组蛋白去乙酰化酶抑制剂SAHA影响U251细胞中p21稳定性的机制

The Effect of HDACs Inhibitor SAHA on p21 Protein Stability in U251 Cells

目前已开发的组蛋白去乙酰化酶抑制剂如SAHA表现出了广泛的临床应用前景。然而,其作用机制有待进一步阐明。该研究旨在探明SAHA对p21蛋白稳定性的影响。运用Real-time PCR、免疫共沉淀、泛素化降解实验、免疫印迹和流式细胞技术分析了SAHA对p21 mRNA和蛋白水平、稳定性和泛素化水平的影响;并分析了SAHA通过调节GSK-3β的活性影响p21蛋白稳定性及细胞周期的进程。结果发现,SAHA不但可以上调p21 mRNA水平,还可以稳定其蛋白水平;而且SAHA通过影响GSK-3β的活性降低p21蛋白泛素化水平,从而抑制其降解,并因此改变细胞周期进程,这表明SAHA可以通过抑制GSK-3β的活性增加p21蛋白的稳定性,维持其蛋白水平从而发挥SAHA的生物学效应。该研究结果将为脑胶质瘤的临床研究提供实验依据。

Previous studies suggest that HDACs inhibitors including SAHA are proming drugs against tumors in clinical trails. However, the mechanism of SAHA against tumor cells still remains to be clarified. In this study, we investigated the effects of SAHA on the expression of p21 mRNA and protein in tumor cells using Real- time PCR and Western blot, and the stability and ubiquitination of p21 protein mediated by GSK-3β in U251MG cells through Western blot and Co-IP assay. Subsequently, cell cycle progress was determinded using FACS in U251MG cells treated with SAHA and transfected with vector, GSK-313KD or GSK-3βCA. We found that SAHA upregulated both p21 mRNA and protein levels, and decreased ubquitination levels of p21 protein and thus en- hanced its stability, and arrested cell cycle progress at GI phase. Our finding confirmed that SAHA enhanced the stability of p21 protein via inhibition of GSK-3β activity, and thus played roles in blocking cell cycle progress, which might provide the evidence for further clinical research against glioma cells.