The Study Of Specific And Not Specfic Inhibition Of K-RAS Expression By Short Hairpin RNA In Human Pancreatic Cancer Cell Line

Pancreatic carcinoma is one of the high malignant tumors that threaten human health,Its high malignancy and difficulty in early diagnosis lead to poor prognosis,The survival rate of five years is poor than 5%.Except for early operation, there is not other effectiven treat measures. And because early diagnose is difficult,only 14% of the patients can be treated by operation. In recent years, the incidence of pancreatic carcinoma has been on the rise with a 3~7-fold increase reportedly, both at home and abroad, within the past decade. According to statistics by Hrudan et al In 1991, pancreatic carcinoma was the fourth leading cause of death in the United States, preceded only by lung cancer, colon cancer, and breast cancer. In China, accompanied with improvement in quality of life and change of diets, the incidence of pancreatic carcinoma has increased from No. 20 in 1980 to No. 5 in mortality. Based on the statistics by Center for Disease Control in Shanghai in 2000, there were 6/100000 new patients per year, with a morbidity of 5.5 that was almost equaled to mortality.In tumorigenesis and progression of pancreatic cancer. K-ras mutation might be the most important factor. In the patients with pancreatic carcinoma, about 80% of their genes have mutations, and most of these mutations are found to be located in codon 12 of exon one. GAT(46%),GTT(32%) and CGT(13%) are the most common form of K-ras mutation at codon 12.Research has shown that restraining the expression of K-ras mutation can restrain the progression of pancreatic cancer cells effectively.RNA interference is a new technology found in recent years to restrain gene expression in recent years, and because of the idiosyncracy and high performance, it becomes the focus in tumor research field. Basing on choppy K-ras gene order, this recearch will build shRNA plasmid carrier:pGenesil-1-P1 and pGenesil-1-M2 Which aim at the idiosyncracy of tow kinds of pancreatic cancer cells. And to observe the treatment of RNA interference for pancreatice carcinoma of K-ras mutation and to validate that if two kinds of nonspecific shRNA order have crossed restraining effect on pancreatic cancer cells,and find a new way for gene theraty of pancreatic cancer.Aim:To investigate the inhibition efficacy of the growth of pancreatic carcinoma cell lines after K-ras shRNA transduction by plasmid and the effect of two shRNAMethord:1 To structure the specific recombination plasmids pGenesil-1-P1 pGenesil-1-M2 for human pancreastic cancer and negative plasmids-----pGenesil-1-HK.2 Culture pancreatic carcinoma cell line. and transduce pGenesil-1-P1,Genesil-1-M2 and Genesil-1-HK into pancreatic carcinoma cell line,3 Etracted the total RNA and protein use the RT-PCR and westernblotting methods to detective the effective of RNAi for pancreatic carcinoma which the k-ras gene mutation4 The cell growth activities were estimated by CCK-8 cell proliferation assay.RESULTS:Order-checking proved the sequence of plasmid was right, the transfection of plasmid was successfully through fluorescence microscope. Results of RT-PCR revealed that cells treated with specific shRNA transfection had lower transcription of K-ras mRNA as compared with the notspecific and control, Western blots indicated a decreased expression, as compared with the notspecific and controls of K-ras protein in the cells transducted by shRNA. Specific transfection plasmid exhibits an anti- proliferative effect in cell proliferation assay.Conclusions:After transfect into the pancreatic cancer cell line , The specific shRNA recombination plasmids can inhibit the mutant k-ras gene mRNA and protein expression, exhibits cell proliferation.but the notspecific transfection can not.