摘要
目的观察转化生长因子-β1(TGF-β1)在肝癌细胞对Wnt信号通路的调节作用。方法对肝癌细胞株HepG2细胞使用5、10、20μg/LTGF-β1重组蛋白诱导,48h后细胞计数检测肝癌细胞增殖。蛋白质印迹法(Western blot)方法检测上述各组上皮-间充质转化(EMT)标志蛋白及Wnt通路中相关蛋白表达。对肝癌细胞进行β-连环蛋白(β-catenin)小干扰RNA(siRNA)沉默,及β-cateninsiRNA沉默加TGF-β1重组蛋白诱导,分别检测EMT标志蛋白及Wnt通路中相关蛋白表达,并进行对比分析。实验数据以SPSS18.0软件进行统计分析,实验图片采用ImageJ软件进行灰度分析,目的蛋白相对表达量以目的蛋白/内参蛋白的比值表示。结果TGF-β1可促进肝癌细胞体外增殖,且与TGF-β1处理浓度呈明显相关(5μg/L实验组P<0.01,10μg/L实验组P<0.01,20μg/L实验组P<0.01)。TGF-β1处理组的肝癌细胞上皮细胞标志蛋白E-钙黏蛋白(E-cadherin)表达下调;间质标志蛋白波形蛋白(Vimentin)和β-catenin表达上调,且与TGF-β1处理浓度呈明显相关(E-cadherin对照组:1.000±0.058;5μg/L实验组:0.937±0.049,P>0.05;10μg/L实验组:0.490±0.046,P<0.01;20μg/L实验组:0.520±0.035,P<0.01。Vimentin对照组:1.000±0.058;5μg/L实验组:1.097±0.042,P>0.05;10μg/L实验组:2.217±0.169,P<0.01;20μg/L实验组:2.467±0.088,P<0.01。β-catenin对照组:1.000±0.115;5μg/L实验组:1.893±0.116,P<0.01;10μg/L实验组:2.327±0.104,P<0.01;20μg/L实验组:2.633±0.078,P<0.01)。β-catenin沉默组上皮标志物E-cadherin的表达上调,间质标志物Vimentin和β-catenin表达下调;而加入TGF-β1可以使β-catenin沉默的肝癌细胞中的E-cadherin的表达降低且Vimentin和β-catenin表达上调(E-cadherin对照组:1.000±0.068;β-cateninsiRNA沉默组:2.420±0.095,P<0.01;β-cateninsiRNA沉默+TGF-β1组:1.187±0.038,P<0.01。Vimentin对照组:1.000±0.113;β-cateninsiRNA沉默组:0.450±0.052,P<0.01;β-cateninsiRNA沉默+TGF-β1组:1.353±0.088,P<0.01。β-catenin对照组:1.000±0.058;β-cateninsiRNA沉默组:0.597±0.038,P<0.01;β-cateninsiRNA沉默+TGF-β1组:0.917±0.033,P<0.01)。结论TGF-β1可促进肝癌细胞体外增殖,且与TGF-β1处理浓度呈明显相关。TGF-β1可诱导肝癌细胞发生EMT样改变,且与TGF-β1处理浓度呈明显相关。TGF-β1可通过Wnt信号通路诱导肝癌细胞发生EMT过程。
Objective To observe the regulation of transforming growth factor-β1 (TGF-β1) on Wnt signaling pathway in hepatoma cells. Methods (1) Hepatoma cell line HepG2 cells were induced with 5, 10, 20 μg/L TGF-β1 recombinant protein. After 48 hours, the cell count was used to detect the proliferation of hepatoma cells.(2) Western blotting analysis was used to detect the expression of related proteins in the epithelial-mesenchymal transition (EMT) and Wnt pathway. The β-catenin was silenced by the β-catenin small interfering RNA (siRNA) in hepatoma cells, with or without TGF-β1 recombinant protein induced to detect the expression of related proteins in EMT and Wnt pathway, and comparative analysis was performed. Results TGF-β1 can promote the proliferation of hepatoma cells in vitro, and the concentration of TGF-β1 treatment is correlated (P<0.01 in 5 μg/L test group, P<0.01 in 10 μg/L test group, P<0.01 in 20 μg/L test group). The expression of E-cadherin was down-regulated in TGF-β1 treatment group;the expression of EMT marker proteins Vimentin and β-catenin was up-regulated and correlated with TGF-β1 treatment concentration (E-cadherin control group: 1.000±0.058;5 μg/L test group: 0.937±0.049, P>0.05;10 μg/L test group: 0.490±0.046, P<0.01;20 μg/L test group: 0.520±0.035, P<0.01. Vimentin control group: 1.000±0.058;5 μg/L test group: 1.097±0.042, P>0.05;10 μg/L test group: 2.217±0.169, P<0.01;20 μg/L test group: 2.467±0.088, P<0.01.β-catenin control group: 1.100±0.115;5 μg/L test group: 1.893±0.116, P<0.01;10 μg/L test group: 2.327±0.104, P<0.01;20 μg/L test group: 2.633±0.078, P<0.01). The expression of E-cadherin was up-regulated in the β-catenin-silenced group, and the expression of interstitial markers Vimentin and β-catenin was down-regulated. The expression of E-cadherin was decreased by the addition of TGF-β1 in hepatoma cells silenced by β-catenin, with up-regulated Vimentin and β-catenin expression (E-cadherin control group: 1.000±0.068;β-catenin siRNA silencing group: 2.420±0.095, P<0.01;β-catenin siRNA silencing+ TGF-β1 group: 1.187±0.038, P<0.01. Vimentin control group: 1.000±0.113;β-catenin siRNA silencing group: 0.450±0.052, P<0.01;β-catenin siRNA silencing+ TGF-β1 group: 1.353±0.088, P<0.01.β-catenin control Group: 1.000±0.058;β-catenin siRNA silencing group: 0.597±0.038, P<0.01;β-catenin siRNA silencing+ TGF-β1 group: 0.917±0.033, P<0.01). Conclusion TGF-β1 can promote the proliferation of hepatoma cells in vitro, and the concentration of TGF-β1 treatment is correlated. TGF-β1 can induce EMT changes in hepatoma cells and it is correlated with the concentration of TGF-β1 treatment. TGF-β1 can induce EMT in hepatoma cells via Wnt signaling pathway, and β-catenin may be a key target.
作者
解刚强
范永刚
王伟
翟景明
姚国良
刘可峰
封冰
张贝克
Xie Gangqiang;Fan Yonggang;Wang Wei;Zhai Jingming;Yao Guoliang;Liu Kefeng;Feng Bing;Zhang Beike(Department of General Surgery,the First Affiliated Hospital of University of Henan Science and Technology,Luoyang 471003,China)
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2019年第6期1033-1035,共3页
Chinese Journal of Experimental Surgery
作者简介
通信作者:张贝克,Email:hopebetter@163.com.
