摘要
目的 研究肝喜片通过微小分子RNA200a/E盒结合锌指蛋白(microRNA200a/zinc finger E-box binding homeobox,miR200a/ZEB)信号通路抑制肝癌细胞HepG2上皮间质转化的机制.方法 培养肝癌细胞HepG2,制备肝喜片含药血清,采用不同浓度(10％、15％、20％)的肝喜片含药血清作用于HepG2细胞,同时设置空白对照组.运用CCK-8实验检测HepG2细胞的增殖,划痕实验、Transwell实验分别检测HepG2细胞的横向迁移和纵向迁移,Western blot检测ZEB 1、ZEB2、E钙黏蛋白(E-cadherin)、波形蛋白(Vimentin)的表达,RT-PCR检测miR200a、ZEB1、ZEB2、E-cadherin、Vimentin mRNA的表达.结果 与空白对照组比较,肝喜片各剂量组的细胞存活率显著降低,且具有明显的时间和浓度依赖性(P＜0.05,P＜0.01);肝喜片各剂量组的横向迁移率及纵向迁移细胞数显著减少(P＜0.05,P＜0.01).与空白对照组比较,肝喜片中、高剂量组miR200a表达升高,肝喜片各剂量组E-cadherin蛋白及中、高剂量组E-cadherin mRNA表达升高,肝喜片各剂量组Vimentin蛋白及中、高剂量组Vimentin mRNA表达下降,肝喜片各剂量组ZEB 1、ZEB2蛋白及mRNA表达下降(P＜0.05,P＜0.01);与肝喜片低剂量组比较,肝喜片中、高剂量组miR200a、E-cadherin蛋白及mRNA表达升高,Vimentin、ZEB 1、ZEB2蛋白及mRNA表达下降(P＜0.05,P＜0.01);与肝喜片中剂量组比较,肝喜片高剂量组miR200a、E-cadherin蛋白及mRNA表达升高,Vimentin、ZEB1、ZEB2蛋白及mRNA表达下降(P＜0.05,P＜0.01).结论 肝喜片可显著抑制肝癌细胞HepG2的增殖和转移,其机制与调控miR200a/ZEB信号通路、促进E-hadherin的表达、抑制Vimentin的表达、逆转上皮间质转化相关.
Abstract
Objective To investigate the mechanism by which Ganxi Pill inhibits epithelial-mesenchymal transition(EMT)in HepG2 cells through the microRNA200a/zinc finger E-Box binding homeobox 1(miR200a/ZEB)signaling pathway.Methods HepG2 cells were cultured and treated with Ganxi Pill-medicated serum at different concentrations(10％,15％,and 20％),with a blank control group set up in parallel.The CCK-8 assay was used to assess the proliferation of HepG2 cells,while scratch assay and Transwell assay were conducted to evaluate the horizontal and vertical migration of the cells,respectively.Western blot was employed to check the expressions of ZEB1,ZEB2,E-cadherin,and vimentin.Additionally,RT-PCR was utilized to measure the mRNA expression levels of miR200,ZEB1,ZEB2,E-cadherin,and vimentin.Results Compared with the blank control group,the cell survival rate in all Ganxi Pill groups was significantly reduced,exhibiting clear time-and concentration-dependence(P＜0.05,P＜0.01).The horizontal migration rate and the number of vertically migrating cells significantly decreased in all Ganxi Pill groups(P＜0.05,P＜0.01).Compared with the blank control group,the expression of miR200a increased in the medium-and high-dose Ganxi Pill groups;E-cadherin protein level was elevated in all Ganxi Pill groups,with its mRNA expression increased in the medium-and high-dose groups.Meanwhile,the protein expression of vimentin was reduced across all Ganxi Pill groups,with its mRNA expression decreased in the medium-and high-dose groups.The protein and mRNA expressions of ZEB1 and ZEB2 were reduced in all Ganxi Pill groups(P＜0.05,P＜0.01).Compared with the low-dose group,the medium-and high-dose Ganxi Pill groups exhibited increased protein and mRNA expressions of miR200a and E-cadherin,along with reduced protein and mRNA expressions of vimentin,ZEB1,and ZEB2(P＜0.05,P＜0.01).Compared with the medium-dose group,the high-dose Ganxi Pill group showed further increases in the protein and mRNA expressions of miR-200a and E-cadherin,while the protein and mRNA expressions of vimentin,ZEB1,and ZEB2 decreased(P＜0.05,P＜0.01).Conclusion Ganxi Pill can significantly inhibit the proliferation and migration of HepG2 cells,and its mechanism might be related to the regulation of the miR200a/ZEB signaling pathway,promotion of the E-hadherin expression,inhibition of the vimentin expression,and reversal of EMT.
维普
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