Molecular Mechanisms Of Inhibitory Effects Of Human Dominant Negative EGFR On Gastric Cancer: An Experimental Study

Objective: We aim at confirming that the expression intensity of epidermal growth factor receptor (EGFR) in gastric cancer tissues is correlated with gastric cancer progression. Then we construct and identify the eukaryotic expression vector(pEGFPN1-DNEGFR) carrying human dominant negative epidermal growth factor receptor(DNEGFR), for the purpose of detecting the expression and the sub-cellular localization of dominant negative epidermal growth factor receptor- enhanced green fluorescence protein (DNEGFR-EGFP) in COS-7 cells transfected transiently with pEGFPN1-DNEGFR.After stable transfection of human gastric cancer cells with pEGFPN1-DNEGFR,we propose to explore the mechanism of the inhibitory effect of DNEGFR-EGFP on endogenous EGFR function, to detect the effect on malignant phenotype of human gastric cancer cells, and to elucidate molecular mechanisms.Methods:(1) Immunohistochemistry PV method was used to detect the expression of EGFR in 60 cases of gastric cancer, and the relationship between protein expression and clinic pathological features was analyzed.(2) The cDNA coding signal peptide, extracellular ligand-binding domain and membrane-spanning region of epidermal growth factor receptor(EGFR) precursor was obtained by reverse transcription-polymerase chain reaction(RT-PCR),then directionally cloned into the empty vector(pEGFP-N1) to construct pEGFPN1-DNEGFR.After being confirmed by PCR amplification assay, double enzyme digestion, DNA sequencing and bioinformatics analysis of nucleotide sequence,pEGFPN1-DNEGFR was transfected transiently into COS-7 cells, mediated by Lipofectamine 2000. The expression of and the sub-cellular localization of DNEGFR-EGFP in COS-7 cells were detected by Western blotting and Laser Scanning Spectral Confocal Microscope, respectively.(3) The pEGFPN1-DNEGFR was transfected stably into human gastric cancer cells (SGC-7901 and NCI-N87), mediated by Lipofectamine 2000. The expression of and the sub-cellular localization of DNEGFR-EGFP in human gastric cancer cells were detected by Western blotting and Laser Scanning Spectral Confocal Microscope, respectively. The influence of DNEGFR-EGFP on endogenous EGFR mRNA level, EGFR protein and its phosphorylation level was detected by RT-PCR and Western blotting, respectively.(4) MTT(methyl thiazolyl tetrazolium) assay, flow cytometry, TUNEL(terminal deoxynucleotidyl transferase)-mediated dUTP nick end-labeling) test, wound healing assay, cell adhesion assay, in vitro invasion assay, HUVEC(human umbilical vein endothelial cell) tube formation assay, subcutaneous xenograft model of nude mouse were used to test the effects of DNEGFR-EGFP on malignant phenotype of human gastric cancer cells, Western blotting,ELISA(enzyme linked immunosorbent assay) were used to test the expressions of relative proteins for the purpose of elucidating molecular mechanisms.Results:(1) The positive expression rate of EGFR was 48.3%(29/60) in gastric cancer tissues, and the positive expression was correlated with depth of invasion, lymph node metastasis,and TNM stage.(2)The pEGFPN1-DNEGFR was constructed successfully, which was confirmed by PCR amplification assay, double enzyme digestion, DNA sequencing and bioinformatics analysis of nucleotide sequence. The expression of DNEGFR-EGFP was verified by Western blotting, and its predominant localization on the cell membrane of COS-7 cells was identified by Laser Scanning Spectral Confocal Microscope.(3) The expression of DNEGFR-EGFP was verified by Western blotting, and its predominant localization on the cell membrane of gastric cancer cells was identified by Laser Scanning Spectral Confocal Microscope. DNEGFR-EGFP decreased the phosphorylation level of EGFR protein, but didn't change EGFR mRNA level and protein level. (4) DNEGFR-EGFP led to G0/G1 arrest and induced apoptosis by down-regulating CDK2,Cyclin D1,pGSK-3β(ser 9) and up-regulating p21,p27,inhibited in vitro growth of human gastric cancer cells in the end; it repressed invasion and angiogenesis of SGC-7901 cells by inhibiting them from secreting MMP-2, MMP-9 and VEGF, and also inhibited adhesion ability and motility of cells. These results indicate that DNEGFR-EGFP reversed malignant phenotype of human gastric cancer cells in part.Conclusion: Immunohistochemistry results indicate EGFR takes part in gastric cancer progression, which lays a theoretical foundation on the application of biologicals targeting EGFR in gastric cancer biotherapy. The mechanisms that DNEGFR-EGFP inhibits endogenous EGFR function by down-regulating endogenous EGFR protein phosphorylation level are elucidated. It is verified that it reverses malignant phenotype of human gastric cancer cells in part, and the molecular mechanisms are elucidated, which lays a solid foundation for further research of"dominant negative strategy targeting EGFR"(we name the EGFR signal pathway blockade strategy as"dominant negative strategy targeting EGFR") in gastric cancer biotherapy.