The Experimental Research Of Cross-inhibition Of K-RAS By Vector-based RNA Interference In Human Pancreatic Cancer Cell Lines

Objective: To construct plasmids vector expressing short hairpin RNA(shRNA) to understand the possibility of cross-inhibition of K-ras gene with different kinds of mutation by vector-based RNAi in human pancreatic cancer cells PANC-1 and CFPAC-1. And to establish certain theoretic groundwork for the use of RNAi technique in the treatment of pancreatic cancer.Methods: To silence the expression of two kinds of K-ras gene mutation, GAT and GTT, we synthesized 2 shRNA sequences and colon these two sequences into pGenesil-1,to construct 2 recombinant K-ras shRNA plasmids, pGenesil-1GAT and pGenesil-1GTT. These two kinds of plasmids were transfected into the human pancreatic cancer cells PANC-1, with K-ras mutation of GAT, and the human pancreatic cancer cells CFPAC-1, with K-ras mutation of GTT. Each kind of cells was divided into 4 groups (special interference group, cross interference group, empty plasmid transfected group and control group). In the special interference group, cells were transfected with the recombinant shRNA plasmid, which was designed for the same mutation of K-ras gene as the cells. In the cross interference group, cells were transfected with the recombinant shRNA plasmid, which was not designed for the same mutation of K-ras gene as the cells. In the empty plasmid transfected group, the cells were transfected with the empty plasmid pGenesil-1KB, and the cells were transfected with the PBS in the control group. RT-PCR and Western blot were used to identify K-ras mRNA and protein expression. And the cell proliferation was analyzed by employing CCK-8 methods. In this way, we detected he interferential effect of the plasmid pGenesil-1GAT in PANC-1 and the plasmid pGenesil-1GTT in CFPAC-1, and the cross-interferential effect of the plasmid pGenesil-1GTT in PANC-1 and the plasmid pGenesil-1GAT in CFPAC-1.Results: 1. Plasmids expressing specific shRNA targeting K-ras gene were constructed successfully,and were certified through agarose gel electrophoresis and gene sequencing. 2. The recombinant plasmids were efficiently transfected into all human pancreatic cancer cells. K-ras mRNA and protein expression were significantly inhibited by mutation-specific shRNA in PANC-1 cells, transfected with pGenesil-1GAT (special interference group), and showed a reduced proliferation after the transfection compared with the empty plasmid group and control group (p<0.05), while the mRNA and protein expression of the PANC-1 cells, transfacted with pGenesil-1GTT (cross interference group), were not inhibited, and the cells proliferated as well as empty plasmid transfected group and control group(p>0.05). Similarly, in CFPAC-1 cells, which was transfected with pGenesil-1GTT(special interference group), the K-ras mRNA and protein expression were reduced sharply and the proliferation of the cells decreased compared with the empty plasmid group and control group(P<0.05), while the K-ras gene of CFPAC-1 cells, transfected with pGenesil-1GAT (cross interference group), was not blocked, and the cells proliferation did not decrease compared with the empty plasmid group and control group(p>0.05).Conclusions: 1. The shRNA sequence targeting K-ras can be inserted into the pGenesil-1 plasmid, and the recombinant plasmids expressing specific shRNA can be transfected into the human pancreatic cancer cell line PANC-1 and CFPAC-1 successfully. 2. Mutation-specific shRNA can specifically inhibit the relevant mutant K-ras genes, and the proliferation of the cells was decreases. So K-ras gene is very important for the cancer cells proliferation, especially in the pancreatic cancer cells. Therefore, it is feasible to treat pancreatic cancer by using RNAi targeting the mutated K-ras gene. 3. There is Even though there was only one nucleic acid different between the 2 K-ras gene mutation, there is no effective cross-silence between each other in present research. This indicated that the plasmid-based RNA interference significantly inhibit the expression of the target gene. And there is no cross-silence in RNAi, which provides more data for the application of RNAi in the treatment of pancreatic cancer.