华支睾吸虫分泌排泄蛋白对肝癌细胞系HepG2细胞增殖、迁移和上皮间质转化的影响

Effect of Clonorchis sinensis excretory/secretory proteins on the proliferation,migration and epithelial-mesenchymal transition of hepatocarcinoma HepG2 cells

目的探讨华支睾吸虫分泌排泄蛋白(CsESP)对肝癌细胞株HepG2增殖、迁移和上皮间质转化(EMT)的影响。方法采用CCK-8实验测定不同浓度的CsESP对肝癌细胞HepG2的活力、细胞增殖能力和细胞毒性作用的影响;细胞划痕实验检测在不同浓度(1、10、20、50、100和200μg/mL)CsESP的作用下(设PBS对照组),HepG2细胞迁移的变化;荧光定量PCR法(RT-qPCR)检测HepG2细胞的癌变相关因子(MMP2和p300)以及EMT基因如E-钙黏蛋白E(E-cadherin)、N-钙黏蛋白(N-cadherin)、波形蛋白(Vimentin)和α-连环蛋白(α-catenin)mRNA的表达水平。结果 CCK-8实验结果显示,在CsESP浓度为10和20μg/mL时,细胞增殖明显,当CsESP浓度超过20μg/mL时,细胞活力降低。细胞划痕实验结果显示,在不同浓度CsESP的作用下,在0、24、48、72 h 4个时间点分别采集照片,并对其相对迁移面积进行统计分析,发现24和48 h 2个时间点,10和20μg/mL组的迁移能力明显高于PBS组和高浓度(50μg/mL)组,差异有统计学意义(P<0.05)。RT-qPCR检测出癌变基因MMP2以及N-cadherin、Vimentin、α-catenin的mRNA水平高于PBS组,差异有统计学意义(P<0.05),而癌变因子p300和E-cadherin水平并无明显变化。结论在CsESP的最适作用浓度下,CsESP可促进癌细胞增殖、癌变、迁移和上皮间质转化。

Objective Explore the effect of Clonorchis sinensis excretory/secretory proteins(CsESP)on the proliferation,carcinogenesis and epithelial-mesenchymal transition of hepatocarcinoma HepG2 cells.Methods HepG2 cells were treated with different concentrations of CsESP(1,10,20,50,100 and 200μg/mL).The proliferation of HepG2 cells was determined by CCK-8 assay.The migration ability of HepG2 cells was assessed through the wound-healing assay.The relative mRN A levels of MMP2,p300,E-cadherin,N-cadherin,Vimentin,α-catenin were determined by RT-qPCR.Results The CsESP could significantly promote HepG2 cell proliferation and the optimal concentration was 10 and 20μg/mL.The vitality of HepG2 cells was significantly reduced when the CsESP concentration exceeded 20μg/mL.The wound-healing assay showed that 10 and 20μg/mL CsESP could obviously promote HepG2 cells healing in different concentrations of CsESP at 24 h,48 h,post-treatment.The mRNA levels of MMP2,N-cadherin,Vimentin,α-catenin were increased in the experimental groups compared with the PBS group(P<0.05);however,the p300 and E-cadherin level were not increased significantly(P>0.05).Conclusion CsESP could promote HepG2 cell proliferation,carcinogenesis,migration,and epithelial-mesenchymal transition.