泛素化修饰对K562/G01细胞BCL6蛋白表达水平的调节及对细胞增殖、凋亡的影响

Effects of Ubiquitination on the Expression of BCL6 Protein,Cell Proliferation and Apoptosis in K562/G01 Cells

目的:探讨泛素蛋白酶系统(UPS)对伊马替尼(IM)耐药细胞株K562/G01中BCL6蛋白水平及耐药细胞增殖和凋亡的影响。方法:应用Western blot技术检测蛋白酶抑制剂MG-132处理CML细胞前后BCL6蛋白水平差异;用RT-PCR及Western blot方法分别检测ML364(泛素特异性蛋白酶USP2抑制剂)处理前后K562/G01细胞株中BCL6及USP2 mRNA和蛋白的表达水平;应用CCK-8法及流式细胞术分别检测ML364和IM单用及联用对K562/G01细胞增殖及凋亡的影响。结果:蛋白酶抑制剂M G132处理后,K562/G01的BCL6蛋白水平显著上升(P<0. 05); K562/G01细胞株的去泛素化酶USP2 mRNA和蛋白表达水平均高于K562细胞株(P <0. 05); M L364处理K562/G01后,USP2和BCL6蛋白表达水平同步下调(P <0. 05);与IM单药组相比,ML364与IM联合用药组K562/G01细胞增殖抑制率及细胞凋亡率均明显升高(P <0. 05)。结论:M L364通过抑制USP2介导的BCL6蛋白去泛素化水平降低K562/G01细胞株BCL6蛋白稳定性,使K562/G01细胞株中BCL6蛋白表达下调,从而增加耐药细胞对IM的敏感性。

Objective:To explore the the effects of ubiquitin-proteasome system(UPS)on BCL6 protein level,proliferation and apoptosis of cell imatinib(IM)-resistant K562/G01 cells.Methods:Western blot was used to detect the expression of BCL6 in K562/G01 cells before and after treatment with protease inhibitor MG-132.The RT-PCR and Western blot respectively were used to detect the mRNA and protein expression levels of BCL6 and USP2 in K562/G01 cells treated with or without ML364(a ubiquitin-specific protease USP2 inhibitor).The effects of IM alone or in combination with ML364 on proliferation and apoptosis of K562/G01 were analysed by CCK-8 method and flow cytometry.Results:After treatment with protease inhibitor MG132,the BCL6 protein level of K562/G01 significantly increased(P<0.05).The mRNA and protein expression level of ubiquitin-specific protease USP2 in K562/G01 cell line was higher than that in K562 cell line(P<0.05).After treatment of K562/G01 with USP2 protease inhibitor ML364,the expression levels of USP2 and BCL6 proteins were dow n-regulated simultaneously(P<0.05).After combination of ML364 and IM,both the proliferation inhibitory rate and the apoptosis rate of K562/G01 cells significantly increased(P<0.05).Conclusion:ML364 decreases the BCL6 protein stability in K562/G01 by inhibiting the USP2-mediated deubiquitination,and dow n-regulate the BCL6 protein experssion,thereby increases the sensitivity of drug-resistant cells to IM.