Relationship Between C-Met Abnormally Expression And Sorafenib Resistance In Hepatocellular Carcinoma And The Establishment Of HCC Animal Model

Part one HGF/c-Met expression characteristics evaluation for patients with HCCObjective To detect the serum HGF concentration and c-Met protein expression in liver of HCC patients.Methods The level of serum HGF was detected by ELISA method. Immunohisto-chemistry was used to detect c-Met protein staining intensity within liver pathology specimens.Results The serum HGF concentration in HCC group, cirrhosis group and normal control group were0.609±0.134ng/ml,0.378±0.116ng/ml, and0.271±0.083ng/ml, respectively. HGF levels in patients with HCC group were significantly higher than that in other two groups (P<0.01). There was a significant difference of HGF levels between different tumor volume (P<0.05). HCC tissues showed positive expression of c-Met staining, but the expressions were weakly in the paracancerous tissues, cirrhotic liver tissue and normal liver tissue (P<0.01). The intensity of c-Met expression closely related to the tumor differentiation and showed a negative correlation (P<0.05).Conclusions1. The serum HGF level of HCC patients was significantly higher than that of liver cirrhosis patients and normal control group.2. The serum HGF levels of HCC were significantly associated with tumor size.3. The c-Met protein staining intensity in HCC tissue was significantly higher than in paracancerous and normal liver tissues.4. The c-Met protein staining intensity was closely related to HCC histological differentiation.Part two The c-Met over-expression cell lines building and sorafenib-resistant features observingObjective To evaluate the effect of c-Met overexpression and subsequent cell signaling abnormalities for sorafenib-resistance of HCC and to find countermeasures.Methods The c-Met gene was transfected to HepG2cells by lentiviral vector, and selected the stably transfected cell lines HepG2-met by puromycin. Evaluated the cell cycle and apoptosis rate for HepG2cells and HepG2-met cells with sorafenib. The c-Met protein and its downstream mTOR signaling protein such as P70S6K,4E-BP1 and their phosphorylation levels were detected by Western Blot, to find out if c-Met overexpression can subsequently lead to mTOR signaling abnormalities. Then observed if the resistance to sorafenib could be reversed by combination with mTOR inhibiting drug rapamycin.Results c-Met mRNA was determined by RT-PCR. The stable transfected cell lines HepG2-met was selected by2.5ug/ml puromycin for about10days. Western blot analysis showed that the c-Met, P70S6K and4E-BP1protein phosphorylation level in HepG2-met cells were significantly higher than in HepG2cells. HepG2-met had more proliferative activity. Sorafenib inhibited the proliferation of HepG2/HepG2-met cells, and the effects enhanced with drug concentration increased. HepG2-met cells had a certain resistance to sorafenib. HepG2-met cells S phase decreased and apoptosis rate increased when cultured with sorafenib combining small doses of rapamycin, the results suggested that the resistance to sorafenib could be reversed by combination with mTOR inhibiting drug.Conclusions1. HepG2-met cells with high level c-Met expression were constructed.2. c-Met expression and phosphorylation levels increased in HepG2-met cells, while its downstream mTOR signaling protein phosphorylation of P70S6K and4E-BP1were as higher than in HepG2cells.3. HepG2-met cells were more proliferatively active.4. HepG2-met cells showed a certain degree of drug resistance to sorafenib.5. Rapamycin inhibited mTOR signaling pathway protein phosphorylation of P70S6K and4E-BP1partially reversing sorafenib resistance of HepG2-met.Part three Establishment of nude mice bearing human liver tumor modelsObjective Xenograft tumor models established by transplantation of human tumor into immunodifficient mice, and made the initial detection of HBV status in tumor.Methods The fresh human liver tumor samples were selected from clinic patients, and implanted in nude mices. Compared the histopathological characteristics between the original liver cancer and the stabilized tumor model by HE staining. And then detected the HBV DNA existing status in tumor model by PCR methods.Results We successfully established four cases of liver cancer animal models. The pathological characteristics of xenograft tumor samples and primary patient tumor specimens were similar. HBV S, C, and X sequences were respectively detected from three cases of liver cancer xenograft model tumor samples by PCR methods.Conclusions1.Four cases of liver cancer xenograft models were established by subcutaneously implanting in the nude mices.2. Xenograft tumor samples and patients’ primary tumor specimens had the similar histomorphometric characteristics.3. HBV DNA can be detected in the HCC model specimens, and it may be important carcinogenic factors.