Effects Of Microrna-122 On Bel-7402/5-fu Cell Apoptosis Induced By 5-fluracil

Objective:This study is to investigate the effects of microRNA-122 and its mechanism on BEL-7402/5-FU cell apoptosis induced by 5-fluracil.Method:MicroRNA microarray analyzed a wide diversity in miRNA expression between BEL-7402 and BEL-7402/5-FU cells. PcDNATM6.2-GW/EmGFP-miR linear vector carrying luciferase gene, which inserted fragment of Pre-miR-122(TM) (miR-122), was transient transfected into BEL-7402/5-FU cells. Transfection efficiency was tested by fluorescence inverted microscope and Quantitative Real-Time Reverse-Transcription PCR (Q-RT-PCR). Cell viability of each group was measured by MTT assay. Cell apoptotic percentage was detected by flow cytometry. Western blot was used to detect the caspase-8, caspase-9 and caspase-3 protein expression.Result:MicroRNA microarray showed that 2 miRNAs were significant (>2-fold) up-regulation (eg.miR-18 and miR-224) and many miRNAs were down-regulation (<0.5-fold) (eg.miR-122, miR-195, miR-199a, miR-125a, et al) in the BEL-7402/5-FU compared with BEL-7402 cells. MTT results showed that miR-122 transfectants had a higher cell inhibition rate than BEL-7402/5-FU cells or negative. MiR-122 promoted the sensitivity of BEL-7402/5-FU to 5-FU. Flow cytometry results demonstrated that miR-122 transfected cells had a higher apoptosis rate than BEL-7402/5-FU cells or negative. Moreover, miR-122 transfected cells had significant move value than BEL-7402/5-FU cells or negative by 10μmol/ml 5-FU for 24 hours. Western bolt results showed that the expression level of pro-caspase-8, pro-caspase-9 and pro-caspase-3 in miR-122 transfected cells were obviously decreased, while the expression level of active caspase-8, caspase-9 and caspase-3 were obviously increased compared with BEL-7402/5-FU cells or negative.Conclusion:1 miR-122 increased cell spontaneous apoptosis induced by 5-FU and sensitized BEL-7402/5-FU cells to 5-FU;2 miR-122 down-regulate pro-caspase-8, pro-caspase-9 and pro-caspase-3 expressions and up-regulate active caspase-8, caspase-9 and caspase-3 expressions in BEL-7402/5-FU cells.