Abstract:

Abstract:Objective
To explore the influence of PI3K/AKT/mTOR and JAK/STAT3 signaling pathway in apoptosis of the hepatocarcioma cells(HCC),and to provide basis for gene therapy for liver cancer.Methods BEL-7402 cells at inlogarithm growth phase were selected and randomly divided into control group,scramble-siRNA group,STAT3-siRNA group,scramble-siRNA +rapamycin(Rapa) group,Rapa group and STAT3-siRNA+Rapa group.The plasmids pGCsi.U6/neoRFP-STAT3 designed for expression of STAT3 siRNA was transfected into BEL-7402 cells by LipofectamineTM 2000.The cells with or without transfection siRNA were treated in wells in the absence or presence of rapamycin.The apoptotic rate was detected  by flow cytometry (FCM) and AnnexinV/PI apoptosis detection kit staining.The morphological changes of the cells were assessed by Hoechst33258 immunofluorescence staining.Simultaneously,the mitochondrial membrane potential(ΔΨm) was visualized by the JC-1 fluorescence staining and inverted fluorescence microscope.Further more,the expression level of active caspase-3 protein was analyzed by Western blotting method.Results The apoptotic rate of BEL-7402 cells in STAT3-siRNA+Rapa group (60.22%±0.87 %) was higher than those in other groups (P<0.05);and the ΔΨm was significantly decreased (27.28%±1.82%,P<0.05).The Hoechst33258 fluoresence staining results showed that the characteristic changes of chromatin condensation and nuclear  fragmentation were observed in STAT3-siRNA + Rapa group.The Western blotting results showed that the protein expression level of active caspase-3 in STAT3-siRNA + Rapa group was significantly higher than those in the other groups (P<0.05).Conclusion Combined treatment of rapamycin and STAT3 gene silencing can significantly promote the apoptosis of BEL-7402 cells and they display synergistic effects.

Key words: mTOR protein, STAT3 gene, RNA interference, rapamycin, apoptosis, hepatocelluar carcinoma