增强人波形蛋白表达可降低肝癌细胞的增殖和侵袭能力

Enhanced expression of human vimentin intermediate filaments in hepatocellular carcinoma cells decreases their proliferative and invasive abilities in vitro

目的探讨波形蛋白（VIM）对肝癌细胞增殖和侵袭能力的影响。方法构建VIM基因质粒载体pcDNA3．1-VIM，将VIM基因稳定转染人肝细胞癌HepG2细胞株，通过逆转录聚合酶链反应（RT-PCR）和Western blot测定VIM的表达，分别用细胞增殖实验和标准的被覆基质胶侵袭小室，测定癌细胞的增殖活性和侵袭程度。结果DNA序列分析和限制性核酸内切酶消化证实重组载体已正确克隆，建立的细胞株HepG2-pV在RNA和蛋白水平均稳定高表达VIM。HepG2-pV细胞增殖能力降低（P〈0．05），软琼脂集落形成率为35．9％±3．9％，明显低于空质粒对照细胞（HepG2-P）和HepG2细胞（均P〈0．05）。HepG2-pV组侵袭细胞数为17±4，明显低于HepG2-P组和HepG2组（均P〈0．05）。结论在体外增强肝癌细胞VIM表达，可以降低其恶性表型。

Objective Expression of vimentin in carcinoma cells is a marker for poor prognosis in patients. The aim of this investigation was to assess the influence of vimentin on the characteristics of carcinoma cells. Methods The full-length vimentin gene open reading frame （ 1401 base pairs） was cloned into the plasmid vector pcDNA 3.1 （ ＋ ）, and these vectors were used to stably transfect the human hepatocellular carcinoma HepG2 cell line. Vimentin gene expression was evaluated by RT-PCR and Western blot. Proliferative activity and invasive potential of tumor cells were determined by the CellTiter 96 aqueous one solution cell proliferation assay and BioCoat GFR Matrigel invasion chamber, respectively. Results DNA sequencing and restriction endonuclease digestion analysis demonstrated that the recombinant vector was correctly cloned. The stable cell line demonstrated a higher vimentin RNA and protein expression. However, both proliferative and invasive abilities of the cells were reduced in vitro （ P 〈 0.05 ）. Conclusion A recombinant plasmid pcDNA3.1-VIM is successfully constructed and a carcinoma cell line HepG2-pV highly expressing vimentin is obtained. Recombinant vimentin protein suppresses the proliferative and invasive abilities of HepG2 cells, suggesting that it might decrease malignant phenotype of tumor cells in vitro. This work makes a foundation for further study on the relationship between vimentin and biological phenotype of carcinoma cells.