和厚朴酚通过UbcH8诱导AML1-ETO蛋白降解

目的：研究和厚朴酚诱导AML1-ETO蛋白降解的可能机制。方法：用10、20、40 μmol/L的和厚朴酚处理Kasumi-1细胞，分别在24 h和48 h后qRT-PCR检测AML1-ETO、UbcH8 mRNA的表达；Western blot法检测蛋白表达；40 μmol/L和厚朴酚处理24 h，基因芯片分析寻找靶基因；构建UbcH8过表达质粒，通过反转录病毒转染Kasumi-1细胞，检测AML1-ETO蛋白；通过短发夹RNA（shRNA）敲除UbcH8的表达，40 μmol/L和厚朴酚处理48 h，同时检测AML1-ETO和UbcH8蛋白；构建异种移植小鼠模型，和厚朴酚处理，检测肿瘤的成瘤性和相关蛋白表达。结果：和厚朴酚减少Kasumi-1细胞中AML1-ETO蛋白的表达，并具有浓度和时间依赖性，但没有影响AML1-ETO mRNA的表达；和厚朴酚处理后的AML1-ETO蛋白稳定性降低；基因芯片分析发现，和厚朴酚将UbcH8 mRNA的表达提高了约4倍；和厚朴酚处理后，Kasumi-1细胞中的UbcH8 mRNA和蛋白的表达量均升高；过表达UbcH8的Kasumi-1细胞中，AML1-ETO蛋白表达量减少；用和厚朴酚处理UbcH8敲除后的Kasumi-1细胞，AML1-ETO的降解被阻断；和厚朴酚抑制了异种移植小鼠模型的肿瘤生长，肿瘤中的AML1-ETO蛋白显著降低，而UBCH8表达升高。结论：和厚朴酚能通过UbcH8降解白血病细胞中的AML1-ETO蛋白。

Honokiol-induced degradation of AML1-ETO oncoprotein through upregulation of UbcH8 in acute myeloid leukemia

Objective: To investigate the possible mechanism of the degradation of AML1-ETO protein induced by honokiol. Methods: Kasumi-1 cells were treated with 10, 20 and 40 μmol/L honokiol for 24 h and 48 h. The mRNA expression of AML1-ETO and UbcH8 and the protein expression were detected by qRT-PCR and Western blot respectively. Microarry analysis was used to select the target gene. To explore the role of UbcH8 in the degradation of AML1-ETO protein induced by honokiol, the expression of UbcH8 was knocked down by small hairpin RNAs (shRNAs) and overexpressed by MSCV-based vector to detect the related indicators. A xenograft mice model was treated by honokiol to detect tumorigenicity and related protein expression. Results: Honokiol decreased the protein expression of AML1-ETO in a time- and concentration- dependent manner, and reduced the stability of AML1-ETO protein, but did not affect the mRNA expression of AML1-ETO in kasumi-1 cell. We identified UbcH8 as the target gene by honokiol through microarray analysis. Honokiol obviously increased the expression of UbcH8 by 4-fold. Furthermore, overexpression of UbcH8 decreased the expression of AML1-ETO and knockdown of UbcH8 by shRNAs prevented honokiol-induced degradation of AML-ETO, suggesting that UbcH8 plays a critical role in the degradation of AML1-ETO by honokiol. Finally, honokiol decreased xenograft tumor size in a xenograft leukemia mouse model. Meanwhile, the protein of AML1-ETO was significantly reduced, while UBCH8 expression was elevated. Conclusion: Honokiol can degrade AML1-ETO oncoprotein through upregulation of UbcH8 in acute myeloid leukemia.