Flap Endonuclease 1 Silencing Is Associated With The Increasing Cisplatin Sensiticity Of Gastric Cancer SGC-7901 Cells

Backgroud:Flap endonuclease-1 (FEN1), which plays a key role in DNA replication and repair, has been proven to be intimately involved in the development and progression of cancer. Our previous study determined that FEN1 may be associated with development and progression of gastric cancer. Additionally, our previous study also proved the downregulation of FEN1 can suppress the proliferation of and induce apoptosis in gastric cancer SGC-7901 cells. Moreover, several FEN1 inhibitors have been discovered to permit sensitization to DNA injury agents. These results may provide a promising treatment method that could enhance the traditional chemotherapeutics for gastric cancer. However, the further searches about the connection of FEN1 with CDDP in gastric cancer are limited. Based on our previous work, this study is to investigate whether FEN1 can be a new target to enhance the sensitivity to cisplatin in gastric cancer, providing experimental basis for FENlas a new treatment target of gastric cancer.Objective:The aim of this study was to determine the role of FEN1 in the chemosensitivity of SGC-7901 cells.Methods:1. The cells were treated with a range concentrations of CDDP and with 30 μM CDDP for time intervals, respectively. These cells were used to explore the FEN1 expression levels induced by CDDP by western blotting.2. FENl was silenced via specific FEN1 targeted siRNA which has been screened in our previous study compared with the negative control group (transfected with NC-siRNA) and Untransfected group. The transfection efficiency of FEN1 siRNA in SGC-7901 human gastric cancer cells was measured by Western blot analysis to evaluate the protein expression of FEN1.3. The cells in FEN1-siRNA+ CDDP, NC-siRNA+CDDP, Untransfected+CDDP groups were treated with increasing concentrations of CDDP for 48 h or with 10 μM CDDP for three time durations to test the viability of SGC-7901 cells to CDDP. The cell viability was measured by MTT cell survival assays.4. The number of apoptotic cells was analysed by flow cytometry using the fluorescein-isothiocyanate-labelled enhanced Annexin V/propidium iodide Apoptosis Detection kit (Invitrogen). And, western blot analysis was used to verify the inhibitory effects of FEN1-siRNA and to analyse the expression levels of Bax, Bcl-2 and Bcl-xl.Results:1.The FEN1 protein levels are significantly enhanced with a range of CDDP concentrations (0,10,20,30,40,50 μM CDDP for 48 h) and with treatment with 10 μM CDDP for three time intervals (24 h,48 h, and 72 h) by western blot analysis (P< 0.05).2. The suppression of FEN1 expression was observed in the SGC-7901 cells transfected with FEN1-siRNA compared with the transfected with NC-siRNA cells and Untransfected cells (P< 0.05).3. By MTT cell counting method, a significant lower survival rate in the FEN1-siRNA group after treatment with different concentrations of CDDP (0,2.5,5,10,20,30,40 and 50 μM) and after treatment for three different time durations (24 h,48 h, and 72 h), compared with the NC-siRNA group and Untransfected group (P< 0.05).4. The results of flow cytometry suggested that apoptosis rate in FEN1-siRNA+CDDP group significantly rised (P< 0.05); while decreased in NC-siRNA+CDDP and Untransfected +CDDP group in SGC-7901 cells. Meanwhile, the expression of Bax was elevated in the FEN1-siRNA+ CDDP cells, compared with the NC-siRNA+CDDP cells and Untransfected+ CDDP cells (P< 0.05). Conversely, the expression levels of Bcl-2 and Bcl-xl were significantly decreased in the FEN1-siRNA+ CDDP SGC-7901 cells compared with the NC-siRNA+CDDP cells and Untransfected+ CDDP cells.Conclusion:FEN1 expression was significantly induced by CDDP in a dose- and time-dependent manner. The targeting of FEN1 in SGC-7901 cells in combination with CDDP treatment significantly inhibited their-proliferation and effectively increased their apoptosis rate. In addition, in the FEN1-siRNA+ CDDP group, the Bax levels were significantly increased, whereas the expression levels of Bcl1-2 and Bcl-xl were effectively decreased compared with the NC-siRNA+CDDP group and Untransfected+ CDDP group. Our research provides a potential chemotherapeutic target that exhibits enhanced sensitivity to CDDP after FEN1 silencing in SGC-7901 cells.