Investigate The Relationship Between The Transcription Factor Snai1and Multidrug Resistance In Gastric Cancer

Objective:To investigate the relationship between the transcription factor Snai1and ERCC1in gastric cancer,as well as the role of Snail and ERCC1.We investigated the effect of cisplatin on human gastric adenocarcinoma cell SGC7901and SGC7901/DDP to confirm the correlation between the expression of Snai1.ERCC1and E-cadherin.Methods:Paraffin-embedded tissue sections from51human gastric cancer cases were processed by immunohistochemistry to investigate the expression Snail and ERCC1proteins.We measured the expression of Snai1,E-cadherin and ERCC1by Western Blot in human gastric adenocarcinoma cell SGC7901which was treated with cisplatin for different concentration and different hours.We observed the characteristics of cells by electron microscopy in human gastric adenocarcinoma cell SGC7901which was treated with cisplatin for different hours.In addition,we extended the study to evaluate the cells’invasion property through the transwell experiment.SGC7901/DDP cell lines stably transfected with amiRNA-Snail plasmid(SGC7901/DDP-amiRNA group)or negative control plasmid (SGC7901/DDP-mock group)were constructed, respectively.Western blotting and immunofluorescence were used to determine the protein expressions of Snail,drug resistance gene ERCC1,and E-cadherin,the down stream target of Snai1in transfected and untransfected cells.Survival rates of cells treated with DDP at different concentrations (0.01,0.1,1.0,and10.0n g/ml) were assessed by CCK-8assay and the50%inhibiting concentration (IC50) of DDP was calculated.Results:26samples (50.98%) in51patients expressed Snai1.24samples (47.06%) in51patients expressed ERCC1.Immunohistochemistry showed that Snail was highly correlated with ERCC1in gastric cancer(r=0.737,P=0.01).Treated with1μ g/ml cisplatin for0,24,48,72h,protein was detected in SGC7901cells,the results showed that the expression of Snail and ERCC1were up regulated with time extension(P<0.01).SGC7901cells were exposed for48h to different concentrations of cisplatin.When treated with low concentration of cisplatin,Snail and ERCC1expression demonstrated increased in SGC7901(P<0.01).with the increase of the concentration of cisplatin,Snai1and ERCC1gene displayed decline status of expression.The morphology of cultured SGC7901cells treated with or without cisplatin for48hours was examined microscopically.Cisplatin induced SGC7901cells were found to lose cell-cell contacts and to acquire fibroblast phenotypes as a result of transient cisplatin treatment.By means of in vitro cell culture,SGC7901was confirmed to have significant lower invasion ability than parent cells SGC7901/DDP by the following invasion assays(P<0.001).we demonstrated reexpression of E-cadherin in SGC7901/DDP-amiRNA cells with ow expression of Snail and ERCC1.(P<0.05).ICso of DDP was significantly lower in SGC7901/DDP-amiRNA group than in SGC7901/DDP-Mock group (P<0.001).Conclusion:Over expression of Snail in gastric cancer is associated with tumour multidrug resistance probably through the increase of ERCC1expression and inducement of the process of EMT. Cisplatin can induce increased expression of Snail and ERCC1, these increase were time and concentration dependent.SGC7901was confirmed to have significant lower invasion ability than parent cells SGC7901/DDP.Silencing of Snail gene by amiRNA-Snail may reverse the resistance of human gastric cancer cell line SGC7901/DDP to DDP.