Investigation Of C-met Expression In Sensitivity Of Gastric Cancer Cells MKN-45to EGFR-TKI

Bakegrounds and Objectives Gastric cancer was one of the tumors with overexpression of EGFR.But most of the clinical and basic researchs showed that gastric cancer may be present primary drug resistance to EGFR tyrosine kinase inhibitors. There were some research results showed that in non-small cell lung cancer c-Met secondary overexpression was responsible for aquired resistance to gefitinib. The overexpression of c-Met in gastric cancer was common, studies have found that hepatocyte growth factor antagonist NK4could improve sensitivity of gastric cancer cells to gefitinib, this reseach prompted that c-Met abnormal expression may affect the sensitivity of gastric cancer to EGFR-TKIs.In this study, the gastric cancer cell MKN-45with overexpression of EGFR and c-Met and without EGFR mutation was selected as the object to explore the possibility of change sensitivity of gastric cancer cell MKN-45to gefitinib by changing c-Met gene expression, the results of this study may provide evidence for combination therapy with gefitinib and C-met inhibitors in gastric cancer.Materials and methods1、Human gastric cancer cell MKN-45were donated by the Pathology Laboratory of Xiangya School of Medicine, Central South University.Culativated the cells with the DMEM complete culture media containing10%fetal bovine serum in the 37℃,80%moisture,5%CO2, saturated humidity incubator.According to the c-Met cDNA sequence to design and synthesis the sequence of three target gene by Shanghai jimai company.2、 Transfected the three c-Met-siRNAs into the Human gastric cancer cell MKN-45mediated by lipofectamineTM2000.Meanwhile,installed the lipofectamineTM2000group, negative control group and blank group. Detected the transfect efficiency under the fluorescence microscope.3、48h after the transfection,detected the c-Met mRNA and protein by RT-PCR and Western blot respectively; Drug sensitivity assay via MTT was to evaluate the growth inhibition of gastric cancer cell MKN-45, and50%inhibition concentration (IC50) was represent to cellular sensitivity to gefitinib treatment;Flow cytometry was used to analyze the effects on apoptosis of c-Met-siRNAs transfection in gastric cancer cell MKN-45. SPSS19.0statistical package for data analysis, all the data were expressed as mean±standard deviation, used the t test to analyze the difference between two groups, used the single factor ANONA analysis to analyze the difference between three or more groups，P<0.05was considered statistically significant.Results1、48h after transfection,the expression of c-Met mRNA and protein detected by RT-PCR and Western-Blot of transfected gastric cancer cell MKN-45decreased significantly compared with all the control groups (P<0.05).2、Apoptosis rate of gastric cancer cell MKN-45was significantly increased after transfection (P<0.05).3、 The IC50of gastric cancer cell MKN-45to gefitinib did not significantly reduced after transfection(P>0.05).Conclusion1、 Successfully lowered the expression of c-Met gene in gastric cancer cell MKN-45by RNA interference technology.2、 Promoted apoptosis in gastric cancer cell MKN-45by successful transfection of c-Met-siRNA.3、 Silented c-Met gene failed to improve the sensitivity of gastric cancer cell MKN-45to gefitinib.