A DNA Vector-based RNA Interference Technology To Suppress Tumor Cell Proliferation

Backgrounds: Cyclin-dependent kinase 2(CDK2), a member of cyclin protein kinases, plays an important role in the eukaryotic cell cycle by controling the systhesis of DNA at the phase of S in cell cycle. Tumor growth can be controlled through inhibiting tumor cell proliferation induced by down-regulating the expression of CDK2. Cervical cancer and Osteosarcoma are the most common maligant tumors in the world. It was found that the expression of cdk2 gene was extremely high both in Hela cells and in U-2 OS cells. RNA interference (RNAi) is a process to inhibit specific gene expression via the degradation of the target mRNA induced by the double strand RNA. Therefore, it is very useful to apply RNAi in suppressing cdk2 expression in cervical cancer cells and osteosarcoma cells, and it will provide a new approach for the treatment of cervical cancer and osteosarcoma. The present study was designed to construct an RNAi plasmid which can effectively suppress cdk2 expression in cervical cancer cells and osteosarcoma cells. The effects of DNA vector-based RNAi on cell proliferation were evaluated. This will provide a new approach for cervical cancer and osteosarcoma gene therapies.Methods: (1) According to the coding sequence of cdk2 gene and the principles of shRNA design, four pairs of sense and antisense oligonucleotides which code different shRNA were synthesized. Then the different annealed double-stranded DNA fragments were inserted into the BS/U6 vectors and the precise sequences of the RNAi plamids were confirmed by sequencing. (2) The transfections of RNAi plasmids into Hela and U-2 OS cells were performed using Lipofectamine 2,000 according to the manufacturer's protocol. The parent plasmid BS/U6 was used as a negative control. (3) To determine whether the RNAi plasmids could suppress the expression of cdk2, the Western blotting and RT-PCR were applied. (4) MTT and Flow Cytometry (FACS) were employed to examine the effects of RNAi plasmids on cell proliferation and cell cycle.Results: (1) These four RNAi plasmids for shRNA-cdk2 were successfully generated after the precise sequences of them were confirmed by sequencing. (2) At mRNA level, compared to the cells transfected with the control vector after three days post-transfection, the cdk2 mRNA levels were significantly decreased both in Hela and U-2 OS cells (73.1%, 86.1%, 88.4% and 77.1% in Hela cells; 76.8%, 84.3%, 84.6%, and 75.8% in U-2 OS cells respectively). (3) At protein level, compared to the cells transfected with the control vector after one day post-transfection, the CDK2 protein levels were also markedly down-regulated in these two tumor cell lines ( 68.2%, 72.7%, 77.3% and 41% in Hela cells; 37.5%, 53.4%, 57.3% , and 33.3% in U-2 OS cells respectively). (4) The cell proliferation was dramatically suppressed by the RNAi plasmids through the MTT analyses. In detail, compared to the cells transfected with the control vector after 3 days post-transfection, the cells proliferation of different RNAi plamid treatments were decreased both in Hela and U-2 OS cells (55.2%,62.3%,61.9% and 45.8% in Hela cells; 34.3%,35.6%,34.9% , and 28.8% in U-2 OS cells respectively). (5) The DNA content analyses by using flow cytometry revealed that transfection of RNAi plasmids induced a significant increase in the percentage of cells at the G1 phase of the cell cycle. The percentage of cell at G1 phase increased by 6.88%, 7.99%, 9.17%, 6.25% in the Hela cells transfected with the four plasmids respectively while the percentage of cells at S phase reduced by 3.65%, 8.57%, 13.16%, 5.12% in them respectively. Meanwhile, the percentage of cells at G1 phase increased by 2.5%, 8.21%, 4.59%, 2.48% respectively and the percentage of cells in S phase in U-2 OS cells decreased by 2.12%, 9.46%, 6.44%, 2.11% respectively.Conclusions: (1) Our study has constructed successfully the siRNA expression plasmids which can effectively induce cdk2 expression in tumor cells. This will provide an experimental foundation for constructing the RNAi plasmids for other genes and a powerful tool to investigate the function of other interested genes. (2) The DNA vector-based RNAi plasmids which can inhibit the tumor cells proliferation will provide a new approach for future clinical treatments in cancers such as cervical cancer and osteosarcoma.