摘要
目的观察竹节香附素A(Raddeanin A,RA)对人结肠癌HCT116细胞株上皮间充质转化(epithelial-mesenchymal transition,EMT)的影响及其可能机制。方法收集结肠癌患者的结肠癌组织标本和正常结肠组织标本,免疫组化实验检测β-连环蛋白(β-catenin)和上皮型钙黏蛋白(E-cad)的表达情况,通过四甲基偶氮唑蓝(MTT)实验检测不同浓度的RA(1、2、4、8、16μmol/L)对HCT116细胞株的增殖抑制情况。运用转化生长因子β1(TGF-β1)蛋白诱导建立HCT116细胞株EMT模型。通过划痕实验及Transwell实验分别观察RA 1、2μmol/L对EMT模型(TGF-β1诱导组)细胞迁移、侵袭能力的影响。免疫蛋白印迹(Western Blot,WB)实验和实时荧光定量聚合酶链式反应(qRT-PCR)实验分别检测β-catenin、E-cad、波形蛋白(Vimentin)、Snail相关蛋白及基因的表达情况。结果免疫组化结果显示,β-catenin蛋白在结肠癌组织中的表达高于正常结肠组织(P<0.05),E-cad蛋白表达减少(P<0.05)。MTT实验结果显示,RA能显著抑制HCT116细胞株增殖(P<0.05),并呈浓度依赖性(P<0.05)。划痕实验及Transwell实验分别证实,经TGF-β1诱导后的HCT116细胞株迁移和侵袭能力均显著增强(P<0.05,P<0.01),且WB和qRT-PCR实验结果显示,E-cad蛋白和基因表达减少(P<0.05,P<0.01),Vimentin、Snail、β-catenin蛋白和基因表达增加(P<0.05,P<0.01),说明造模成功。划痕实验显示,RA 1、2μmol/L作用于EMT模型细胞后,划痕愈合率较TGF-β1诱导组降低(P<0.05,P<0.01),且RA 2μmol/L组低于RA 1μmol/L组(P<0.05),提示细胞迁移能力减弱;Transwell实验显示,RA 1、2μmol/L可抑制穿膜细胞数,与TGF-β1诱导组比较差异有统计学意义(P<0.05,P<0.01),且RA 2μmol/L组低于RA 1μmol/L组(P<0.05)。WB及qRT-PCR实验显示,RA 1、2μmol/L组Wnt/β-catenin通路相关因子β-catenin,以及EMT相关因子Vimentin、Snail蛋白和基因表达较TGF-β1诱导组显著下降(P<0.05,P<0.01),E-cad蛋白和基因表达较TGF-β1诱导组升高
Objective To explore the effect of Raddeanin A(RA)on epithelial-mesenchymal transition(EMT)of colon cancer(CC)HCT116 cell and its possible occurrence mechanism.Methods The expression ofβ-catenin and epithelial cadherin(E-cad)of CC tissue samples and normal colon tissue samples were determined using immunohistochemistry(IHC).The inhibition of HCT116 cells proliferation by different concentration RA(1,2,4,8,16μmol/L)was detected with methyl thiazolyl tetrazolium(MTT)assay.Transforming growth factor beta1(TGF-β1)induced HCT116 cells to establish EMT model,Scratch test and Transwell assay were used to verify the effect of RA 1 and 2μmol/L on cell migration and invasion in TGF-β1 group of EMT model.The expression of laboratory-related genes and the proteins(β-catenin,E-cad,Vimentin,Snail)were detected by real-time quantitative polymerase chain reaction(qRT-PCR)and Western blot(WB)techniques.Results IHC results showed increased expression ofβ-catenin in the CC tissue compared with the normal colon tissue(P<0.05),while decreased expression of E-cad(P<0.05).The results of MTT assay showed that RA could significantly inhibit the proliferation of HCT116 cells and with concentration-dependence manner(P<0.05).Both Scratch test and Transwell test confirmed that the migration and invasion abilities of HCT116 cells induced by TGF-β1 were significantly enhanced(P<0.05,P<0.01);the results of WB and qRT-PCR experiments showed that the expression of E-cad protein and gene decreased(P<0.05,P<0.01).The protein and gene expressions of Vimentin,Snail andβ-catenin increased(P<0.05,P<0.01),indicating that the modeling was successful.Scratch experiment showed the healing rate retarded following RA 1 and 2μmol/L acted in TGF-β1 group(P<0.05,P<0.01),and RA 2μmol/L group was lower than RA 1μmol/L group(P<0.05),suggesting the cell migration ability was weakened.In Transwell assay,the difference in transmembrane cells number was statistically significant between groups(P<0.05,P<0.01),and RA 2μmol/L group was lower than RA 1μmol/
作者
孟春芹
刘先勇
Meng Chunqin;Liu Xianyong(Department of Integrated Traditional Chinese and Western Medicine,The Affiliated Jiangning Hospital of Nanjing Medical University,Nanjing,Jiangsu 211100)
出处
《河北中医》
2022年第1期100-106,共7页
Hebei Journal of Traditional Chinese Medicine
基金
南京市中医药青年人才培养计划项目(编号:JNYYRC202007)
2019年南京医科大学校级科研项目(编号:NMUB2019238)
2019年南京医科大学康达学院科研发展基金项目(编号:KD2019KYJJZD023)
2019年江苏省“双创博士”项目(县级医院创新类)(编号:NJYYSC201903)。
