Effect Of Vimentin Expression On Malignant Phenotype In Hepatocellular Carcinoma Cells

Background&Objective Clinically, expression of vimentin intermediate filaments in carcinoma cells was inclined to a biomarker for patients' poor prognosis. However, the function of vimentin on oncobiology has been in dispute. The aim of this investigation is to determine the relationship of vimentin expression with malignant phenotype in hepatocellular carcinoma cells and its potential mechanism. Methods The full-length vimentin gene open reading frame (ORF, 1401 base pairs) was cloned into the plasmid vector pcDNA3.1(+), and these vectors were stably transfected into HepG2 human hepatocellular carcinoma cells. Vimentin gene expression was evaluated by RT-PCR and flow cytometry assay; the proliferative activity and invasive potential of tumor cells were determined by the CellTiter 96 AQueous One Solution Cell Proliferation Assay and the BioCoat GFR Matrigel Invasion Chamber, respectively; and the cell cycle was detected by the flow cytometry. To assess potential differences in tumorigenicity, the wild-type cells and transfected cells were subcutaneously inoculated into nude mice. The expression levels of correlation factors and apoptosis in transplanted tumors were evaluated by using immunohistochemistry and TUNEL assay. Recombinant plasmid pcDNA3.1-VIM as a gene vaccine was intramuscularly injected into the mice for testing the effect of immunotherapy in tumor-beating mice (MethA and Sp2/0-Ag14). Results DNA sequencing and restriction endonucleases digestion analysis demonstrated that the recombinant pcDNA3.1-VIM vector was correctly cloned. The HepG2-pV cells demonstrated a higher RNA and protein levels of vimentin expression. However, in HepG2-pV cells, both proliferative and invasive abilities were reduced in vitro, moreover, inhibition of growth velocity in nude mice transplanted tumor model, and augmented apoptosis of transplanted tumor. The cell cycle of HepG2-pV cells was arrested during the period of DNA synthesis. The immunostaining of vimentin, Bcl-2 and Caspase-3 was heavy, but PCNA and CD44 were light staining among the fourteen primary antibodies in the transplanted tumor tissue of HepG2-pV cells. Immunotherapy of tumors with pcDNA3.1-VIM as a xenogeneic homologous gene vaccine was ineffective in mesenchymal tumors of mice. Conclusion Recombinant plasmid pcDNA3.1-VIM is successfully constructed and a carcinoma cell subline HepG2-pV highly expressing vimentin is obtained. In HepG2 cells, enhanced expression of vimentin partly reverses malignant phenotype of tumor cells in vitro and in vivo. Overexpression vimentin results in augmented apoptosis, arrested cell cycle and the expression changes of proteins associated with tumor growth. This study suggests that vimentin is likely to function with other proteins and controls the transcription or translation of other genes.