Study in the Effect of EZH2 Gene Targeting siRNA on Cell Death in HL-60 Cell Line

Objective: To study small interfering RNA (siRNA) targeting EZH2 gene effect on cell proliferation, apoptosis and histone modulation in HL-60 cells line.

Methods: (1) Three siRNA segments targeting EZH2 gene were designed and transfected into HL-60 cells by Lipofectamine^TM 2000. The optimal segment was screened by RT-PCR. (2) Cell growth inhibited by EZH2 siRNA was determined by WST-1. Cell apoptosis was measured by Flow cytometry. (3) The expression of procaspase-3, procaspase-9, Bcl-2, and Bax was detected by Western blot. The levels of histone H3 lysine27 dimethylation and trimethylation and histone acetylatiom of H3 were evaluated by Western blot.

Results: (1) The efficiency of transfection with EZH2 siRNA for 6 hours was (82 ±2.97)%. The sequence of optimal segment of EZH2 siRNA was sense 5'- GAGGGAAAGUGUAUGAUAATT -3'，antisense 5'- UUAUCAUACACUUUC CCUCTT -3'。 (2) EZH2 siRNA suppressed the proliferation of HL-60 cells. (3) EZH2 siRNA downregulates the expression of procaspase-3, procaspase-9, Bcl-2 and upregulates the expression of Bax and induced cells apoptosis. (4) EZH2 siRNA suppressed the protein and mRNA of EZH2 in HL-60 cells. The levels of histone H3 lysine27 dimethylation and trimethylation was down regulated and histone acetylatiom of H3 was up regulated.

Conclusions: (1) Lipofectamine^TM2000 transfected siRNA for EZH2 gene into HL-60 cells has high efficiency. (2) EZH2 siRNA inhibits cell growth and induces cell apoptosis in HL-60 cell line. It might be a new therapeutic target in human leukemia. (3) EZH2 siRNA downregulates the levels of histone H3 lysine27 dimethylation and trimethylation and upregulates histone acetylation of H3. The mechanism of EZH2 effect on histone acetylation need to be further studied. RNAi technology is expected to become a new tool for targeted gene therapy of acute leukemia, and EZH2 might be one of the targets of gene therapy for leukemia.