摘要
本研究旨在通过构建过表达及敲低Ct-OATP1B3基因的质粒,探讨Ct-OATP1B3对人(Homo sapiens)非小细胞肺癌细胞株H1975和PC9增殖、周期、凋亡、侵袭的影响及其机制。用RT-qPCR法检测各组细胞中Ct-OATP1B3、E-cadherin、N-cadherin和Vimentin mRNA的表达量,Western blot法检测各细胞中Ct-OATP1B3、GAPDH、Tubulin、Na^(+)/K^(+)ATPase、E-cadherin、N-cadherin和Vimentin的蛋白含量。采用CCK-8法检测各组细胞的增殖能力,流式细胞仪检测各组细胞的周期和凋亡变化,Transwell小室法检测各组细胞的侵袭能力。结果显示,Ct-OATP1B3在非小细胞肺癌细胞株H1975和PC9中的mRNA和蛋白含量明显高于正常肺上皮细胞BEAS-2B的,且在PC9细胞中的表达水平相对最高。H1975细胞用Ct-OATP1B3过表达质粒转染后,与空载组相比,过表达组中Ct-OATP1B3的mRNA和蛋白水平明显升高(P<0.01),细胞增殖活力增加(P<0.01),侵袭能力增加(P<0.01),周期和凋亡无明显变化,E-cadherin的mRNA和蛋白水平下调(P<0.01),N-cadherin和Vimentin的mRNA和蛋白水平上调(P<0.05,P<0.01)。PC9细胞用Cst-OATP1B3敲低质粒转染后,与阴性对照组相比,敲低组中Ct-OATP1B3的mRNA和蛋白水平明显下降(P<0.01),细胞增殖活力降低(P<0.05),侵袭能力减弱(P<0.01),周期与凋亡无明显变化,E-cadherin mRNA和蛋白水平上调(P<0.01),N-cadherin和Vimentin的mRNA和蛋白水平下调(P<0.01)。本研究提示,Ct-OATP1B3对人非小细胞肺癌细胞株H1975和PC9的增殖和侵袭有明显影响,对周期和凋亡无明显影响,其机制可能与调节上皮间充质转化(epithelial mesenchymal transition,EMT)进程有关。
This study aims to investigate the effect of Ct-OATP1B3 on the proliferation,cycle,apoptosis and invasion and its mechanism in human non-small cell lung cancer cell lines H1975 and PC9 by constructing the Ct-OATP1B3 gene overexpression and knockdown plasmids.The expression of Ct-OATP1B3,E-cadherin,Ncadherin and Vimentin mRNA in each group of cells was detected by RT-qPCR,and the protein contents of Ct-OATP1B3,GAPDH,Tubulin,Na^(+)/K^(+)ATPase,E-cadherin,N-cadherin and Vimentin were detected by Western blot.The CCK-8 method was used to detect the proliferation ability of each group of cells,the flow cytometry was used to detect the cycle and apoptosis changes of each group of cells,and the Transwell chamber method was used to detect the invasion ability of each group of cells.The results showed that the mRNA and protein level of Ct-OATP1B3 in non-small cell lung cancer cell lines H1975 and PC9 was significantly higher than that of normal lung epithelial cells BEAS-2B,and was relatively highest in PC9 cells.After transfection of H1975 cells with the Ct-OATP1B3 overexpression plasmid,compared with the empty group,the Ct-OATP1B3 mRNA and protein levels increased significantly in the overexpression group,the cell proliferation activity and invasion ability increased(P<0.01),but no significant changed in cell cycle and apoptosis.E-cadherin mRNA and protein levels were down-regulated(P<0.01),N-cadherin and Vimentin mRNA and protein levels were upregulated(P<0.05,P<0.01).After transfection of H1975 cells with Ct-OATP1B3 knockdown plasmid,compared with the negative control group,Ct-OATP1B3 mRNA and protein levels in the knockdown group decreased significantly and cell proliferation activity was reduced(P<0.05),invasion ability was weakened(P<0.01),but cell cycle and apoptosis was unchanged,E-cadherin mRNA and protein levels were up-regulated(P<0.01),and mRNA and protein levels of N-cadherin and vinmentin were down-regulated(P<0.01).The results of our study indicated that Ct-OATP1B3 had a significant effect on the prolifera
作者
郑玉洁
李亚霁
吴兰香
Zheng Yujie;Li Yaji;Wu Lanxiang(Institute of Life Sciences,Chongqing Medical University,Chongqing,400042)
出处
《基因组学与应用生物学》
CAS
CSCD
北大核心
2022年第9期2039-2050,共12页
Genomics and Applied Biology
基金
国家自然科学基金项目(81473938)资助
