plasmid vectors were constructed and transfected B cell lymphoma Raji cells. Influence of Bcl-2 gene expression and growth inhibition of Raji cells induced by Bcl-2 expression vectors was observed in vivo and in vitro, which will provide an experimental foundation for the further investigation of B cell lymphoma gene therapy.METHODS:â—‹1 Structure of Bcl-2 mRNA was analyzed by software to select targeted interference sites. Bcl-2 shRNA recombinant plasmids named pGenesil-1-Bcl-2-544 and pGenesil-1-Bcl-2-1009 and negative recombinant plasmid named pGenesil-1-NC were constructed. After sequencing and restrictive enzyme digestion identification, recombinant plasmids were extracted for Raji cells'transfection.â—‹2 The following experiments were divided into four groups: blank control group, pGenesil-1-Bcl-2-544 group, pGenesil-1-Bcl-2-1009 group and pGenesil-1-NC group.â—‹3 At 48h, cell apoptosis and relative amount of Bcl-2 mRNA were detected to observe Bcl-2 gene inhibition effect of Raji cells transiently transfected with respective interfering recombinant plasmid.â—‹4 Stable Raji cells transfected with respective recombinant plasmid were obtained by G418 selection. Expression of Bcl-2 mRNA and protein was estimated by using RT-PCR, FCM and Western-blotting and cell proliferation ability was evaluated by cell count.â—‹5 Models of nude mice bearing B cell lymphoma were established by injecting BALB/c mice with respective stable transfection Raji cells. To study the growth inhibition of transplanted B cell lymphoma tumor induced by Bcl-2 shRNA, tumorigenic time, tumor size and tumor weight were observed, Bcl-2 protein expression was detected by using immunohistochemistry and cell ultra structure was observed under transmission electron microscope.RESULTS:â—‹1 The results of sequencing and restrictive enzyme digestion confirmed that recombinant plasmids of pGenesil-1-Bcl-2-544, pGenesil-1-Bcl-2-1009 and pGenesil-1- NC were successfully constructed.â—‹2 The transient experimental results at 48 h indicated that the apoptotic index was 4.70 and 3.78 times in pGenesil-1-Bcl-544 group and pGenesil-1-1009 group respectively when compared with blank control group (p< 0.05) and the inhibition rate of Bcl-2 gene on Raji cells was 31.91% and 47.61% respectively compared that of control group (p<0.05).â—‹3 Stable Raji cells transfected with various recombinant plasmids were selected with G418. FCM results showed that the percentage of stable transfection cells was above 97% in each group. The inhibition rate of Bcl-2 mRNA was 72.71% and 45.50% in pGenesil-1-Bcl-2-54 group and pGenesil-1-Bcl-2-1009 group respectively. Compared with the control group, statistically significant differences were observed (vs control, p<0.05). The inhibition rate of Bcl-2 protein detected by FCM was 73.37% and 59.67% respectively (vs control, p<0.05), and the result of Western blotting corresponded to that of FCM. The Comparison results of cell proliferation rate between each groups indicated that the proliferation ability of stably transfected cells was decreased obviously.â—‹4 The average tumorigenic time was significantly delayed respectively in pGenesil-1-Bcl-2-544 group and pGenesil-1-Bcl-2-1009 group, and the average tumor sizes and weights also significantly decreased respectively. The immunohistochemical results further confirmed the Bcl-2 protein expression was significantly reduced in the two interfering groups. A certain proportion of cell apoptosis was found under transmission electron microscope in the two interfering groups.â—‹5 The results also showed that the interfering effect of pGenesil-1-Bcl-2-544 against Bcl-2 gene was significantly superior to pGenesil-1-Bcl-2-1009. The inhibition effect of tumor cells' proliferation in pGenesil-1-Bcl-2-544 group was also more obvious than that of Genesil-1-Bcl-2-1009 group. No obvious effect of interfering Bcl-2 gene was observed between pGenesil-1-NC group and control group, and also no significant effect of Raji cell proliferation was found in these groups.CONCLUSION: Bcl-2 shRNA recombinant plasmid vectors named pGenesil-1-Bcl-2-544 and pGenesil-1-Bcl-2-1009 were successfully constructed. The two interfering recombinant plasmids could interfer the Bcl-2 gene expression of Raji cells and inhibit the proliferation of B-cell lymphoma Raji cells in vitro and in vivo. 5'-GTACATCCATTATAAGCTG-3' and 5'-CATCGCCCTGTGGATGACT-3' could be used as the RNAi targets specific to Bcl-2 gene in B-cell lymphoma Raji cells.