盐酸普鲁卡因通过DNA去甲基化作用抑制结肠癌细胞恶性进展机制研究

目的： 研究盐酸普鲁卡因（procaine hydrochloride，PCA）对结肠癌HCT116、SW480细胞增殖、侵袭、迁移和凋亡等功能表型的影响，探讨PCA在调节抑癌基因TBX5甲基化状态及其表达水平中发挥的作用。方法： 采用不同浓度PCA （0.5，1，2，4，8 mmol·L^-1）对HCT116、SW480细胞进行不同时间的处理，CCK-8试剂盒检测细胞增殖活性，筛选最佳药物浓度及作用时间；在最佳药物浓度及作用时间下，PCA对人结肠上皮FHC细胞进行处理，CCK-8法检测用药前后FHC细胞活性；平板克隆实验、流式细胞技术、细胞划痕实验、Transwell侵袭实验分别检测PCA对结肠癌细胞增殖活性、凋亡、迁移及侵袭能力的影响；亚硫酸氢盐扩增子测序（BSAS）、qRT-PCR检测PCA作用前后TBX5甲基化状态和表达水平的变化。结果： PCA抑制2种结肠癌细胞的增殖，最佳用药浓度为2 mmol·L^-1，最佳用药时间为48 h （P<0.05）。与空白对照组比较，PCA显著抑制2种结肠癌细胞的集落形成能力、迁移能力及侵袭能力且诱导细胞凋亡（P<0.05）。与FHC细胞相比，HCT116、SW480细胞呈现高甲基化状态；与空白对照组相比，PCA处理后HCT116、SW480细胞中TBX5的甲基化程度降低，mRNA表达水平升高，差异有统计学意义（P<0.05）。结论： 盐酸普鲁卡因可能通过逆转抑癌基因TBX5甲基化，提高其表达水平从而抑制结肠癌细胞增殖、迁移和侵袭等恶性进展。

Mechanism of procaine hydrochloride suppressing malignant progression of colonic cancer cells through DNAdemethylation

OBJECTIVE To explore the effects of procaine hydrochloride (procaine hydrochloride, PCA) on the proliferation, invasion, migration and apoptosis of colonic cancer HCT116 and SW480 cells and understand the role of PCA in regulating the methylation status and expressing tumor suppressor gene TBX5.METHODS HCT116 and SW480 cells were treated with different concentrations of PCA for different durations.The proliferating activity of HCT116/SW480 cells was detected by CCK-8 kit and the optimal drug concentration and acting time were selected.Under the optimal drug concentration and acting time, PCA was added to human colonic epithelial FHC cells and the activity of FHC cells was detected by CCK-8 method.The effects of PCA on the proliferation, apoptosis, migration and invasion of colonic cancer cells were detected by plate cloning test, flow cytometry, cell scratch test and Transwell invasion assay.And the changes of TBX5 methylation status and expression level pre/post-PCA dosing were detected by bisulfite extender sequencing (BSAS) and quantitative real-time polymerase chain reaction (qRT-PCR).RESULTS PCA suppressed the proliferation of two kinds of colonic cancer cells and the optimal concentration and time were 2 mmol·L^-1 and 48h respectively.As compared with blank control group, PCA significantly suppressed colony formation, migration and invasion of two kinds of colonic cancer cells and induced apoptosis.As compared with FHC cells, HCT116/SW480 cells showed hypermethylation.As compared with blank control group, the methylation degree of TBX5 decreased in HCT116/SW480 cells and the expression level of mRNA rose in PCA-treated HCT116/SW480 cells.CONCLUSION Procaine hydrochloride suppresses the malignant progression of colonic cancer cells, such as proliferation, migration and invasion, by reversing the methylation of TBX5 and up-regulating its expression.