Down-regulation Calreticulin In Differentiation Of Human Leukemic HL-60Cells Induced By Diallyl Disulfide

Objective: To study down-regulation calreticulin (CRT) in differentiation of humanleukemic HL-60cells induced by diallyl disulfide (DADS), and investigate themolecular mechanism of differentiation in HL-60cells induced by DADS.Methods: The morphology changes was investigated by Giemsa staining. Theexpression of calreticulin was detected with siRNA, QPCR, Western blot and flowcytometry. The proliferation was detected by MTT.Results:1. The Morphological change of HL-60cells after treated with DADSMorphological observation exhibited that the cells have abundant cytoplasm,nucleus becoming small and the ratio of nucleus to cytoplasm shrinking after72hoursof exposure to DADS at concentration of1.25mg·L-1.2. The expression of CRT after in HL-60cells treated with DADSQPCR amd Western blot results showed that the expression of CRT can reduce thein mRNA and protein level in a time-dependent decrease after DADS treated HL-60cells (P<0.05).3. The experimental of CRT gene was silenced by RNA interference.QPCR result showed that the expression of CRT mRNA in siRNA group wassignificantly lower than in control group at48h and72h after RNA interference,respectively (P<0.05). And the expression of CRT mRNA in DADS+control groupand DADS+siRNA group was significantly lower than in control group and in siRNAgroup (P<0.05). Flow cytometry showed that the expression of CRT protein in siRNAgroup was significantly lower in control group at48h and72h after RNA interference,respectively (P<0.05). And the expression of CRT protein in DADS+control groupand DADS+siRNA group was significantly lower than in control group and in siRNAgroup, respectively (P<0.05). 4. Effect of proliferation in HL-60cells after CRT by RNA interference.MTT exhibited that the proliferation of HL-60cells in siRNA group wassignificantly inhibited than in control group, and in DADS+control group andDADS+siRNA group was significantly inhibited in a time-dependent model than incontrol group and in siRNA group for24h,48h,72and96h, respectively (P<0.05).5. The experimental of CD33and CD11b after CRT by RNA interference.Flow cytometry showed that the expression of CD33in siRNA group wassignificantly decrease and CD11b was significantly increase than in control group(P<0.05), and the expression of CD33were significantly decrease and CD11b weresignificantly increase in DADS+control group and DADS+siRNA group than incontrol group and in siRNA group at48h and72h after RNA interference,respectively (P<0.05).Conclusion:1. DADS can induce the differentiation of HL-60cells through down-regulation ofexpression of calreticulin.2. Silent calreticulin can induce the differentiation of HL-60cells, and enhance thedifferentiation of DADS inducing HL-60cells.