The Influence Of Down-Regulation EIF4E Gene Expression By ShRNA On Biological Behaviors Of Human Breast Cancer MDA-MB-231 Cell Line

Objective: As an important regulatory factor of cell growth and degree, eukaryotic translation initiation factor 4E is closnesisiely related to tumorigenesisi.This study was to investigate the effects of EIF4E small interfering RNA(siRNA) on biological behaviors of Human breast cancer cell Line MDA-MB-231.Methords: Targeting EIF4E shRNA was to design and synthesis ,and it was clon into pGPU6/GFP/Neo eukaryotic expression vector.The pGPU6/GFP/Neo-EIF4E recombinant expression plasmid was constructed ,and was identifyed by enzyme restriction and sequence analysis.The recombinant expression plasmid was transfected into breast carcinoma cell line MDA-MB-231 by LipofectamineTM2000.Reverse transcription polymerase chain reaction,western blotting, Immunnofluorescence and Immunohistochemistry were used to detect the expressions of EIF4E,CyclinD1,VEGF-C,MMP-2, MMP-9 mRNA and protein,respectively; MTT assay,cell colony formation assay and Flow cytometr were performed to detect the effects of siRNA expression plasmid on the proliferation,cell colony formation rate and cell cycle of human breast cancer MDA-MB-231 cells;Hochest33258 attaining was used to determine cell apoptosis rate;Cell scratch experiment and transwell experiment were used to detect the invasion and migration of MDA-MB-231 cells, respeatively.Results: Recombinant expression plasmid pGPU6/GFP/Neo-EIF4E was successfully and it was stably transfected into MDA-MB-231 cell lines. The expressions of EIF4E,VEGF-C,Cyclin D1,MMP-9,MMP-2 mRNA and protein were decreased markedly after transfection (P<0.05).The growth of MDA-MB-231cells was inhibitied and the cell colony formation rates were significantly decreased.Cell apoptosis with nuclear chromentation and cell membrane were observed under Hochest33258 staining,the apoptosis rate in pGPU6/GFP/Neo-EIF4E was higher than control group (P<0.05).Flow cytometry showed the more G1 phase cells(71.3±0.467 vs 53.1±0.431)% and the less S phase cells(16.17±0.581 vs26.47±0.376)%(P<0.05). The invasion and migratin of stable tranfection MDA-MB-231 cells were significantly lower than control group cells (Invasive cells: blank control group 341.7±21.47,recombinant expression plasmid 79.43±11.21,p<0.05;migratory cells: blank control group 639±47.34, recombinant expression plasmid 298.23±20.01,p<0.05). Cell scratch experiment showed,group of recombinant expression plasmid cell migratin ability decreased significantly(P<0.05).Conclusions:Recombinant expression plasmid pGPU6/GFP/Neo-EI F4E could down-regulation EIF4E expression in human breast cancer MDA-MB-231cells and suppress its proliferation,invasion and migration to some extent,promote the cell apoptosis,increase a large number of cell to stay at G1 stage,decrease a lot of cells develop into S stage,the molecular mechanism may be related to silenced the expression of VEGF-C,Cyclin D1,MMP-2,MMP-9,suggesting that the EIF4E siRNA may be an effective method to treat breast carcinoma.