The Effect Of RNAi Technology Ihibiting C-Met Gene And PI3K/AKT Signaling Pathway On Gastric Cancer MKN-45Cells

BackgroundsThe gastric cancer is one of the most common malignant gastrointestinal tumors in the world. Surgical resection、chemotherapy and radiotherapy are the main treatment of gastric cancer. However, the clinical effect is still unsatisfactory. Seeking the new treatment of gastric cancer especially gene therapy is imperative.The study reveals that the expression of c-Met gene in gastric cancer is high. The combination of c-Met protein and its ligand hepatocyte growth factor(HGF) which activates downstream signaling pathway of PI3K/Akt strongly stimulates turner cell proliferation、invasion、metastasis and angiogenesis. RNAi technology can specificly silence the target gene mRNA. Now it has become the hotspot of gene therapy of cancer.In this experiment, we used RNAi technology to silence c-Met oncogene of gastric cancer MKN-45cells, observed c-Met and downstream PI3K/Akt signaling molecules expression of gastric cancer MKN-45cells before and after transfection from the two levels of gene and protein, providing the theoretical basis for gene therapy of gastric cancer.Materials and methods1、Human gastric cancer MKN-45cells were donated by the Xiangya Hospital pathological Laboratory of the Central South University. Culativated the cells with the DMEM complete culture media containing10%fetal bovine serum in the37℃,80%moisture,5%CO2, saturated humidity incubator. According to the c-Met cDNA sequence to designed and synthesized the sequence of3target gene by Shanghai Jikai company.2、Transfected the three c-Met-siRNAs into the Human stomach carcinoma cell MKN-45mediated by lipofectamineTM2000.3、48h after the transfection, detected the expression of c-Met and downstream signaling molecules by RT-PCR and Western-blot from two levels of gene and protein. 4、Used SPSS17.0to analyze the results, the cutoff for statistical significance was set as P<0.05.Results1、The RT-PCR show that the expression level of c-Met gene is obviously lower than the control group (P<0.05), however the expression level of PI3k and AKT gene is not obviously lower than the control group (P>0.05)2、The Western blot show that the expression level of c-Met、p-PI3k and p-AKT protein is obviously lower than the control group (P<0.05), however the expression level of PI3k and AKT protein is not obviously lower than the control group (P>0.05)Conclusion1、The three c-Met-siRNAs could successfully silence the c-Met gene of stomach carcinoma cell MKN-45, the c-Met mRNA and protein expression were inhibited greatly after transfection.2、After silencing the c-Met gene, the expression level of phosphorylated molecules of the PI3k/AKT downstream signaling pathway were greatly inhibited, but the silence of c-Met gene showed no significant impact on non-phosphorylated signaling molecules.