Construction Of ShRNA Expression Vector With Dual-promoter And Its Application In RNA Interference To Inhibit Tumor Cells Proliferation

ObjectiveRNA interference(RNAi) is a gene silencing mechanism which is triggered by endogeneous or exogeneous double-stranded RNA. It has high specificity and has been widely used in the study of tumor therapy. However, expression vector in previous reports could only transcript to generate one specific small interfering RNA(siRNA) and scilence single gene expression, which is therefore difficult to treat polygenic diseases. We constructed shRNA expression vector with dual-promoter aimed at hTERT and Bcl-2 gene, which have high expression in most tumor cells. By detecting the change of biological phenotypes after this vector was transfected into Hela cells, we observed and studied the gene silencing effect and inhibition effect of this vector. Moreover, the modeling tool carrier could lay the foundations for future studies on RNA interference.Methods1. Eukaryotic expression vector pcDNA3.1(+) was modificated. The CMV promoter and its transcript terminator were cut; Complete gene expression cassettes and replication origin were reserved. After modification, dual-promoter, which were obtained by amplifing human genomic DNA were inserted into the vector.2. According to the gene sequences of hTERT and Bcl-2 and shRNA design principles, single stranded DNA (ssDNA) encoding shRNA were chemically synthesized. Corresponding ssDNA were then annealed and respectively connected to the downstream of the two U6 promoters.The dual-promoter shRNA vector pdPRO-TB was therefore constructed. Control plasmid pdPRO-T(shRNA recombinant plasmid targeted hTERT) and pdPRO-B (recombinant plasmid targeted Bcl-2),negative control plasmid pdPRO were constructed in the same way.3. The following experiments were divided into six groups:blank control group, empty liposome group, negative control group(pdPRO group),pdPRO-TB group, pdPRO-T group and pdPRO-B group. Plasmids in each group were transfected into Hela cells simultaneously and each group concluded 6 pores. The inhibitory effect of cell growth and proliferation were analyzed by MTT method. Results1. The results of sequencing and restrictive enzyme digestion confirmed that pdPRO-TB, pdPRO-T, pdPRO-B and pdPRO were all successfully constructed.2.48 hours after transfection, compared to normal Hela cells, suspension cells in pdPRO-TB group increased apparently and cell growth in this group also slowed. Similar changes occurred in pdPRO-T group and pdPRO-B group, however, not as obvious as pdPRO-TB group. Cells in negative control group had no change and the bottom of the cell culture plate was almost plastered by cells.3. The MTT result showed that time factor and group factor both had influence on cells inhibitor(CI)% (F=287.107,391.590, P<0.01)and there were interaction between time and group factor(F=41.592, P<0.01). CI% in pdPRO-TB group, pdPRO-T group and pdPRO-B group were apparently higher than in empty liposome group and negative control group, while comparison of CI% had no significant difference between empty liposome group and negative control group. The inhibitory effect appeared at 24h after transfection and reached peak at 48h, then it turned weak at 72 hours.ConclusionThe shRNA expression vector pdPRO-TB could inhibit Hela cell proliferation most significantly and the two control plasmids(pdPRO-T and pdPRO-B) also had inhibition effect on cell proliferaton. These phenomena implied siRNA which targeted to hTERT and Bcl-2 had synergistic effect on gene silencing and provided experimental evidence for studying the interaction of the two genes in promoting tumor proliferation. Besides, this modeling tool carrier suggested that the shRNA expression vector with dual promoter maybe a effective tool in treating tumor in a polygenic collaborative way.