Study Of The Relationship Between Ubiquitin Proteasome System And Autophagic Death In Acute Myeloid Leukemia

Objective:The Ubiquitin-proteasome system(UPS)is another important protein degradation pathway in eukaryotes,which plays an important role in various cellular functions and homeostasis.Ubiquitin carboxyterminal hydrolases(UCH)and Ubiquitin(Ub)are members of the UPS family,and more and more studies have confirmed that UCH and Ub are closely related to the development of certain tumors contact.Autophagy is a pathway for the degradation of intracellular proteins that are commonly found in eukaryotic cells.It is mainly responsible for the degradation of some organelles and long-lived proteins present in the body.Studies have shown that there is a close relationship between autophagy and ubiquitin proteasome systems.SQSTM1 /P62(sequestosome 1)degrades polyubiquitinated proteins selectively by macrophages and proteasomes.In this study,the effects of autophagy receptor rapamycin and autophagy inhibitor 3-MA on HL-60 cells were investigated before and after treatment.The effects of autophagy system on ubiquitin proteasome system and P62 were investigated.Secondly,the effect of the ubiquitin proteasome system on autophagy and P62 was investigated by observing the differences between the two groups before and after treatment with the bacteriocin inhibitor bortezomib.Thirdly,bortezomib and rapamycin were used in combination to observe the interaction between ubiquitin proteasome system and autophagy and the effect on P62.Fourthly,the expression of LC3Ⅱ,UCH-L3 and P62 mRNA in peripheral blood of patients with acute myeloid leukemia was detected,and the relationship between acute myeloid leukemia and autophagy and ubiquitin protease and P62 were further studied.Hoping to provide a new direction for the monitoring and treatment of leukemia.Methods:1.Add autophagy inducer rapamycin and autophagy inhibitor 3-MA into HL-60 cells,TransDetest Cell Counting Kit(CCK8)detect the proliferation of HL-60cells;Flow Cytometry detect the cell apoptosis;The morphology of autophagosomes was observed under transmission electron microscope;RT-PCR observe the mRNA expression of LC3Ⅱ,UCH-L3 and P62;Western-blot detect the protein expression level of LC3Ⅱ,UCH-L3,P62 and Ub.2.Add the ubiquitin proteome inhibitor bortezomib into HL-60 cells,TransDetest Cell Counting Kit(CCK8)detect the proliferation of HL-60 cells;Flow Cytometry detect the cell apoptosis;The morphology of autophagosomes was observed under transmission electron microscope;RT-PCR observe the mRNA expression of LC3Ⅱ,UCH-L3 and P62;Western-blot detect the protein expression level of LC3Ⅱ,UCH-L3,P62 and Ub.3.HL-60 treatmented with bortezomib,a ubiquitin proteasome inhibitor,in combination with autophagy-inducing agent rapamycin.The morphology of autophagosomes was observed under transmission electron microscope;RT-PCR observe the mRNA expression of LC3Ⅱ,UCH-L3 and P62;Western-blot detect the protein expression level of LC3Ⅱ,UCH-L3,P62 and Ub.4.Extract peripheral blood mononuclear cells from acute myeloid leukemia patients,RT-PCR observe the m RNA expression of LC3Ⅱ,UCH-L3 and P62 of both primary group and healthy people.Results:1.Autophagy-inducing agent rapamycin and autophagy inhibitor 3-MA on cell growth inhibition rate of time and concentration-dependent;The percentage ofapoptotic cells were increased with time;the formation of autophagosome was observed under electron microscopy in rapamycin group.In Rapamycin treatment group,the mRNA expression of LC3 II,UCH-L3 were up-regulated and P62 was down-regulated by RT-PCR,the protein expression of LC3 II,UCH-L3,Ub were up-regulated and P62 was down-regulated by Western blotting.In 3-MA treatment group,the mRNA expression of LC3 II,UCH-L3 were down-regulated and P62 was up-regulated by RT-PCR,the protein expression of LC3 II,UCH-L3 and Ub were down-regulated and P62 was up-regulated by Western-blot,The difference was statistically significant(P <0.05).2.The inhibition of bortezomib on cell growth was time-dependent and concentration-dependent;The percentage of apoptotic cells were increased with time;The formation of most autophagosome was observed under electron microscope.After treated with bortezomib,the mRNA expression of LC3 II,P62 were up-regulated and UCH-L3 was down-regulated by RT-PCR,the protein expression of LC3 II,P62were up-regulated and UCH-L3,Ub was down-regulated by Western blotting.The difference was statistically significant(P <0.05).3.Bortezomib and rapamycin were togeher treated HL-60 cells,the formation of autophagosome were observed under electron microscopy,the mRNA expression of LC3Ⅱ,P62 were up-regulated and the mRNA expression of UCH-L3 was down-regulated by RT-PCR.The protein expression of LC3Ⅱ,P62 were upregulated and the protein expression of UCH-L3,Ub were down-regulated by Western blotting.Bortezomib and 3-MA were together treated HL-60 cells,the formation of autophagosomes were observed under electron microscopy,but less than the number of bortezomib and rapamycin co-treatment group;The difference was statistically significant(P<0.05).4.The expression of LC3 II was up-regulated,the expression of UCH-L3 was down-regulated,the expression of P62 was down-regulated in the early stage of acute myeloid leukemia compared with the healthy control group,the difference was statistically significant.(P <0.05).Conclusion:1.The expression of UCH-L3,P62,Ub in acute myeloid leukemia cells induced by autophagy of HL-60 cells changed from the gene and protein level,while the autophagy inhibitor 3-MA inhibited this change in expression,suggesting that both the ubiquitin proteasome system and P62 may be involved in the autophagy process of rapamycin-induced acute myeloid leukemia cells.2.The level of autophagy was increased after that HL-60 cells were treated by the ubiquitin proteasome inhibitor bortezomib,suggesting that inhibition of ubiquitin proteasome system may promote autophagy.3.The levels of autophagy in the treatment of HL-60 with bortezomib and rapamycin were higher than that of rapamycin alone,indicating the synergistic effect between autophagy and ubiquitin proteasome system.4.The expression of LC3 II,UCH-L3 and P62 in peripheral blood of patients with acute myeloid leukemia showed that the occurrence of acute myeloid leukemia may be the result of autophagy,Ubiquitin proteasome and P62.