The Regulation Effect Of SiRNA Targeted Human Oncogene Bmi-1 And Its Downstream Target Gene Screen

Objective: To explore the regulation effect of siRNA targeted human oncogene Bmi-1 and its downstream target gene.Methods: Three siRNAs targeting human Bmi-1 gene were synthesized chemically in vitro, transfected into INK4a gene naturally deleted A549 cells and INK4a gene generally expressed K562 cells by LipofectimineTM2000. The transfection efficiency was detected by fluoromicroscopy. MTT assay was used to assess the proliferation of the cells. The IC50 in the two kinds of cells was calculated. The change of cell cycle was analyzed by flow cytometry. The Bmi-1 expression on mRNA level was detected by semi-quantative RT-PCR; The Bmi-1, P16 and hTERT expressions on protein level were tested by Western Blotting.Results: (1) The transfection efficiency of LipofectamineTM2000 for Bmi-1 siRNA was up to 85%; (2) Among the three designed siRNAs, only the suppressive effect of Bmi-1 siRNA1 on the A549 and K562 cells correlated with its concentration and had no cell specificity; (3) The IC50 of Bmi-1 siRNA1 was 83.69 nmol/l and 87.69 nmol/l in A549 cells and K562 cells respectively; (4) The percentage of G1 phase was significantly increased in K562 cells, however not in A549 cells; (5) The double time of the two kinds of cells was prolonged; (6) The Bmi-1 expression on mRNA level in A549 and K562 cells was suppressed by up to 53.88% and 58.94% at 24h after transfection respectively; (7) The corresponding decrease of Bmi-1 expression on protein level in A549 and K562 cells was found and the suppressive percentage was 68.86% and 70.24% at 48h after transfection respectively; (8) Bmi-1 siRNA1 inhibited the proliferation of K562 cells through up-regulating P16 protein; (9) Bmi-1 siRNA1 down-regulated the hTERT expression in A549 cells.Conclusions: (1) LipofectamineTM2000 which was used for Bmi-1 siRNA transfected into A549 and K562 cells had high transfection efficiency and was convenient; (2) The proliferation of A549 and K562 cells was suppressed significantly by Bmi-1 siRNA1 without cell specificity; (3) The expression of Bmi-1 on mRNA level in A549 and K562 cells could be suppressed by Bmi-1 siRNA1; (4) The change of cell cycle was significant in K562 cells, but not in A549 cells; (5) The INK4a gene was a critical down-stream target for Bmi-1 in K562 cells; (6) There might be a Bmi-1-hTERT gene regulation pathway in INK4a gene naturally deleted A549 cells.