The Effects Of RhoGDI2 Silence For The Differentiation In Human Leukemia HL-60 Cells Induced By Diallyl Disulfide

Objective: Diallyl disulfide(DADS) is a allyl sulfur compound derived from garlic. Research show that, DADS can induce differentiation of acute myelocytic leukemia HL-60 cells, and analysis of differential expression proteins in differentiation of HL-60 cells induced DADS by proteome technique show down-regulation of Rho GDP dissociation inhibitor 2(RhoGDI2). RhoGDI2 is an important member of the Rho GDI family, and abnormal expression in a wide wariety of tumor cells. The Objective of this experiment is to detect the effect of RhoGDI2 silence for the differentiation in human leukemia HL-60 cells induced by diallyl disulfide, to confirm that RhoGDI2 may be a key target molecules thatv differentiation HL-60 cells, and develop a new generation of drugs to cure leukemia.Methods: Western blotting and RT-PCR were performed to detect the expressions of RhoGDI2 after treated with DADS. The eukaryotic expression vector encoding human RhoGDI2 gene sequence was transfected into HL-60 cells to establish a Silence cell line, use Western blotting and RT-PCR to detect the RhoGDI2 protein and m RNA expression level in HL-60 cells after transfection. We detection the effects of proliferation, differentiation,migration and invasion in HL-60 cells after treated with DADS by CCK-8 experiment, NBT experiment, immunofluorescence and Transwell experiment, and use ATRA as the positive control. Western blotting and RT-PCR were performed to detect the expressions of RhoGDI2, Rac1, Pak1 and LIMK1 after treated with DADS.Results: 1. The expression of RhoGDI2 in HL-60 cells after treated with DADS Western blot and RT-PCR show that, the expression of RhoGDI2 protein and m RNA were inhibited in a time dependent model, after HL-60 cells were treated with DADS for 0h, 12 h, 24 h and 36h(P<0.05). 2. structure HL-60 cells with low expression of RhoGDI2 pc DNA?6.2-GW/Em GFPmi R-RhoGDI2 interfering plasmid were constructed by Invitrogen company. We transfect HL-60 cells with 4 groups of interference plasmid, and green fluorescent can be found by fluorescence microscope, Western blot and RT-PCR show that, compared with the negative transfected group, the expression of RhoGDI2 protein and m RNA in interfering group were decreased, and the most prominent effect of each group was the number one interfering plasmid(P<0.05), so we chose it as subjects in the next experiments. 3. The effects of RhoGDI2 silence for the differentiation in HL-60 cells induced by DADS NBT experiment showed that, The differentiation ability of interference group were higher than untransfected group and negative transfected group(P<0.05); The differentiation ability of each group were higher than untreated group after treated with 1.25mg·L-1 DADS(P<0.05); There is no difference between the group treated with DADS and the group treated with ATRA(P>0.05). CCK-8 experiment showed that, The reproductive activity of interference group were decreased, compared with untransfected group and negative transfected group(P<0.05); The reproductive activity of each group were lower than untreated group after treated with 1.25mg·L-1 DADS(P<0.05); There is no difference between the group treated with DADS and the group treated with ATRA(P>0.05). Transwell experiment showed that, The migration and invasion ability of interference group were lower than untransfected group and negative transfected group(P<0.05); The migration and invasion ability of each group were lower than untreated group after treated with 1.25mg·L-1 DADS(P<0.05); There is no difference between the group treated with DADS and the group treated with ATRA(P>0.05). Immunofluorescence showed that, the expression of CD11 b protein were up-regulated and the expression of CD33 protein were decreased in each group, after treated with DADS 24 h. 4. The effects of RhoGDI2 silence and DADS for the Rac1/LIMK1 pathway Western blot showed that compared with untransfected group and negative transfected group, the expression of RhoGDI2, Rac1, Pak1 and LIMK1 protein were decreased in interference group(P<0.05); The expression of RhoGDI2, Rac1, Pak1 and LIMK1 protein in each group were lower than untreated group after treated with DADS 24h(P<0.05); There is no difference between untransfected group and negative transfected group(P>0.05).Conclusion: 1. RhoGDI2 silenced can promote the differentiation in HL-60 cells; 2. RhoGDI2 silenced can enhance the effect for the differentiation in HL-60 cells induced by DADS; 3. DADS can induct the differentiation of HL-60 cells through down-regulated of RhoGDI2 that inhibiting Rac1/LIMK1 signaling pathways.