Objective To investigate the effects of human recombinant hepatocyte growth factor(rh-HGF) on the expression of c-Met in intima of allograft vessels after cardiac transplantation in rats. Methods Heterotopic heart transplantation were established in abdominal cavity with eighty Wistar rats and forty SD rats. Donors’ cardiac grafts from Wistar rats were transplanted to SD rats(allograft) or Wistar rats(isograft).Sixty recipient rats were divided into three groups, control group：20 Wistar rats were injected with normal saline 1ml/kg·d intraperitoneally after transplantation; cyclosporine A (CsA) group:20 SD rats were injected with CsA 5mg/kg·d intraperitoneally on operation day; rhHGF group:20 SD rats were injected with rh-HGF 500μg/kg·d and CsA 5mg/kg·d intraperitoneally on operation day. The cardiac grafts were harvested at the 15th day and 60th day after transplantation. The crosssection of vascular tissues were used for immunohistochemistrical staining of c-Met, and investigated the expression of c-Met messenger ribonucleic acid (mRNA ) in intima of allograft vessels by reverse transcriptionpolymerase chain reaction(RT-PCR). The pathologic changes of allograft coronary vessels were observed with histopathological method. Results The allograft coronary arteries showed minimal intimal thickening, the endothelium and internal elastic lamina remained almost intact in rh-HGF group after transplantation.The expression of c-Met and c-Met mRNA in intima of allograft vessels after transplantation in rhHGF group were significantly higher than those in CsA group and control group（expression of c-Met at 60d: 1.85±0.26 vs. 0.96±0.10, t=8.491,P=0.000;1.85±0.26 vs. 0.58±0.03, t=13.725,P=0.000; expression of c-Met mRNA at 60d: 192±0.22 vs. 0.88±0.07, t=11.940,P=0.000;1.92±0.22 vs. 0.42±0.02,t=19.206,P=0.000）. Conclusion rh-HGF may prevent the progression of cardiac allograft vasculopathy through upregulating the expression of c-Met to stimulate endothelial cell repair and growth.
ObjectiveTo study the expression of c-Met in colorectal carcinoma cells and the effect of hepatocyte growth factor (HGF) on proliferation and invasion of colon carcinoma cells SW480. MethodsReal-time PCR and Western blot methods were respectively used to detect the expressions of c-Met mRNA and protein in the different colorectal carcinoma cells in order to screen the high c-Met expression cells. The SW480 cells were incubated with different concentrations (0, 20, 40, and 70 ng/mL) HGF. MTT assay and Transwell test were used to evaluate the effects of proliferation and invasion in the SW480 cells. Results①The c-Met was expressed in each colorectal carcinomar cells, especially highly expressed in the colon carcinoma cells SW480 in vitro.②MTT assay showed that the HGF could promote the proliferation of SW480 cells in a dose-dependent manner with some extent.③Transwell test showed that the HGF could increase the invasion of SW480 cells. ConclusionThe c-Met is highly expressed in colorectal carcinoma cells and HGF could promote proliferation and increase invasion of colorectal carcinoma cells in vitro.
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.
ObjectiveTo study the mechanism of the effect on invasion and metastasis of colorectal cancer by down-regulating c-Met gene.Methodsc-Met genes were knocked down in SW480 cells, differential genes were screened by gene chip, functional cluster analysis of differential genes was carried out, and IPA was used to analyze the interaction network of cell signal pathway and related differential genes, as well as the ralationship between related genes and upstream regulatory molecules. The related genes in the suppressed signal pathway were selected for qPCR verification.ResultsAfter knockdown of c-Met, the number of up-regulated genes and down-regulated genes in SW480 cells was 399 and 286, respectively. Cluster analysis showed that c-Met knockdown had a great effect on the gene expression level of SW480 cells, IPA pathway analysis showed that HGF signaling pathway was suppressed, and after c-Met knockdown, IPA interaction network suggested that AKT2, PIK3CA, and MAP2K4 in HGF pathway were down-regulated, and qPCR verified that the above genes were also down-regulated, which was consistent with the results of microarray.Conclusionc-Met may affect the invasion and metastasis of colorectal cancer through the regulation of AKT2, PIK3CA, and MAP2K4 in HGF pathway.