HDAC6对人肝癌HepG2细胞迁移和侵袭的影响及其机制

目的:明确HDAC6对人肝癌HepG2细胞迁移和侵袭的影响及其机制。方法:应用Western blot技术检测正常肝细胞系LO2和肝癌细胞系HepG2细胞中 HDAC6的表达。应用HDAC6抑制剂Tubastatin A抑制HepG2细胞中HDAC6的表达，运用Western blot及荧光定量PCR技术检测HepG2细胞中转化生长因子β1（transforming growth factor-β1,TGF-β1）和上皮间充质转化（epithelial mesenchymal transition，EMT）相关分子标志物（N-cadherin,β-catenin,Vimentin）的表达；用TGF-β1抑制剂SB431542刺激HepG2细胞后，检测HepG2细胞中 EMT标志蛋白的表达，应用TGF-β1刺激HepG2细胞后观察HepG2细胞的形态变化。应用过表达HDAC6的质粒P3-HDAC6、P3-HDAC6+TGF-β1分别作用于HepG2细胞，通过Western blot检测HepG2细胞中EMT相关分子标志物的表达，应用划痕及Transwell技术检测HepG2细胞处理前后的迁移和侵袭能力变化。结果:肝癌细胞系HepG2细胞内HDAC6的蛋白表达量明显低于正常肝细胞系LO2（P＜0.05）。 HepG2细胞Tubastatin A组中N-cadherin、β-catenin、Vimentin、TGF-β1的mRNA和蛋白表达水平相较于空白对照组明显增高（P均<0.05）。SB431542组中EMT标志蛋白表达水平相较于空白对照组明显降低（P均<0.01）。 TGF-β1刺激HepG2细胞后细胞变得分散且狭长。同时发现P3-HDAC6+TGF-β1组的细胞迁移和侵袭能力在48 h后明显强于P3-HDAC6组且弱于空白对照组（P均<0.05）。结论:HDAC6可通过下调TGF-β1的表达从而抑制HepG2细胞的EMT进而抑制HepG2细胞的迁移和侵袭。

Effect of HDAC6 on migration and invasion of hepatocellular carcinoma HepG2 cells and its mechanism

Objective:To investigate the role of HDAC6 in the migration and invasion of HepG2 cells and its potential mechanism.Methods:Western blot was used to detect the expression of HDAC6 in normal hepatic cell line LO2 and HCC cell line HepG2.The HDAC6 inhibitor Tubastatin A was used to inhibit the expression of HDAC6 in HepG2 cells.Using Western blot and PCR technique to detect the expression of transforming growth factor 1 (TGF-β1) and epithelial mesenchymal transformation（EMT）related markers（N-cadherin，β-catenin，Vimentin）in HepG2 cells.The expression of EMT related markers was detected after the stimulation of HepG2 cells by the TGF-β1 inhibitor SB431542.Morphological changes of HepG2 cells were observed by stimulating HepG2 cells with TGF-β1.P3-HDAC6,P3-HDAC6+TGF-β1 were applied to HepG2 cells,respectively.Expression of EMT-related molecular markers in HepG2 cells was detected by Western blot,and changes in migration and invasion ability of HepG2 cells before and after treatment were detected by wound healing and Transwell assays.Results:The protein expression of HDAC6 in HepG2 cells was significantly lower than that in LO2 cells (P＜0.05).The mRNA and protein expression levels in the Tubastatin A group of HepG2 cells were significantly higher than those in the control group (P＜0.05).The expression level of EMT related markers in SB431542 group was significantly lower than that in the control group (P＜0.01).HepG2 cells become dispersed and elongated when stimulated by TGF-β1.Meanwhile,it was also found that the cell migration and invasion ability of P3-HDAC6+TGF-β1group was significantly stronger than that of P3-HDAC6 group and weaker than that of the control group 48h later (P＜0.05).Conclusion:HDAC6 inhibits the EMT of HepG2 cells and further inhibits the migration and invasion of HepG2 cells by down-regulating the expression of TGF-β1.