Objective:To investigate the effect of miR-122 on apoptosis and drug sensitization in 5-fluracil-resistant human liver adenocarcinoma cell line BEL-7402/5-FU and to determine the inhibitory effect of the miR-122 on the gene expression in cells BEL-7402/5-FU.Method:We have used microarray to analyze the miRNA expression between BEL-7402 and BEL-7402/5-FU cells.Computation softwares were used to determine the predicted accessible miRNA binding sites.MiR-122 may be bound to 3'UTR of Bcl-2 family mRNA.So their interaction can be validated. MiR-122 and negative miRNA expression vectors were constructed and stably transfected into BEL-7402/5-FU cells.Drug sensitivity of the cells to 5-fluracil (5-FU)were analyzed with MTT and flow cytometry.Real-time PCR was used to detect miR-122,Bcl-XL,Bcl-2 and P53 mRNA expression.Western Blotting was used to detect Bcl-2,Bcl-XL and P53 protein expression.Result:MTT results showed that miR-122 transfectants had a higher cell inhibition rate than negative transfected cells and untreated cells after treated with 0.1,1,10,100μmol/ml 5-FU(23.40±1.83%vs.8.85±1.50%, 8.41±1.56%;24.00±1.67%vs.10.43±1.82%,9.29±2.94%;40.5±5.91 %vs.12.01±1.10%,12.61±3.43%;65.54±6.10%vs.42.65±2.49%,41.13±1.91%;P<0.05),IC50value of 5-FU in miR-122 transfected cells had significant lower value than that of transfected cells and untreated cells (21±1.4μmol/ml vs.206±14.8μmol/ml,223±14.4μmol/ml,P<0.05).Moreover, Flow cytometry results demonstrated that miR-122 transfected cells had increased apoptosis rate than negative transfected cells and untreated cells (5.6±0.35%vs.1.0±0.52%,2.2±0.48%).MiR-122 transfected cells had significant more value apoptosis rate than that of untreated cells after addition value of 5-FU(1 and 10μmol/ml 5-FU)for 24 hours(25±1.22%vs. 7.6±0.86%,9.2±0.78%;42±2.0%vs.9.22±0.9%,11.9±1.0%; P<0.05).The mRNA and protein expression level of Bcl-2 and Bcl-XL in miR-122 transfectants were obviously reduced comparing with untreated cells.Moreover,miR-122 increased the activity of P53.Conclusion:MiR-122 can sensitize BEL-7402/5-FU to 5-FU and increase BEL-7402/5-FU spontaneous apoptosis,specifically down-regulate Bcl-2 and Bcl-XL expressions,and active P53 in BEL-7402/5-FU cells. MiR-122 may be a potential therapy agent against human liver adenocarcinoma. |