曲古霉素A通过调控Ku70乙酰化水平对结肠癌HCT116细胞上皮间充质转化和侵袭转移的影响

Effects of trichostatin A on EMT,invasion and metastasis of colon cancer HCT116 cells mediated by Ku70 acetylation

目的探讨曲古霉素A(TSA)通过调控Ku70乙酰化水平对结肠癌HCT116细胞上皮间充质转化(EMT)和侵袭转移的影响。方法体外培养HCT116细胞,采用MTT法筛选并确定TSA的最佳给药浓度;采用Western印迹(WB)检测对照组(DMSO处理组)和TSA处理组细胞中Ku70及其乙酰化水平以及EMT相关蛋白E-钙黏蛋白(cadherin)、N-钙黏蛋白、Snail和Slug的表达情况;采用Transwell-迁移和侵袭实验观察TSA介导Ku70乙酰化对HCT116细胞侵袭和转移能力的影响。结果 MTT检测结果表明TSA可明显降低HCT116细胞的活性(P<0.05);WB结果显示,TSA可明显上调HCT116细胞中acetyl-Ku70的蛋白水平并抑制其EMT,表现为上调上皮细胞标志蛋白E-钙黏蛋白的表达,并下调EMT标志蛋白N-钙黏蛋白及EMT相关转录因子Snail和Slug的表达(P<0.05);此外,Transwell-迁移和侵袭实验结果表明,TSA可明显抑制HCT116细胞的迁移和侵袭(P<0.05)。结论 TSA可能通过上调HCT116细胞中Ku70乙酰化水平,降低Ku70与DNA的亲和力,从而抑制肿瘤细胞DNA的损伤修复能力,发挥抑制结肠癌细胞EMT和迁移侵袭的作用,为临床抗结肠癌转移治疗提供新的思路和作用靶点。

Objective To investigate the effect of trichostatin A(TSA)on epithelial-mesenchymal transition(EMT),invasion and metastasis of colon cancer HCT116 cells by regulating the level of Ku70 acetylation. Methods MTT assay was used to detect the effect of TSA on cell viability of colon cancer HCT116 cells. Western blot(WB)was used to detect the expressions of Ku70,acetyl-Ku70 and EMT-related proteins E-cadherin,N-cadherin,Snail and Slug in the control(DMSO-treated)group and TSA-treated groups. Transwell-migration and invasion assays were used to observe the effect of Ku70 acetylation on invasion and metastasis abilities of colon cancer HCT116 cells after TSA treatment. Results MTT assay results showed that TSA could significantly reduce the activity of colon cancer HCT116 cells(P<0.05). WB results showed that TSA could significantly up-regulate the expression of acetyl-Ku70 and inhibit the EMT of HCT116 cells by upregulating the expression of epithelial cell marker E-cadherin and down-regulating the expression of mesenchymal cell marker N-cadherin and other EMT-related transcription factors,such as Snail and Slug(P<0.05). In addition,Transwellmigration and invasion assays results showed that TSA could also significantly inhibit the migration and invasion of colon cancer HCT116 cells by promoting Ku70 acetylation(P<0.05). Conclusion TSA may significantly reduce the affinity of Ku70 to DNA by significantlyup-regulatingtheacetylationlevelofKu70 incoloncancerHCT116 cells,therebyinhibitingthe EMT,migration and invasion of colon cancer HCT116 cells. This study can provide a new treatment strategy for clinical anti-colon cancer metastasis.