【Abstract】ObjectiveTo investigate the effects of endogenous photodynamic therapy (PDT) on intracellular cAMP and cGMP concentrations of human colon carcinoma cell lines SW480. MethodsSW480 cells were divided into control group, light group, δaminolevulinic acid （ALA） group (ALA group) and endogenous PDT group (ALAPDT group). Intracellular cAMP and cGMP concentrations of each group were detected by radioimmunoassay at 30, 60, 90 and 120 min after irradiation. ResultsThere was a significant increase in intracellular cAMP concentration of ALAPDT group at 30 min after irradiation (P＜0.001) and sequent decrease, but intracellular cAMP concentrations of ALAPDT group at 60, 90 and 120 min after irradiation had no statistical difference than the other groups (Pgt;0.05). Intracellular cGMP concentration of different time point of each group was not significantly different. ConclusionThese results indicate that the cytoprotection of SW480 cell are produced by an instantaneous increase in the intracellular cAMP concentration while endogenous PDT is killing SW480 cell.
Objective
To investigate effect of hepatocyte growth factor (HGF) after lentivirus-mediated RNA interference (RNAi) targeting c-Met on invasion of colonic carcinoma cell line SW480.
Methods
The experiment was assigned into 3 groups: NC group, the normal cells were infected by the shRNA negative control virus (the NC-20 andNC-40 represented the negative group which were added 20 ng/mL and 40 ng/mL respectively HGF after being infected); KD group, the normal cells were infected by the shRNA-c-Met target virus (the KD-20 and KD-40 represented the interfered group which were added 20 ng/mL and 40 ng/mL HGF respectively after being infected; KD1, KD2, KD3, and KD4 represented the different RNAi targets for the purpose gene); CON group, the normal cells were not infected by any virus. The lentiviral vector shRNA-c-Met was constructed and verified by polymerase chain reaction (PCR) and DNA sequencing. The SW480 cells were infected with the shRNA-c-Met after packed with lentivirus plasmid. Fourty-eight hours transfection later, the c-Met mRNA of the transfected SW480 cell was detected by real time PCR and the c-Met protein was examined by Western blot. Seventy-two hours after transfection, the cell apoptosis was detected by flow cytometry and the invasions in the different cells with stable transfection were detected by Transwell test.
Results
The RNAi sequence targeting c-Met gene was successfully inserted into the lentiviral vector. The shRNA-c-Met transfection resulted in an obviously reduced expression of c-Met mRNA in the SW480 cells. The efficency of gene knock down of the KD4 (the cells with No.4 target spot knocked down) was 81.4%. The shRNA-c-Met tansfection resulted in an obviously reduced expression of c-Met protein in the SW480 cells. After transfection, the apoptosis rate of the KD group was significantly higher than that in the NC group (P<0.001) or the CON group (P<0.001). The invasion ratios in the NC group, NC-20 group, and NC-40 group were significantly higher than those in the KD group (P<0.001), KD-20 group (P=0.015), and KD-40 group (P=0.017), respectively; which in the NC-20 group and NC-40 group were increased as compared with the NC group (P<0.001,P<0.001), and in the NC-40 group was increased as compared with the NC-20 group (P=0.005). The invasion ratios in the KD-20 group and KD-40 group were increased as compared with the KD group (P<0.001,P<0.001), and in the KD-40 group was increased as compared with the KD-20 group (P=0.014).
Conclusion
Lentivirus-mediated RNAi targeting c-Met could effectively suppress expression of c-Met in SW480 cells and could reduce invasion of HGF on SW480 cells with knocked down c-Met.
