Cancer stem cells play important roles in the development of tumors also are important targets to therapy of cancer. Former researches had confirmed the pre-leukemia transcription factor 3 (PBX3) was involved in maintaining the characteristics of liver cancer stem cell. We found that PBX3 is an extremely unstable protein with a short half-life in hepatocellular carcinoma cells. Unstable proteins are believed to be susceptible to degradation by ubiquitin-proteasome system. However, when we treated hepatoma cells using the proteasome inhibitor MG132, found the levels of PBX3 protein and mRNA were significantly downregulated, suggesting that PBX3 protein is not degraded by the ubiquitin-proteasome system. Our study aims to investigate the mechanism of MG132 regulation of PBX3. We observed that the levels of miR-424, let-7c, miR-222, miR-200b were upregulated when hepatoma cells were treated with MG132, and this increase was negatively correlated with the levels of PBX3. Using the miRWalk algorithm, previous studies have predicted that these miRNAs target the PBX3 gene. Thus, we investigated the mechanism by which the proteasome inhibitor MG132 regulates these miRNAs. It has been reported that the Argonaute2 protein is an important component of the RNA-induced silencing complex (RISC), and it can regulate the levels of certain miRNAs. Consequently, we also investigated whether the proteasome inhibitor regulates related miRNAs by stabilizing Argonaute2. Using co-infection, co-immunoprecipitation (Co-IP), and western blot assays, we found that MG132 stabilizes the expression of the Argonuate2 protein by inhibiting its degradation via the ubiquitin-proteasome pathway. In summary, the PBX3 protein, which is closely linked to the stemness of hepatoma cells, does not undergo degradation by the ubiquitin-proteasome system (UPM).

癌症干细胞在肿瘤的发展中起着重要作用，也是癌症治疗的重要靶标。以前的研究已经证实白血病前转录因子3（PBX3）参与维持肝癌干细胞的特性。我们发现PBX3是一种极不稳定的蛋白，在肝细胞癌细胞中的半衰期很短。不稳定蛋白被认为易于被泛素-蛋白酶体系统降解。但是，当我们使用蛋白酶体抑制剂MG132处理肝癌细胞时，发现PBX3蛋白和mRNA的水平显着下调，表明PBX3蛋白不会被泛素-蛋白酶体系统降解。我们的研究旨在研究MG132调节PBX3的机制。我们观察到miR-424，let-7c，miR-222，MG132处理肝癌细胞后，miR-200b上调，并且这种增加与PBX3的含量呈负相关。使用miRWalk算法，以前的研究已经预测到这些miRNA靶向PBX3基因。因此，我们研究了蛋白酶体抑制剂MG132调节这些miRNA的机制。据报道，Argonaute2蛋白是RNA诱导的沉默复合物（RISC）的重要组成部分，它可以调节某些miRNA的水平。因此，我们还研究了蛋白酶体抑制剂是否通过稳定Argonaute2来调节相关的miRNA。使用共同感染，共同免疫沉淀（Co-IP）和蛋白质印迹试验，我们发现MG132通过抑制其通过遍在蛋白-蛋白酶体途径的降解来稳定Argonuate2蛋白的表达。综上所述，