Down-regulation Cofilin1Induced Differentiation In HL-60Cells By Diallyl Disulfide

Objective: To study the molecular mechanism of diallyl disulfide (diallyl disulfide,DADS) inhibiting proliferation, migration and invasion as well as inducting ofdifferentiation on leukemic HL-60cell by reducing the expression of cofilin1.Methods: The effects of DADS on expression of cofilin1and Rac1-Rock1-LIMK1signal pathway in HL-60cells were detected by RT-PCR, western blotting andImmunocytochemistry. The influence of the ability of migration in HL-60cell afterthe effect of DADS was detected through Transwell experiment.ThemiRNA-containing plasmid targeting cofilin1pcDNA6.2-GW/EmGFPmiR-cofilin1transient transfected HL-60cell. The expression of cofilin1was also detected byRT-PCR, Western blotting after the effect of DADS and interference with cofilin1.The role of DADS and HL-60cell proliferation, differentiation and the ability ofinvasion were tested by MTT colorimetric, NBT, Transwell experiment after theeffect of DADS and interference with cofilin1.Results:1. The expression of cofilin1was inhibited after treated with DADS in HL-60cells.RT-PCR, Western blot results showed that the expression of cofilin1wasinhibited in a time-dose dependent model after treated with1.25mg·L-1DADS for0、12、24、48h in HL-60cells (P<0.05).Immunohistochemical results showed that the protein expression of cofilin1wasstrongly positive in cytoplasm in control group, however, the expression levels weresignificantly decreased after treated with1.25mg·L-1DADS for12、24、48h. Theresults showed that the protein expression of cofilin1was time dependent decreasedafter after treated with1.25mg·L-1DADS in HL-60cells.2. The ability of migration was inhibited by DADS in HL-60cells.Transwell expriment revealed that the ability of migration was inhibited in time dependent in HL-60cells after treated with1.25mg·L-1DADS for0、12、24h(P<0.05).3. Effect of DADS in HL-60cells after cofilin1was silenced by RNA interference.pcDNA6.2-GW/EmGFPmiR-cofilin1-miRNA interfering vector runningagarose electrophoresis gel extracted plasmid extraction results indicated high purityand can be seen under the inverted fluorescence microscope, cells transfected withgreen fluorescent hair.RT-PCR result showed that compared with negative transfected group,cofilin1gene expression was decreased,the gene expression in group1were the most obvious.Western blot showed that the cofilin1protein expression of transfectedcofilin1-miRNA1-4of HL-60cells compared with negative transfected groupdecreased. The most obvious expression inhibition was the first transfected group.cofilin1-miRNA1plasmid were used as subjects in follow-up experiments.4. Effect of DADS on proliferation, differentiation and invasion in HL-60cells aftercofilin1was knocked down by RNA interference technology.MTT colorimetric assayed that the proliferation of transfected cofilin1-miRNAafter treated with DADS for24、48h was reduced compared to untransfected groupand negative transfection group(P<0.05), and thus the ability of proliferation was thebiggest inhibited in the cofilin1-miRNA group treated with DADS.NBT results showed that the abilities of differentiation of transfectedcofilin1-miRNA were higher than negative transfection group after treated with1.25mg·L-1DADS for48h. No difference was found between the1.25mg·L-1DADS andARTA group(P>0.05).Transwell invasion showed that the number of cancer cells which passedthrough the Matrigel coated membrane was reduced in the DADS treatment group andcofilin1-miR group(P<0.05). No difference was found between the untransfectedgroup and negative transfection group(P>0.05).It indicated that the mechanism ofDADS inhibiting the ability of invasive was related with downregulating of cofilin1expression in HL-60cell.5. DADS influenced the Rac1-Rock1-LIMK1-cofilin1signaling pathwaysRT-PCR, Western blot results showed that the expression level of Rac1, Rock1, LIMK1were lowed after treated with DADS for0、12、24、48h(P<0.05) in HL-60cell.Conclusion:1. DADS can induct the differentiation of HL-60cells by down-regulating theexpression of cofilin1.2. Silencing cofilin1gene can enhance the ability of inducting the differentiation ofHL-60cells by DADS.3. DADS can induct the differentiation of HL-60cells though inhibitingRac1-Rock-LIMK1-cofilin1signaling pathways that down-regulated and inactivedcofilin1.