Inhibitory effects of Beclin 1 gene expression on the activities of autophagy and proliferation/apoptosis of colon cancer cell line HCT116 by RNA interference

Purpose To investigate the effects of downregulation of autophagy regulatory molecules Beclin 1 expression induced by RNA interference on the autophagy-related gene mTOR/ULK1/LC3-B and proliferation/apoptosis of HCT116 cells.Methods HCT116 cells were transiently transfected with four specific eukaryotic expression vectors carrying shRNA targeting Beclin 1 and the negative control vectors,respectively.The most effective Beclin 1-shRNA was screened by RT-PCR and Western blot.The levels of mTOR/ULK1/LC3-B's mRNA and protein expression were detected by RT-PCR and Western blot.Cell proliferation and apoptosis were determined by CCK-8 detection kit and flow cytometry(FCM).Results(1) The expression of Beclin 1 gene at mRNA and protein levels was significantly lower in HCT116 cells transiently transfected with BECN1-shRNA,BECN1-1245 was the most effective,and it led to the highest inhibitory rate(80%).(2) The mRNA and protein levels of ULK1 and LC3-B were both reduced in HCT116 cells transiently transfected with BECN1-1245 48 hours later than those in the negative control cells(P≤0.05),whereas no difference was found in the expression of mTOR between the two groups(P 0.05).(3) At 24,48 and 72 hours after transfection,BECN1-1245 group showed a lower survival rate compared with the negative control group detected by CCK-8 assay.(4) The apoptotic rate of the HCT116 cells transfected with BECN1-1245 48 hours later was higher than those of the control cells evaluated by FCM(P ≤0.05).Conclusion Beclin 1 plays an important role in regulating autophagy in HCT116 cells,inhibition of Beclin 1 gene expression can reduce the activity of autophagy,inhibit cell proliferation and induce apoptosis.Therefore,Beclin 1 gene may be a new target of gene therapy for colorectal cancer.