Effect Of Specific SiRNA Targeting Against C-Src Gene In Pancreatic Carcinoma Cells

Pancreatic adenocarcinoma is the fourth most common cause of cancer death.Metastasis to the lymphatics, liver, and vessel walls leads to widespread disease, resulting in a severe wasting condition that accounts for deaths in advanced pancreatic cancer. Clinically chemoresistance is one of the major causes for chemotherapeutic treatment failure in pancreatic adenocarcinoma patients. Standard chemotherapeutic agents only have marginal effect on patient survival. Because of the high mortality associated with pancreatic adenocarcinoma, it is essential that therapeutic regimens be developed to inhibit tumor growth, increase chemosensitivity of chemotherapeutics, and restrain angiogenesis of tumors.The progression of pancreatic adenocarcinoma has been associated with deregulation of several signaling molecules.One of the potential therapeutic targets receiving considerable recent attention is activation of c-Src, a nonreceptor protein tyrosine kinase. c-Src regulate diverse biological processes by associating with multiple signaling and structural molecules. Overexpression of c-Src occurs in many solid tumors, often at later stages of disease, and can be predictive of poor prognosis.RNA interference(RNAi)was a kind of gene silence post transcription. It could trigger monitor program post transcription, and induce definete single strand mRNA degradation. The main functionary mechanism was that dsRNA catalyzed into 21-23nt small fragment by nuclease, then break down corresponding mRNA with the same sequence in definite site and definite space. At present, it was successfully used in the research of gene function and the correlation of upper and downstream moleucle from singal transmission system, and it might provide a new trategy for tumor therapy. RNAi might bring new breakthrough for functional genomics, gene therapy and most other territory.Objective: To project and construct definite RNA interfere plasmid vector targeted to c-Src mRNA, to construct stable transfection cell lines,to investigate the interfere efficiency of c-Src in the cell lines and the effect on the tumor character, and to investigate the feasiability that RNAi technology prevented and treated the pancreatic adenocarcinoma.Methods: SiRNAs(small interfering RNA, siRNA) for c-Src gene was designed and synthesized, then be transfected into pancreatic adenocarcinoma cells line PANC-1 by positive ion liposome LipofectamineTM 2000. Transfect efficiency was assessed by flow cytometry. Use RQ-PCR(Real-time Quantitative Polymerase Chain Reaction) to detect the expression of c-Src andβ-actin mRNA, and then screening the most effective siRNA. Expression of c-Src protein was detected by western blot. Two single strand DNA templates were designed according to the sequence of siRNA which has confirmed to be effective to knockdown c-Src gene. Make the blank plasmid linearization by use of restriction enzyme, and then the insertion element was inserted into the blank plasmid by T4 ligase. Enzyme cutting, and sequencing was performed to check whether c-Src siRNA plasmid be constructed successfully or not. Transfect PANC-1 cells with c-Src siRNA plasmid and negative control plasmid by LipofectamineTM 2000 respectively. As a reporter gene, Green Fluorescence Protein(GFP) was evaluated by flow cytometry and fluorescent microscopy to estimate the transfection efficiencies and expression efficiencies when the positive cell clones were selected with G418 and after the transfected monoclone cells were choosed. To verify the effect of suppressing c-Src expression on PANC-1 cells gemcitabine chemoresistance by MTT and FCM , to determine VEGF levels of culture supernatants by ELISA. In a nude mouse model, tumor growth of transfected cells were studied, c-Src expression and microvessel density in tumor tissue were measured by immunohistochemistry.Results:①The transfection rate of liposome used in PANC-1 cell line is 96.3%,the most effective siRNA could suppress expression of c-Src gene and the suppression rate is 86.1%.②Enzyme cutting and sequencing confirmed that the c-Src siRNA plasmid is successfully constructed.③PANC-1 cells were transfected with pNRNA/pSrcs and grown in selective media containing G418, expression of phosphorylated Akt were decreased in pSrcs transfected cells(p<0.05).④MTT cytotoxicity assay and FCM show: gemcitabine-induced apoptosis was markedly increased after transfection of c-Src specific siRNA plasmid(p<0.05).⑤Expression levels of VEGF were reduced significantly(p<0.05) relative to control and Blank.⑥In mice injected transfected cells, c-Src expression and MVD is decreased in tumors produced from c-Src siRNA plasmid transfected cells (p<0.05).Conclusion: Using pGPU6/GFP/Neo plasmid, we successfully designed and constructed specific RNA interference plasmid vector aimed c-Src gene mRNA. And we transfected the PANC-1 cells stably with c-Src siRNA plasmid. In vitro and vivo, pSrcs down-regulate c-Src gene and phosphorylated Akt in PANC-1 cell line and enhance gemcitabine chemosensitivity, c-Src gene silencing could inhibit angiogenesis of tumors. Together with the results presented here, these data suggest the possibility that c-Src represents an important candidate for targeted therapy in pancreatic cancer.