FAK抑制剂PF573228负调控CD15s抑制肝癌细胞侵袭的作用机制研究

FAK inhibitor PF573228 inhibits proliferation and invasion ability of hepatocellular carcinoma cells by negatively regulating CD15s expression

目的探讨黏着蛋白激酶(FAK)抑制剂PF573228对肝癌(HCC)细胞的抑制效果以及作用机制。方法采用CCK-8法及Transwell小室实验检测不同浓度PF573228(0、0.01、0.1、1、10μM)对HCC细胞系(HepG2和SMMC-7721)相对活力及侵袭能力的影响。对HCC细胞转染pcDNA-CD15s质粒进行过表达处理,分为pcDNA-CD15s组、PF573228组、PF573228+pcDNA-CD15s组,采用荧光定量PCR和Western blot法检测CD15s相对表达量及钙黏蛋白E(E-cadherin)、波形蛋白(Vimentin)、FAK、磷酸化FAK(FAK397)及CD15s蛋白表达水平。结果随着PF573228浓度增加,两种HCC细胞相对活力均逐渐降低,差异均有统计学意义(均P<0.05)。HCC细胞侵袭数随PF573228药物浓度的升高而降低(均P<0.05);PF573228组与pcDNA-CD15s组相比,细胞侵袭数显著降低,而PF573228+pcDNA-CD15s组与PF573228组相比,细胞侵袭数有所回升(均P<0.05)。与H-pcDNA-EGFP组、S-pcDNA-EGFP组比较,H-pcDNA-CD15s组、S-pcDNA-CD15s组中CD15s相对表达量均明显增加(均P<0.05)。E-cadherin蛋白表达水平随PF573228药物浓度的升高而增加,而Vimentin蛋白表达水平随药物浓度的升高而降低(均P<0.05);而FAK397及CD15s蛋白表达水平均随PF573228药物浓度的升高而不断下降(均P<0.05),不同浓度组FAK蛋白表达水平比较差异无统计学意义(P>0.05);与PF573228组比较,PF573228+pcDNA-CD15s组E-cadherin蛋白表达水平有所降低,Vimentin蛋白表达水平有所增加(均P<0.05)。结论PF573228可抑制肝癌细胞增殖和侵袭,同时具有浓度依赖性,这种现象是通过负调控CD15s介导的。

Objective To investigate the anti-tumor effect and its mechanism of FAK inhibitor PF573228 on hepatocellular carcinoma(HCC)cells.Methods The effects of different concentrations of PF573228(0,0.01,0.1,1,10μM)on the relative viability and invasion ability of HCC cell lines(HepG2 and SMMC-7721)were detected by CCK-8 method and Transwell chamber experiments.Over-expressing pcDNACD15s plasmid transfected into HCC cells,and the experimental group was divided into pcDNA-CD15s group,PF573228 group and PF573228+pcDNACD15s group.The relative expression of CD15s and the expression levels of E-cadherin,Vimentin,FAK,phosphorylated FAK(FAK 397)and CD15s protein were detected by fluorescence quantitative PCR and Western blot methods Results With the increase of PF573228 concentration,the relativeviability of the two HCC cells decreased gradually,and the difference was statistically significant(all P<0.05).The number of HCC cell invasion decreased with the increase of PF573228 drug concentration(all P<0.05),the number of cell invasion decreased significantly in the PF573228 group compared with the pcDNA-CD15s group,and the number of cell invasion increased compared with the PF573228 group(all P<0.05).Compared with H-pcDNA-EGFP group and S-pcDNA-EGFP group,the relative expression of CD15s in H-pcDNA-CD15s group and S-pcDNA-CD15s group was significantly increased(all P<0.05).E-cadherin protein expression increased with the increase of PF573228 drug concentration,however the expression level of Vimentin protein decreased with the increase of drug concentration(all P<0.05).The expression levels of FAK397 and CD15s protein decreased with the increase of PF573228 drug concentration(all P<0.05).And there was no significant difference in FAK protein expression between different concentration groups(P>0.05).Compared with PF573228 group,the expression of E-cadherin protein in PF573228+pcDNA-CD15s group decreased,and the expression level of Vimentin protein increased(all P<0.05).Conclusion PF573228 can inhibit the proliferation and invasion of hepatocellular carcinoma cells in a dose-dependent manner,which is mediated by negative regulation of CD15s.